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Welcome
Welcome traveller to St. John’s, the City of Legends, and North America’s oldest city. Perched on the sides of the hills surrounding St. John’s harbour, and often wreathed in the misty fog that drifts in off the North Atlantic, St. John’s is steeped in history… and the paranormal… Dark alleys and laneways wind through the heart of the historic downtown area, and in the shadows lurk the shades of yesteryear.
Walking through the town, one is surrounded by the memories of public hangings, duels, and horrific murders, passing over forgotten cemeteries and unmarked graves, past buildings known to be visited by those who have passed over to the other side… Vengeful lovers, murdered soldiers, and mysterious fires await those who are brave enough to explore the secrets that lie in wait in St. John’s darkest corners.
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j.
0
Solve 4*f - 2*x = 9*f + 27, 5*x + 108 = f for f.
3
Solve -5*u - 30 = -5*l, 40010*u - 40011*u + 2*l = 11 for u.
-1
Solve -4*z = -q - 6, q + z = 6281 - 6282 for q.
-2
Solve 3*n = -2*m + 11, -5*n = 74*m - 77*m - 12 for m.
1
Solve 3 = -c, 3*d = c + 44 - 56 for d.
-5
Solve 0 = -355*b + 356*b - 2*m - 7, 5*b - 4*m - 5 = 0 for b.
-3
Solve 12*w - 1015 = 2*g - 959, -17 = 4*g - 5*w for g.
2
Solve -12*c - 5 = -5*g, -3*c = 33*g - 38*g - 40 for g.
-11
Solve 13*p + 5*r = 23 + 2, -5*p - 5 = -r for p.
0
Solve -3*r - 1040 = 2*v - 1053, v + 1 = r for v.
2
Solve -93*s + 88*s + j = -21, -s = 3*j - 17 for s.
5
Solve 4*p + 5 = 3*g, 3*p - 3*g + 288 = 285 for p.
-2
Solve 3*h = o + 3, 6*o + 14*h - 50 = 15*h for o.
9
Solve 0 = 2*k + 172*j - 175*j + 7, 0 = 2*k - j - 3 for k.
4
Solve -933*t + 929*t - 4*v + 24 = 0, -15 = 5*t - 4*v for t.
1
Solve 3*x = 12*m + 45, 15 - 13 = -m + 2*x for m.
-4
Solve -11*i = 5*k - 8*i - 5, 3*i = -10*k + 6 + 4 for k.
1
Solve -5*d + 202*z - 26 = 205*z, -2*d + 3*z = 2 for d.
-4
Solve -2*h = -5*d - 5*h, 31*h - 29*h - 13 = d for d.
-3
Solve -3*y + 119 = 26*l + 246, 4*y = 3*y - 6*l - 29 for y.
1
Solve 21 = q - 5*b, 4*q - 8*b + 0*b - 24 = 0 for q.
-4
Solve 21 - 18 = -4*u - 5*y, u = 4*y + 15 for u.
3
Solve -x - 4*r + 17 = 0, -4*x + 221*r - 37 = 216*r for x.
-3
Solve -2147*t + 2148*t - 5*h + 15 = 0, -t - 35 = -15*h for t.
-5
Solve 5*r + 2*w = -20, 272*w - 16 = 4*r + 267*w for r.
-4
Solve 8*q + 3*t = 3*q - 76, -3*t - 36 = -3*q for q.
-5
Solve 0 = 2*b + 2*c + 14, -2*b - 117 = -5*c - 96 for b.
-8
Solve 203 - 183 = -5*k - 2*m, 18*m = -5*k + 60 for k.
-6
Solve 0 = 3*k - 245*q + 243*q - 26, -4*k - 5*q + 73 = 0 for k.
12
Solve -j + 102237*d - 102236*d + 25 = 0, 6*j + 3*d = -93 for j.
-2
Solve 0 = -276*c + 274*c - 5*i - 23, -3*c - 2*i = 7 for c.
1
Solve 3*f - 10 = -b, 2*f - 44 = -39*b + 29*b for b.
4
Solve 4*p - 2*r + 4 = 0, 177 - 168 = 5*p + r for p.
1
Solve 2*h - 7*w - 10 = -2*w, -4*w - 36 = 4*h for h.
-5
Solve 4*w = 2*c + 6, -349*c - 5 = -344*c - 5*w for c.
1
Solve t - 1 = c, 5*c - 277*t + 3 = -273*t for c.
1
Solve 2 = -3*b - 2*j, 5*j = b + 155 - 126 for b.
-4
Solve -236*j + 40 = 4*m - 240*j, 3*j = -15 for m.
5
Solve -41 = -5*o + 5*s - 11, 0 = 7*s - s + 24 for o.
2
Solve -i + 17 = 3*g, 3*i - 28 - 2 = -8*g + 17 for g.
4
Solve 740*z = -5*o + 741*z + 4, 0 = 2*o + 2*z - 4 for o.
1
Solve 67*z = 64*z + 3*u - 24, -5*z = 2*u - 30 for z.
2
Solve -3*n + 38 - 11 = 6*q, 20 = 4*q + 4*n for q.
4
Solve b - 1696 + 1707 = -3*g, 5*b = 3*g - 1 for g.
-3
Solve -3*f + 200*o = 201*o + 7, 2*f = -3*o for f.
-3
Solve -7*m = -2*o - 31 + 28, -m = -1 for o.
2
Solve -3*v = 2*p + 3, -37*v - 4*p + 6 = -35*v for v.
-3
Solve -57 = -3*l - 5*y, -359*l + 362*l - 5*y = -33 for l.
4
Solve -53*z - 13 = -48*z - 3*j, 2*j + 18 = -2*z for z.
-5
Solve -2*x - 18 + 28 = 0, x = 4*u + 2*x - 5 for u.
0
Solve 4*i = -4*q + 4, 11000*q = -i + 11003*q - 15 for i.
-3
Solve -3*a - 500*b = -497*b - 39, 0 = -3*a + 4*b - 24 for a.
4
Solve -3*g + 3*n - 170 = -191, 0 = 3*g + 4*n + 14 for g.
2
Solve 3*s = -f - 6, 4*s = 9*f - 18*f - 8 for s.
-2
Solve 0 = -2050*n + 2045*n + 2*l - 19, -3*l = 3*n + 3 for n.
-3
Solve 0 = -4*u - m + 16, 0 = 62*u - 57*u - 3*m + 14 for u.
2
Solve -5*g = 3*x - 2, 3*g + 422 - 424 = -2*x for g.
-2
Solve -4*m + 20 = -5*b, 0 = b - 4*m + 12 + 8 for b.
0
Solve -2*p = -4*k + 6, 5*p + 15019 = 5*k + 15009 for p.
-1
Solve -2*k + 17 = 5*o, 3*o = 5*k - k + 31 for k.
-4
Solve 0 = 6*r + 3*x + 24 + 39, 51 = -2*r + 5*x for r.
-13
Solve -2541*y + 4*f + 100 = -2536*y, 40 = 2*y - 2*f for y.
20
Solve -v = 3*b + 11, 191 - 208 = 2*v + 5*b for v.
4
Solve 2*j - 46 = 9*r + 9 - 14, 4 = -2*j for r.
-5
Solve 3*l + 60*z - 55*z + 15 = 0, l = -5*z - 5 for l.
-5
Solve -4*t + 79 = 7*j, -3*t + 13 = -3*j - 38 for t.
18
Solve -92303*r + 18 = -92299*r - 6*q, -3*r + 3*q = -21 for r.
12
Solve -2*a + 2*h = h, 0 = a + 6*h + 13 for a.
-1
Solve 237*l + 2*d = 239*l + 14, 4*l + 10 = d for l.
-1
Solve 5*x - 26 = 3*q + 48, -5*x = 5*q - 90 for x.
16
Solve -2*d + 6 = 3946*b - 3945*b, -5*d - 3*b + 16 = 0 for d.
2
Solve 5*u = 5*h + 40, -68*h = 2*u - 6*h + 112 for u.
6
Solve 4*v + 30 = -5*y, 4*y - 7*v = -11*v - 28 for y.
-2
Solve 5*z = 10, 124*z = r + 69*z + 66*z - 27 for r.
5
Solve g = -3*b + 7, 2*g + 2*b - 44 = -38 for g.
1
Solve c - 4 = 0, -153*v + c = -154*v + 7 for v.
3
Solve -3*q + 3*v - 3 = 0, -11*v + 176 - 135 = 4*q for q.
2
Solve 5*b + 25 = -c, 148*b - 20 = 152*b - c for b.
-5
Solve 2 = -302*b + 304*b, -n = 2*b - 6 for n.
4
Solve 2*x - 21 = 5*g, 4*g + 249 = -2*x + 279 for x.
13
Solve 2*y = -2*o - 10, -62 = 641*y - 640*y + 20*o for y.
-2
Solve f = -24*z + 25*z + 7, 0 = 2*z + 4 for f.
5
Solve 32*c - 34*c + 5*d = -36, 5*c = -5*d + 125 for c.
23
Solve 2523*c - 2526*c = 4*h + 3, -5 = -2*c - 5*h for c.
-5
Solve 34*r = 39*r - 5*l, r = -4*l - 15 for r.
-3
Solve -5*j = 4*m + 34, 0 = 5*j + 5*m - 19243 + 19278 for j.
-6
Solve 2*q + 5*g + 4*g - g - 24 = 4*g, 0 = g - 5 for q.
2
Solve 29 = 5*v + 583*d - 585*d, -13 = -4*v + 5*d for v.
7
Solve 2*o + 12 = -2*y, 4*y - 2930*o + 36 = -2931*o for y.
-10
Solve -2*f - 14 = -2402*x + 2407*x, 4*x + f = -10 for x.
-2
Solve 4*q - 9*a + 3202 = 3164, -3 = -q - 4*a for q.
-5
Solve -2*n + 5*g + 15 = 0, g + 5 - 2 = -4*n for n.
0
Solve 25 = 4*k + v, 23*k = 18*k + 4*v + 5 for k.
5
Solve 0 = p + 2*f + 1, -419*f + 15 = 6*p - 414*f for p.
5
Solve -2*p - z + 5 = 10, -p + 2 = -4*z for p.
-2
Solve 17 = -7*s - 18, -5*n - 6 = -10*n + 6*n + 2*s for n.
4
Solve -2*j = -12, 5*j = -11*a - 1398 + 1472 for a.
4
Solve 2*s + 42 = -3*y, -60 = 222*s - 226*s + 3*y for s.
3
Solve 5*w = -4*c - 34, 78*w - 76*w + 4*c = -28 for w.
-2
Solve -2*j + 4*j - 3*y = -24 - 13, -35*j - 5*y - 130 = 0 for j.
-5
Solve -2*h = 3*q + 13, 5*h - 15 = -23*q + 25*q for h.
1
Solve -5*j - 515*m = -510*m + 65, 4*m = -20*j - 52 for j.
0
Solve -3*g + 38*o + 158 = 0, g - 18 = 7*o + 12 for g.
2
Solve 4*u - 2*q = -16, -4*u - 250 = -q - 236 for u.
-3
Solve -3*m + 19 = -18*u + 20*u, 4*u = -m + 13 for u.
2
Solve 2*y = -r - 6, -2*r - 25 = y - 7*r for y.
-5
Solve 0 = 4*y + 3*p + 25, -2*p + 43 - 61 = 4*y for y.
-1
Solve 2*r + 52 = -7*i, 418335*i + 5*r = 418340*i + 5 for i.
-6
Solve 8*p - 25 - 5 = -3*k, 3*p - k = 7 for p.
3
Solve 12 = -4*q - 0*q - 4*c, 5*q = 2*c + 20 for q.
2
Solve -y + 196492*o - 196496*o + 31 = 0, -2*y + 5*o - 42 = 0 for y.
-1
Solve -2*z + 3*v = 5, -6*v + 4*v = 2*z for z.
-1
Solve -3*r - 17 = m + 3*m, -567 + 560 = -m + 3*r for m.
-2
Solve -2*d - 225 = -2*f - 229, 2*f - 4*d - 6 = 0 for f.
-7
Solve -20*d + 24*d + 20*z = -24, -2*z + 12 = 4*d for d.
4
Solve 4*x - 32 = 3*y, -59*y + 55*y = -6*x + 46 for y.
-4
Solve 2*s + 12 = 86*j - 88*j, s - 14 = -5*j for j.
5
Solve -15 = -4*r + 178*z - 167*z, r = 3*z + 4 for r.
1
Solve -4*k + 2*y = -12, 0 = 4*y - 8*y for k.
3
Solve -3*s = 2*z + 2, 152 = -z - s + 150 for z.
-4
Solve -227 = -9*z + 5*o, -95*z + 2*o + 46 = -93*z for z.
28
Solve 0 = -3*w + 4*b - 11, -7717*b - 2 = -7718*b for w.
-1
Solve -3*x = 24, -4 = 1377*j - 1381*j + 2*x for j.
-3
Solve 5*o + r - 31 = 0, 136*o - 4*r + 36 = 134*o for o.
4
Solve 9*r = -18, -534*n - r - 87 = -539*n for n.
17
Solve q - 216*b + 220*b + 10 = 0, -3*b = 15 for q.
10
Solve 28*b = 3*d + 29*b + 9, 3*b + 13 = -2*d for d.
-2
Solve -5*p - x = -9, 0 = -19238*p + 19242*p + x - 7 for p.
2
Solve -11*p - 5*b + 1 = 4*p - 11*p, 0 = p + 21*b + 59 for p.
4
Solve 3*g - 5*n = -2*n + 3, -n - 4 = 2 for g.
-5
Solve -5*d - 6 = 4*q - 33, -4*d + 15*q = 33 for d.
3
Solve -4*v - 4 = -2*g, 2*g + 3277 = -3*v + 3260 for v.
-3
Solve 11*j + 66 = 0, 0 = -5*d - 2*j - 92 + 105 for d.
5
Solve 0 = -4*x + 3*l - 1095 + 1084, 4*l - 18 = 2*x for x.
1
Solve 56*q - 12 = 61*q - 2*b, 3*q - 5*b = -49 for q.
2
Solve 5*c + 9*h = 0, 13*c - 8*c = 2*h for c.
0
Solve 31*z + 6 = 35*z - 3*l, l + 2 = -4*z for z.
0
Solve -224*s - 11 = 3*u - 226*s, -u = 4*s - 15 for u.
-1
Solve 0 = -5*o + 20, 19 = -10376*v + 10379*v + 4*o for v.
1
Solve -8 = -p + 3*s, 60*s - 6 = p + 64*s for p.
2
Solve -26785*w - 20 = -26789*w - 2*i, w + 4*i = 40 for w.
0
Solve 5*s - 14 = 2*y, -2*s - 630 = y - 632 for s.
2
Solve 0 = -2*p - 2*c - 26, p + 13 = -2*c - 10 for p.
-3
Solve 3*w = -z + 14, -1 = z - 1151*w + 1149*w for z.
5
Solve 4*p - 144 = -2*v - 154, 20 = -2*p + 4*v for p.
-4
Solve 5*o + 5*u - 27 = 8*u, -28 = 7*u for o.
3
Solve 17 + 63 = -2*a + 14*s, 35 =
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Wayne Helgason saw tears in the eyes of Liberal prime ministers Jean Chrétien and Paul Martin when they spoke of his peoples’ plight. It was clear as day, he said, that improving the lives of aboriginals was more than political — for them, it was personal
Weeks ago, Tanya Buchanan committed to a few holiday parties and prayed she’d be able to find a babysitter —not an easy task in an Italian neighbourhood where there’s an apparent dearth of teens and every family has a nonna. The Toronto mother has desperately tried to find a babysitter in the area —just a […]
The former Supply Depot No. 1 of the Canadian Armed Forces is 800,000 square feet in size, and designed to withstand the force of a nuclear blast, which means it is, apparently, big enough and solid enough to weather the bad craziness of roller derby players pouring into it from all over the world this […]
The road to Attawapiskat is not paved. When there is a “road,” it is made of ice and runs atop a frozen James Bay. For the 2,000 Cree aboriginals living in the fly-in Ontario community, winter means access to the rest of the world.
The words spoken in the old cut-stone courthouse were quiet, meek and in boundless contrast to the leering swagger and antagonism displayed in 2007 during the height of the fiery native occupation in Caledonia, which was when the assault being settled here Friday actually took place
Roller Derby, a sport revived in Texas after lying dormant since the 1970s, pits teams of women who skate around an oval, with one skater on each team trying to pass the others — and the other players trying to prevent that from happening
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I just wanted to surf for some news about the state elections in germany and i´m going to drive mad about the masses of pops, flash, videos for all and everything. I mean every site, every picture, every yt video comes with a flash, a pop-up, an advertising premovie or something esle. this doesn´t make any fun anymore. Will there never be an end?
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[Fenofibrate improves energy metabolism and attenuates isoproterenol induced acute myocardial ischemic injury in rats via PPAR alpha activation].
To observe the effects of peroxisome proliferator-activated receptor (PPAR) alpha agonist Fenofibrate (FF) on energy metabolism and histology in isoproterenol (Iso) induced acute myocardial ischemic injury model. Male Wistar rats were randomly divided into control group, Iso group (5 mg/kg, i.p.) and FF group (80 mgxkg(-1)xd(-1) per gavage for 7 days, then Iso 5 mg/kg, i.p. n = 30 each). Twenty-four hours post Iso, heart weight/body weight ratio, myocardial histopathological changes (HE staining), serum and myocardial free fatty acids (FFA) levels, the myocardial protein expression of PPARalpha (Western blot) were determined. Compared with the control group, pathological myocardial injuries were observed under light microscope in Iso treated hearts and FF pretreatment could significantly attenuate these changes [necrotic area: 0 vs (10.00 +/- 3.00)% vs (7.36 +/- 2.60)%], the heart weight/body weight ratio, FFA in serum (501.17 +/- 43.69 vs 939.53 +/- 69.51 vs 736.53 +/- 70.30 micromol/L) and myocardium (62.01 +/- 9.19 vs 140.59 +/- 19.34 vs 116.28 +/- 14.03 micromol/L) were significantly increased while myocardial protein expressions of PPARalpha (251.57 +/- 10.95 vs 191.97 +/- 10.74 vs 215.08 +/- 9.61) was significantly downregulated in the Iso group and FF pretreatment could significantly attenuate these changes (all P < 0.05). Our data suggested that the FFA utilization was decreased in Iso induced acute myocardial ischemic injury and FF could attenuate Iso induced myocardial damage via activating PPARalpha signaling pathway.
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If you’re a brunette then you know that brown is never simply brown. It has a variety of hues hidden in the colors including caramels, honeys, coffees and chestnut colors. A chic bob with bangs can be the perfect way to show off your luscious locks.
Sounds weird, right? But actually, itâs not! This is probably one of the most common fruits found in India and that is also probably why we donât really relish it as much. But before throwing the banana peel into the bin, read this post to know
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With morning temperatures approaching 90 degrees one day in July 2015, a migrant laborer walking down rows of corn began to experience symptoms of heat exhaustion, including difficulty breathing and extreme nausea.
The laborer was working near Boone, Iowa, for an independent contractor with the St. Louis-based Monsanto Co., which hires such contractors annually to recruit and oversee teams of migrant farmworkers in the cornfields of the Midwest.
The farmworker was doing tasks related to detasseling – the practice of lopping off corn tassels, which enables growers to produce lucrative high-yield hybrid corn seed.
Each year, seed-corn companies like Monsanto bring in thousands of laborers to produce the hybrid seeds, most of which are genetically modified.
The companies sell the seeds to farmers worldwide, in what has become an $11-billion GMO corn industry. The farmers grow the seeds into corn for sale as food, ethanol, livestock feed and components of a range of industrial products, from fireworks to ceiling tiles.
In a two-year investigation of GMO seed-corn production, the Midwest Center for Investigative Reporting found repeated allegations of labor violations over the past decade against Monsanto, its counterpart DuPont Pioneer, other seed companies and the companies’ contractors.
A review of federal documents, lawsuits and Monsanto records – and interviews with advocates and experts – shows that the allegations include broken recruiting promises, minimum-wage violations, improperly withheld pay and substandard living conditions in seed-corn production of Monsanto and Pioneer.
NEEDED MORE WATER
As a way to easily locate workers in emergencies, Monsanto requires its detasseling crews to stay within the rows they are assigned to work in. But that morning, the worker finished his water and needed more. Instead of walking the long way down his row of corn, he crossed over other rows of corn to get water more quickly.
After learning about the shortcut, Monsanto’s contractor, Eleuterio Cortes, allegedly fired the worker. The worker would not speak about his experience for this story and requested anonymity to protect his employment prospects. But an Iowa Legal Aid attorney, Jessica Taylor, complained to Monsanto on his behalf.
According to a Sept. 22, 2015, letter Taylor sent the company, Cortes said he acted because the worker violated company safety policies and “couldn’t handle the job.” The letter also alleged Cortes was not properly registered as a contractor with U.S. Department of Labor.
In response to the letter, Mary Tonkin, Monsanto’s senior counsel at the time, wrote Taylor that the worker was not “terminated” but quit — and that “Mr. Cortes has provided services for Monsanto for several years and has always provided a copy of his registration.”
She added: “We do not have a copy of the registration and are checking to see what happened.”
She offered to pay the worker $500 for the “oversight.”
But after Taylor sent additional registration information from the U.S. Department of Labor, Tonkin conceded that it was “more likely (the worker) was terminated than quit” — and wrote that Cortes, the contractor who allegedly fired the worker, had submitted misleading paperwork to the company.
“I am very disappointed that the documentation provided to Monsanto by Mr. Cortes is false,” Tonkin wrote. “That seriously undermines Monsanto’s view of his credibility.”
Monsanto’s legal team had already handled a case involving Cortes’ license two years earlier.
According to a 2013 lawsuit filed against Monsanto by Texas RioGrande Legal Aid and settled out of court later that year, Cortes in 2010 recruited a crew of 22 migrant workers to the same Monsanto job in Boone, Iowa, then denied them promised housing and pay — and also was without a license at the time. A review of U.S. Department of Labor records revealed no evidence that Cortes was licensed at the time of that job or the 2015 job.
To resolve the second complaint, in 2015, Monsanto paid the worker $1,250.
Tonkin stated in a letter to the legal aid lawyer: “We have placed Mr. Cortes on our do not use list.”
Yvette Vela
Nila Cantu, at her home in South Texas, was among the detasslers who complained about a Monsanto labor contractor who recruited the workers to a 2012 job in Iowa. (Yvette Vela)
HIRED AGAIN
By the next year, however, Cortes was back on a 2016 Monsanto list of active contractors, which legal aid workers gave the Midwest Center for Investigative Reporting. According to Taylor, Cortes did indeed work at the Monsanto site at Boone, Iowa, that year.
Monsanto would not comment directly on Cortes or whether the company employed him in 2016, although it did respond to requests for comment for this story. Cortes could not be reached for comment despite repeated attempts.
But it would not be the first time the company brought in a contractor who had previously been the subject of labor complaints in similar work.
In the past decade, Monsanto approved the hiring of three contractors — Cortes, Hermilo Cantu, Jr., and Abel Cuello — after the contractors had previously triggered complaints related to their work in seed-corn production, according to a review of Department of Labor records and complaints to legal aid offices, including eight lawsuits naming the company over the decade.
Monsanto is not the only target of labor allegations related to corn detasseling. A review of more than a decade of complaints to legal aid offices, including more than a dozen lawsuits, show labor-related allegations filed against DuPont Pioneer and other seed-corn companies, along with the companies’ contractors.
The Midwest Center’s review looked closely at five detasseling-related complaints against Monsanto, including four lawsuits, in which the workers who complained were not prevented from discussing the cases and workers in some of the cases agreed to be interviewed.
But according to legal aid lawyers, both Monsanto and Pioneer generally operate the same way in one important respect: They do not explicitly accept liability for all their contractors’ actions.
Monsanto and legal aid
Over the past decade Monsanto has faced numerous labor complaints, including lawsuits, related to its use of contractors to recruit and supervise workers to produce the company’s seed-corn.
This has put it into regular contact with legal aid clinics representing the workers.
In an email, Monsanto spokesperson Charla Lord wrote “[we] work collaboratively with legal aid groups to address any worker concerns. Over the last several years we have sought their input to continuously improve in this area. We meet regularly with legal aid group representatives so we can update them on activities and listen to their thoughts or concerns.”
She added that the company also invites legal aid representatives to Monsanto work sites “so they can meet our workers and distribute materials pertaining to their legal aid services. This is done during our safety training at the beginning of each detasseling season and again at the start of harvest activities.”
Legal aid lawyers largely confirmed such access in recent years, saying the company generally does quickly address complaints.
“Monsanto has gotten religion on the need to monitor their labor contractors effectively,” says Tom Thornburg, a former attorney with the Michigan-based legal aid clinic Farmworker Legal Services. “They say, ‘we want to hear from you if you have issues. We don’t want these to blow up in litigation.’ I don’t know what caused it, but perhaps some degree of frustration over complaints and lawsuits.”
Legal aid clinics are part of the congressionally subsidized Legal Services Corporation. The earlier Monsanto settles, Thornburg and his counterparts at other clinics pointed out, the less the taxpayer-funded legal aid offices have to spend in resources and court time—and the sooner cases are closed for low-income workers who have toiled on company jobs, most of whom can ill afford any broken promises on wages.
“Workers who have rights that need vindication wait less time,” Texas RioGrande Legal Aid attorney Daniela Dwyer says. “To Monsanto’s credit, they negotiate more effectively than any other seed-corn company.”
But Miguel Keberlein-Gutierrez, attorney with the Illinois legal aid clinic LAF, said the company’s quick responses, and strategy to preempt litigation where possible, do not offset the costs of having to address complaints in the first place.
“While settling cases in general saves resources from the point of the court system,” he wrote, “it has to be weighed against these companies refusing to change their exploitative ways.”
Partly because legal aid clients have little money and inadequate resources to wage a long legal battle, Keberlein-Gutierrez wrote, detasseling cases routinely settle, which means they cannot set precedents or change workplace or industry standards.
“[Seed-corn companies] are aware of how difficult it is to litigate these cases,” Keberlein-Gutierrez wrote, “and history is on the employer’s side when it comes to this specific industry. [Detasseling cases] are little more than a nuisance for these companies—and thus they have little incentive to change.”
In the end, legal aid clinics working on seed-corn labor cases are “constantly doing outreach to workers and bringing complaints to the company’s attention” and as a result “we end up using more resources long-term on a system that doesn’t change.”
Greg Schell, an attorney who has represented migrant farmworkers for more than 30 years at Florida Legal Services and, more recently, at Texas Rio Grande Legal Aid, said Monsanto and Pioneer “have declined to assume key employer-like responsibilities.”
For one thing, Schell said, the two companies delegate to their contractors the responsibility for keeping track of hours for payment, typically an employer’s responsibility.
The ability to log the hours worked by farmworkers, he said, allows contractors the chance to underpay workers to keep money for themselves.
“The heart of the problem is that timekeeping is not accurate,” he said. “If the companies kept the time and did so accurately, people would get paid more.”
In practice, according to Schell and other legal aid lawyers, the companies have not explicitly acknowledged a 2007 appeals court ruling holding seed-corn companies responsible for the actions of contractors who recruit and supervise workers.
The seed-corn company targeted in the case, Reyes v. Remington Hybrid Seed Co., “must be deemed the workers’ employer for events that occurred in the fields under its management or in its offices,” the court said.
Monsanto and Pioneer, the two largest seed-corn companies, have settled numerous allegations like those at issue in Reyes.
Both declined to comment on the Reyes ruling.
In response to similar litigation, the companies generally deny wrongdoing or claim insufficient knowledge to respond to the allegations. The settlements normally explicitly stipulate no admission of wrongdoing on the part of the defendant.
Monsanto and DuPont Pioneer declined to respond in detail to most questions about complaints related to their use of contractors.
Monsanto spokeswoman Charla Lord would not comment directly on extensive written questions about allegations regarding the company’s use of the contractors, despite repeated attempts since 2015.
SAFETY ‘TOP PRIORITY’
But she wrote in an email in 2015 that the company regards safety as “our top priority.”
“That includes guaranteeing the safety of all the products we produce and ensuring the safety of all our workers around the world,” she wrote.
Lord wrote that the company is committed to “workers who work in farm fields” and has pledged to provide them with “a safe and respectful work environment.”
She also pointed out that the company works closely with legal aid to settle any complaints quickly, a practice several legal aid lawyers confirmed.
In 2015, then-Pioneer spokeswoman Jane Slusark declined to address extensive questions about the company’s use of contractors, saying “we do not comment on active litigation.”
In late 2016 Pioneer spokeswoman Susan Mantey declined to respond to the same questions and additional ones about the company’s use of contractors, saying, “DuPont Pioneer follows all state and federal laws regarding migrant worker housing, and we take the living conditions of our workers seriously.”
In a June 2017 email, she added: “DuPont Pioneer is committed to the core values of respect for people, safety, environmental stewardship, and commitment to the highest ethical standards.”
Spokesman Paul Bertels of the National Corn Growers Association, which represents 300,000 corn farmers and lists seed-corn producers like Monsanto and Pioneer as “industry partners,” also declined to comment directly on the use of contractors and complaints against them and the companies that employ them.
“The NCGA does not have a position regarding FLCs,” Bertels wrote in an email, referring to the acronym for the Department of Labor’s designation of Farm Labor Contractors.
USING MIDDLEMEN
In recent decades, complaints have arisen as large agricultural businesses have increasingly hired contractors to serve as middlemen between agricultural companies and migrant workers.
Legal aid lawyers and labor advocates say the current labor-contracting system too often results in abusive – and sometimes dangerous – conditions in the fields.
“Hundreds of thousands of farmworkers out there are subjected to labor abuses,” said Bruce Goldstein, president of Farmworker Justice, a national advocacy group. “Almost any time we talk to farm workers, we find they’re suffering illegal conditions, including in genetically modified seed-corn. Labor contracting situations (lead to) rampant violations of their rights.”
While there are reports of worse conditions in other U.S. crops, labor allegations are better documented in genetically modified seed corn, according to legal aid attorneys and labor specialists. That documentation provides a window into the use of contractors and migrant farmworkers nationwide.
NUMBER OF COMPLAINTS
Over the past decade, legal aid clinics that represent the low-wage workers in Illinois, Indiana, Iowa, Michigan and Texas estimate that they have fielded a combined total of about 50 labor complaints, including lawsuits, related to GMO seed-corn production.
Legal aid lawyers and farmworker advocates said the numbers hardly reflect the scale of illegal treatment of migrant farmworkers or violations of federal law in the cornfields.
“These complaints are the tip of the iceberg,” said Daniela Dwyer, an attorney with Texas RioGrande Legal Aid.
For one thing, Dwyer and others said, legal aid clinics cannot file civil complaints on behalf of the majority of farmworkers nationwide.
Since the mid-1990s legislation in Congress, which subsidizes legal aid, has effectively prevented legal aid organizations from representing some legal immigrants and all undocumented workers.
But undocumented workers now represent more than half of farmworkers nationwide, according to Philip Martin, a University of California-Davis professor of agricultural economics and an expert in farm labor statistics.
In addition, workers eligible for legal aid also remain reluctant to voice grievances, Dwyer said. Among other reasons, she said, they fear losing their wages, forfeiting scarce work the subsequent year, or being stranded with no funds to get home.
Even workers who describe problems or register initial complaints are hard to track down for necessary vetting and are often “traumatized.”
“It’s hard just getting clients to talk, for any reason,” Dwyer said.
Miguel Keberlein Gutiérrez — a Chicago-based lawyer with the LAF legal aid clinic who has represented hundreds of farmworkers in cases against Monsanto, DuPont Pioneer and other companies — estimated that farmworker complaints to legal aid offices represent as little as 10 percent of the violations that take place in seed-corn production each year, an estimate echoed by legal aid counterparts in two other states.
“In 15 years,” Keberlein Gutiérrez said, “I have yet to speak with a (detasseler) who hasn’t revealed suffering some type of violation.”
The Midwest Center for Investigative Reporting’s review of detasseling-related allegations made to legal aid offices has turned up numerous cases involving DuPont Pioneer, Monsanto and contractors for both companies — sometimes involving up to 30 or more members of a particular work crew.
The cases, ranging from informal attorneys’ letters to formal litigation, include more than a dozen suits filed over the past 10 years, almost invariably involving workers’ claims of being misled at recruitment, not paid the days or hours they’re promised, and housed in substandard facilities — in surroundings where they don’t know anybody, don’t speak the language and have little recourse but to accept the terms.
“Plaintiffs were in desperate economic need of work,” stated one typical allegation, from the 2013 lawsuit against Cortes. “And being thousands of miles from their homes back in Texas and lacking money and the means of return transportation, Plaintiffs were left with no real choice.”
RISE IN CONTRACTORS
At the root of the rising complaints, farm labor advocates and legal aid attorneys say, is an increasing use of contractors.
From 2001 to 2015, the number of farm labor contractors and crew leaders nationwide increased nearly 20 percent to more than 173,000, with their wages rising 40 percent, according to the Quarterly Census of Employment and Wages.
Midwest Center for Investigative Reporting graphic
The annual wages paid to farm labor contractors grew by 40 percent between 2000 and 2015. Source: U.S. Bureau of Labor Statistics
“As the workforce went from local teens to Spanish speakers,” he wrote in an email, “there was a need for bilingual go-betweens (and) risk absorbers”—including those who could absorb the risk of immigration raids. “The uptick in farm labor contractors occurred alongside rising illegal migration in the 1990s.”
While statistics for the prior decade are more elusive, growers started relying more on contractors after Congress passed anti-immigration legislation in the late 1980s, which brought the threat of immigration crackdowns, according to the farm labor statistician Martin.
In seed-corn production, Martin said, the change intensified an earlier trend: In the 1980s, high school students who had traditionally done detasseling throughout the Midwest (and still do in some counties) began finding more non-farm summer work.
By the 1990s, the two trends converged, as seed-corn companies began hiring more contractors, or crew leaders, to recruit migrant farmworkers from South Texas.
Problems have arisen, legal aid lawyers said, primarily because crew leaders must work within strict profit margins to recruit and employ the workers.
Seed-corn companies, the lawyers said, typically arrange to pay a contractor a lump sum, but reserve the option to require extra work, with no guarantee of compensating the extra time it takes, to complete the work to specification.
One Monsanto contractor, for example, had to perform work “in accordance with Company quality standards,” according to a 2012 contract that Midwest obtained from the records of a lawsuit filed by the contractor against Monsanto.
Under such contractual terms, Dwyer and Schell said, companies can require contractors to direct their crews to make extra passes in the field — and leave the contractors to cover the cost of paying the crew for the extra work.
MATH SKILLS AND HONESTY
This might not otherwise cause problems for the workers if the company took full responsibility for the workers, Dwyer said. She pointed out that Pioneer now issues checks to crew members directly, but said that both Monsanto and DuPont Pioneer allow the contractors to log the hours of their crew, which she said is often where the alleged trouble starts.
“The number of hours could be dictated by the seed-corn company, but it’s not,” she said. “Monsanto (relies) upon the math skills or the honesty of the crew leader to make sure people are properly paid or given a stub.”
Often the crew leaders are economically vulnerable, too, just “one step from being farmworkers themselves,” said Schell, the attorney at Texas RioGrande Legal Aid. “There is a real incentive for the labor contractors to skim from the workers. The fee paid the contractors by Monsanto and the other seed companies (is) insufficient in many instances for the contractors to pay the workers the wages required by law, pay all taxes and the like, and still make a profit.”
Given their constraints and responsibilities, Schell said, crew leaders can often end up looking to wrest a profit from their hires by committing “cost-cutting violations,” including “violating minimum-wage laws.” (Federal minimum wage is governed by the federal 1938 Fair Labor Standards Act.)
According to Schell and other labor lawyers, the 2007 ruling on the Reyes v. Remington Hybrid Seed Co. case would put the onus on the companies for such alleged violations.
But companies generally take the position that independent contractors are responsible for abiding by the law in recruiting and supervising workers.
“(The) Farm Labor Contractor is the employer of these workers,” Monsanto’s Lord wrote in an email. “In some circumstances Monsanto may be the statutory co-employer.”
Lord would not elaborate on which circumstances the company would be a co-employer or explain the company’s responsibility for recruitment of workers.
LITTLE MATERIAL CONSEQUENCE
But if they fail to explicitly acknowledge liability for their contractors, as legal aid attorneys say the companies do, Monsanto and its counterparts face little material consequence.
Because Congress has prevented legal aid clinics from filing class-action suits since the mid-1990s, they said, the companies typically pay out only $500 to $2,500 per worker in individual settlements.
“Monsanto seems to have built in an amount of roughly $500 per person as an average settlement amount from what we’ve gathered over the years,” Keberlein Gutiérrez, the Illinois Legal Aid attorney, said, adding that the figure often rises in negotiations — but not a lot. “It’s hard for clients to resist any settlement offer because a family of four only makes an average of $14,000 a year.”
Jim Kessler, immigration branch chief for the agency’s Wage and Hour Division, and Michael Kravitz, the division’s communications director, agreed only to an off-the-record interview for this story.
But according to results of a Freedom of Information Act request, the agency’s detasseling-related fines of seed-corn companies run in the tens or hundreds of dollars per worker — and fines for violations in the cornfields in the past decade cost Monsanto only $850.
“You show Monsanto on paper that they have an obligation (under Reyes) that they need to know what their contractors do and police them – that they’re liable – and they will simply settle,” said Thornburg.
“The next season, their crew leaders will repeat the violation — and they’ll settle again.”
By maintaining distance from the behavior of their contractors, and by effectively capping the fees they pay the contractors, the companies invite behavior that spurs consistent complaints each year, said Schell, Thornburg and other attorneys.
“Monsanto has in place substantial oversight mechanisms which superficially should provide substantial protection against wage theft,” said Schell. “But glaring holes in this oversight structure all but guarantee a ‘see no evil, hear no evil’ posture (on) wage problems.”
Many wage problems, according to the Midwest Center’s review of the complaints, start at the places of recruitment in south Texas where contractors allegedly violate farmworker law requiring full disclosure of work terms.
“BAIT AND SWITCH”
In anticipating tight margins and the companies’ strict specifications, contractors often recruit workers by a practice of “bait and switch,” said Bill Beardall, a University of Texas at Austin law professor and migrant labor advocate who helped draft the 1983 federal Agricultural Worker Protection Act.
“Companies lure workers hundreds and even thousands of miles” on the false promises of the contractors, Beardall said. “They use a series of illegal practices that push the risk for unreliability of contractors onto the workers themselves.”
As a result, according to Beardall, Schell and other labor lawyers, the companies sometimes end up paying settlements on their use of contractors.
But by arranging to pay contractors a lump-sum amount, usually per acre, the lawyers said, Monsanto and DuPont Pioneer keep annual wage expenses and liabilities low and predictable — boons in a highly volatile, weather-sensitive industry.
“We’ve attempted to address issues with the seed-corn companies,” said Dwyer. “But season after season we get the same legal complaints, against multiple crew leaders,” she said. “It’s the contractors’ business plan and the seed-corn companies are wedded to it. It’s broken promises as a business plan.”
DETASSELING-RELATED COMPLAINTS
Hybrid corn has been commercially available since before the 1930s and not all hybrid corn is genetically modified.
Since the late 1990s, genetically modified technology has transformed the field, in which now GMO corn seeds account for more than 90 percent of acreage in the United States and about $11 billion in annual sales, according to the National Corn Growers Association.
The association does not keep track of the number of farm labor contractors used in seed-corn production – or on the numbers of workers involved.
But according to Martin, the agricultural labor specialist, GMO hybrid corn attracts scores of contractors and thousands of migrant workers to the fields each growing season.
In recent years, seed-corn companies have increasingly begun shifting to H-2A foreign guest workers, who are recruited legally within other countries, including Mexico and its Central American neighbors to the south. They receive higher-than-minimum-wage pay, but do not have the same protection under the Agricultural Worker Protection Act.
But for now, according to Keberlein Gutiérrez, the Illinois-based lawyer with the legal aid clinic LAF, much seed-corn production still depends on migrant workers recruited from bus stations, parks, and other sites in the Rio Grande Valley.
WORSE CONDITIONS IN OTHER CROPS
The use of contractors doesn’t always go badly. Sometimes wage-related paperwork is maintained, housing is adequate, workers are paid their due, and seed-corn companies hire scrupulous contractors who keep promises.
Indeed, Schell and others point out work in other crops nationwide generally involves even worse labor conditions than corn.
For one thing, Schell and Martin said, pay in GMO corn looks generous compared to other crops.
“Many (detasselers) make minimum wage,” he said. “In Florida, I’m used to representing orange harvesters making the equivalent of $2 or $3 an hour.”
With the higher pay, detasseling jobs are more likely to be filled by documented workers, who have more protection under relevant federal laws and access to legal aid clinics.
The greater access of documented workers, Schell and Martin said, partly explains why work in seed corn has left a relatively full record of allegations.
In addition, legal aid offices may sue GMO seed-corn companies disproportionately more than other growers because “the seed companies are deep pockets,” as Martin put it in an email. “There are probably worse offenses in fruit picking on small farms, etc., but no deep pockets for suits and less publicity.”
Compared to smaller seed-corn companies, Monsanto and DuPont Pioneer also hire the most workers, said Keberlein Gutiérrez — and those workers have greater access to legal aid.
“The bigger operations run more work sites and labor camps [where] it is often easier for us to do outreach (and) get information on when workers are around,” he said.
By contrast, legal aid offices have fielded relatively few complaints against smaller companies.
But legal aid attorneys said the smaller companies use contractors in similar ways, with similar results.
“Some of the smaller ones may actually be worse,” Gutiérrez said, “because they operate even more out of the spotlight.”
MONSANTO GUIDELINES AND SAFETY TRAINING
Monsanto provides guidance, training and some support to its work crews.
“In all situations,” Lord, the Monsanto spokesperson, stated in an e-mail, “Monsanto provides workers with safety training, personal protective equipment, workers’ compensation insurance and in some location, housing. We also audit payroll records to make certain all time is recorded and the workers are paid legally and what they have been promised….”
Lord also wrote that the company trains “all Monsanto employees who interact with field workers on the topic of respect for all workers.”
To facilitate fair treatment of workers, Monsanto issues each contractor a one-page set of guidelines that clarify “expectations Monsanto holds for all Farm Labor Contractors” on issues ranging from pay to violent threats.
A version of the document that Lord provided to the Midwest Center in 2015 states: “Monsanto works hard to select Farm Labor Contractors (FLC’s) who share our commitment to worker safety, respect and creating a great place to work.”
Lord would not comment on how the company enforces the document or how it selects labor contractors – including the extent to which the company uses background checks.
But company, court and federal documents show that the Monsanto hired three contractors – Cortes, Abel Cuello, Jr., and Hermilo Cantu, Jr. – after they had previously triggered labor-related complaints, ncluding litigation.
Cortes did not respond to multiple letters to a known address or calls to his last known phone number.
Cuello and Cantu could not be reached for comment either, despite multiple letters and calls to multiple addresses and phone numbers, obtained through multiple sources.
Pat Geneser, Monsanto Migrant Seasonal Labor Manager, did not respond to the Midwest Center’s requests to reach these detasseling contractors or any others used by the company.
Monsanto senior counsel Mary Tonkin retired in September.
Her replacement, Jennifer Scheessele, was copied on the last two rounds of written questions to Lord, the Monsanto spokeswoman. Neither party responded to the questions, which included an inquiry about the reported re-hiring of Eleuterio Cortes.
But Taylor, the Iowa Legal Aid lawyer, said Cortes was indeed re-hired at Boone, Iowa, in 2016.
She said his performance again brought up “a number of concerns … from the workers” on which her office is “looking into filing complaints.”
While declining to provide details pending an investigation, Taylor said that the concerns involved wages and other “pretty typical (labor) issues” — and meant, for the workers detasseling on that Monsanto job, “things didn’t work out as promised.”
For the 2017 season, Taylor said, it was too early to tell whether Cortes would be once again contracting in Boone, Iowa, but she said that earlier in 2017 he reportedly had been working for Monsanto in a different capacity in Huxley, Iowa — a report that Lord would not corroborate.
Taylor added in an email: “Monsanto has not given us the list of FLCs and housing locations (as) they have in the past. They are relying more and more on H-2A workers contracted through other agencies, and have told us we need to contact them directly for that information.”
Laird Townsend, an award-winning editor and freelance reporter whose work has appeared in major national newspapers and magazines, is the director of Project Word, a fiscally sponsored project of Investigative Reporters and Editors (IRE). Project Word administers the Freelance Investigative Reporters and Editors program (FIRE), which provides unaffiliated reporters with stipends and reporting services.
We can have impact on your life when we publish an IowaWatch investigation. You don’t have to guess when we’ll publish our next story. We’ll tell you in an email. Take advantage of this service and subscribe now to our newsletter.
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Across the Zodiac eBook
The girl retired for a few minutes, and reappeared
in a cloak and veil like those of her new companions,
but of comparatively cheap materials. As we passed
the threshold, Eveena gently and tacitly but decisively
assigned to her protegee her own place beside
me, and put her right hand in my left. The agitation
with which it manifestly trembled, though neither
strange nor unpleasing, added to the extreme embarrassment
I felt; and I had placed her next to Eunane in the
carriage and taken my seat beside Eveena, whom I never
permitted to resign her own, before a single spoken
word had passed in this extraordinary courtship, or
sanctioned the brief and practical ceremony of marriage.
I was alone in my own room that evening when a gentle
scratching on the window-crystal entreated admission.
I answered without looking up, assuming that Eveena
alone would seek me there. But hers were not the
lips that were earnestly pressed on my hand, nor hers
the voice that spoke, trembling and hesitating with
stronger feeling than it could utter in words—
“I do thank you from my heart. I little
thought you would wish to make me so happy. I
shrank from showing you the letter lest you should
think I dared to hope.... It is not only Velna;
it is such strange joy and comfort to be held fast
by one who cares—to feel safe in hands as
kind as they are strong. You said you could love
none save Eveena; but, Clasfempta, your way of not
loving is something better, gentler, more considerate
than any love I ever hoped or heard of.”
I could read only profound sincerity and passionate
gratitude in the clear bright eyes, softened by half-suppressed
tears, that looked up from where she knelt beside
me. But the exaggeration was painfully suggestive,
confirming the ugly view Enva had given yesterday of
the life that seemed natural and reasonable to her
race, and made ordinary human kindness appear something
strange and romantic by contrast.
“Surely, Eunane, every man wishes those around
him happy, if it do not cost too much to make them
so?”
“No, indeed! Oftener the master finds pleasure
in punishing and humiliating, the favourite in witnessing
her companions’ tears and terror. They
like to see the household grateful for an hour’s
amusement, crouching to caprice, incredulously thankful
for barest justice. One book much read in our
schools says that ’cruelty is a stronger, earlier,
and more tenacious human instinct than sympathy;’
and another that ’half the pleasure of power
lies in giving pain, and half the remainder in being
praised for sparing it.’ ... But that was
not all: Eveena was as eager to be kind as you
were.”
“Much more so, Eunane.”
“Perhaps. What seemed natural to her was
strange to you. But it was your thought
to put Velna on equal terms with us; taking her out
of mere kindness, to give her the dowry of a Prince’s
favourite. That surprised Eveena, and it puzzled
me. But I think I half understand you now, and
if I do.... When Eveena told us how you saved
her and defied the Regent, and Eive asked you about
it, you said so quietly, ’There are some things
a man cannot do.’ Is buying a girl cheap,
because she is not a beauty, one of those things?”
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Q:
Force div to have the size of background image
When using a background image on a Div I am not able to display the full height of the image, neither using height:auto; or height: 100%. Why so?
How can I solve the problem?
I have created a JS Fiddle here: https://jsfiddle.net/2d0npz2v/5/
body, html {
height: 100%;
margin: 0px;
}
.imageContainer2 {
background-image: url("http://i.imgur.com/AWi7r5m.jpg");
background-position: center top;
background-size: 100% auto;
height: 100%;
}
<div class="imageContainer2"></div>
UPDATE
Just for clarity, the image needs to be 100% width, and auto height (not cut at the bottom).
This example works but it's using the "content" property instead of the "background-image": https://jsfiddle.net/j47a6x7s/. I am looking for the same behaviour but without using content.
A:
Well, the reason why this is?
In your working example you use content. A content has it's own height and uses up space, which the other elements on the page have to respect.
With the background-image solution, you use a background, which does not use space. The .imageContainer2 element can not know about the height of the background-image AND adapt itself to it.
This very problem was addressed here: How to get div height to auto-adjust to background size?
Just check, if the workaround is suitable for you
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Inhibition of established tumor growth in syngeneic mice by local inoculation of engineered mouse myeloma cells secreting a recombinant fusion protein RM4/TNF.
Mouse myeloma cell line VKCK/RM4-TNF secreting the recombinant fusion protein RM4/TNF was used to study the relationship between tumor necrosis factor (TNF) secretion of tumor cells and its tumorigenicity, and to study the potential mechanisms responsible for the antitumor immune response. To evaluate tumorigenicity, 5 x 10(5) viable TNF-secreting VKCK/RM4-TNF and non-TNF-secreting VKCK tumor cells were subcutaneously injected into BALB/c mice. Tumor progression or regression was evaluated 2 weeks after tumor inoculation. Our animal studies showed that RM4/TNF secretion by VKCK/RM4-TNF tumor cells curtailed its tumorigenicity in BALB/c mice and induced a long-term, protective immune response against a subsequent challenge of the parental VKCK tumor cells. Our animal studies in T-cell subset-depleted BALB/c mice and in T-cell-deficient nude mice demonstrated that both CD4+ and CD8+ T cells play a major role in the reduction of tumorigenicity. In addition, our results further showed that local inoculation of irradiated VKCK/RM4-TNF cells secreting TNF was able to significantly inhibit the established VKCK tumors in syngeneic mice. This study thus highlights the potential utility of engineered tumor cells secreting RM4/TNF in cancer gene therapy.
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Topics
01 April 2011
Pioneer buzzing before Musikmesse
Skratchworx has posted a rumor about Pioneer launcing a product named DJM-T1. In the setup list of controllers for Traktor Pro 2 the mixer is listed under the DDJ-T1, so this is very viable. This might be an old codeword for DJM-900 NEXUS which is sertified for Traktor, but I don't think so.
Pioneer has responded in their forum that their showing off some new gear at Musikmesse in Frankfurt April 6th.
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THE APOCALYPTIC VISION IN THE FICTION OF WALKER PERCY (LOUISIANA, SOUTHERN, RELIGION, TWENTIETH-CENTURY)
Abstract
The novels of Walker Percy comprise books of revelations, His protagonists begin as seers, usually in a quite physical fashion. By suddenly performing an act of attention to the moment at hand, they experience an apocalyptic unveiling of mysteries and disclosure of secrets. As the world and time come back to these seers, they come to themselves and recover consciousness. In Percy's demythologized and partially realized version of apocalyptic eschatology, this instant of sight and insight signifies the imminent end of their old order. No longer objectified or abstracted, his visionaries gain a new cosmos through their new way of seeing.^ Percy may individualize the apocalypse, but he does not neglect its social dimension. While his protagonists seek clearer and more complete vision throughout the course of the novel, they pronounce judgment on an entire age for being lost in the cosmos. As a result, Percy's eschatological prophets foresee approaching doom. Their challenge is to find a new order amid the ruins of the world and of their own life. The revelation of love, the logos of Percy's fiction, provides the context in which his apocalyptists may finally discover their identity and perhaps come to God.^ Awareness of the divine end of the world grants these seers their own tentative visions of the Second Coming. Beginning with an end and ending with a beginning, Percy's novels progress from catastrophe to the possibility of a new creation. But the last pages of his fiction always promise that there is more yet to be seen. The deliberately open-ended conclusions enact the watching and waiting essential to hope in the God whose last day is always now and alway coming. This resolution with its gaze toward the future marks the true beginning of the book in the life of the reader, for Percy envisions his novels as apocalypses themselves. His fictions of the eschaton find their end in the reader's revelation. ^
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Dezmine Wells is a big, fast, strong college basketball player -- a 6-foot-4 guard built like a prototypical linebacker, who plays the game like one, too. He was a top-50 recruit who only scratched the surface of his potential in his first season at Xavier, but averaged nearly 10 points and five rebounds a game (along with a 108.1 offensive rating and 37 percent from beyond the arc) in a largely tumultuous year.
After a prosecutor and grand jury's forceful rebuttal of Xavier's decision to expel Wells, his services would have been warmly welcomed by any of the nation's best teams, including the defending national champion Kentucky Wildcats, which hosted Wells last week.
Instead, Wells, as he announced via what appears to be the iPhone Notes app at 10 a.m. ET Tuesday morning, chose rebuilding Maryland:
"The last couple of weeks have been the toughest in my life for my family and I," Wells' announcement read. "I've learned that it is a major responsibility that comes with being a student-athlete at all times. I'm thankful God has blessed me with a second opportunity to continue my education. I would like to thank UK, UM, UMD and UO for their recruitment and interest in me as a student-athlete. After careful consideration with my family, I've decided to attend the University of Maryland."
Those school acronyms are a little confusing, but that's why we're here: Wells chose Maryland over Kentucky, Memphis and Oregon. The decision came after a little more than a week's worth of predictable and understandable recruiting efforts. As bonus talents go -- as players who drop in your lap and immediately improve your program's short-term outlook -- Wells is a keeper, a rare opportunity to land a proven college player with a ready body in the final week of August.
Xavier's strange decision made Wells the most desirable free agent in recent transfer history. Of course a solid collection of schools went after him. That was hardly a surprise.
What was surprising was Wells' decision. Maryland coach Mark Turgeon toppled two recruiting powerhouses -- Kentucky and Memphis -- and an Oregon program with some of the best facilities in the country (and oodles of playing time to boot). Wells won't pay immediate dividends (he'll almost certainly have to sit out the customary transfer year, barring any sort of precedent-setting hardship transfer waiver ruling by the NCAA).
But he sets the Terps up for a rather massive 2013-14 season. Turgeon is inserting two top-100 players into his lineup in 2012 -- top-10 center Shaquille Cleare and top-20 small forward Jake Layman. He's bringing back a batch of promising young players, including sophomores Nick Faust and Alex Len. And that's before Turgeon, who has massively expanded Maryland's recruiting footprint since taking the job last summer, truly shores up his 2013 class. (Could the Harrison twins be next?)
Taken as a whole, Wells' decision essentially gives Maryland the equivalent of a top-20 recruit -- if not better -- for the 2013-14 season. Combined with the rest of Turgeon's assets, and the players he can go out and get now, it now seems certain that by Year 3 the new Maryland head coach will be able to showcase serious progress toward the program's goal of returning to the national elite.
There was nothing textbook about Wells' arrival, but when a 6-foot-4 ready-made beast of a college baller lands in your lap, you don't question the fates. You just keep moving forward.
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Can the nephron be spared?
The search continues for the best role for mTOR inhibitor drugs in renal transplantation-principally to avoid or minimize the nephrotoxicity of the CNI class of immunosuppressive agents. The Spare the Nephron Trial describes the popular approach of early conversion from a CNI to the mTOR agent sirolimus for patients maintained on mycophenolate mofetil and steroids. At 1-2 years the glomerular filtration rate (GFR) was superior for the sirolimus group, with a loss of tolerability for about 20%.
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On Saturday, 3 March 2012 at 19:02:23 UTC, Chuck Allison wrote: > FYI: > > TDPL is a required text for CS 4450, Analysis of Programming Languages, at Utah Valley University starting Fall 2012. We'll study ML and D (and Prolog if time allows). Slightly off topic digression: Which ML? I ask as an OCaml partisan (and D fan!) who will obviously suggest that you go with that language, since it is probably the more widely used ML variant. -- Brian
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Q:
jQuery slideToggle not working in Internet Explorer 8
I'm using jQuery to slide out a div containing text on click of a div, but it's not working in Internet Explorer 8, while appearing fine in Firefox and Chrome. It's pretty basic, so I've no idea what I'm missing.
$(document).ready(function() {
$("#help").click(function(){
$('.flyout').slideToggle();
});
});
There's a jsFiddle of the code here - http://jsfiddle.net/tetsujin1979/yj5h3van/3/
A:
It is a IE8 bug,
just apply the following css to the block(s) element(s) with the margin in conflict (which disappears)
overflow : hidden
I don't know if this solution could be automated by jQuery
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Q:
What corresponds to a Zoom value of false in Aspose Cells?
I'm refactoring some Excel Interop code to utilize Aspose Cells. One of the legacy lines of code related to printing a sheet is:
xlSheet.PageSetup.Zoom = false;
Aspose Cells does have a [Sheet].PageSetup.Zoom property, but it is an int, not a bool.
What corresponds to false - 100?
UPDATE
In response to the answer, the legacy (Excel Interop) code is:
xlSheet.PageSetup.FitToPagesWide = 1;
xlSheet.PageSetup.FitToPagesTall = 10;
xlSheet.PageSetup.Zoom = false;
So, the original developer was setting the zoom vals and then saying disregard them? Or am I to understand that setting zoom to false does coincide with setting those values, and as long as I set the FitToPages* properties in Aspose, setting PageSetup.Zoom to false is moot/redundant?
A:
Please note, in Excel Interop the PageSetup.Zoom = false means no zoom has to be applied to the worksheet, and the FitToPagesWide and FitToPagesTall properties control how the worksheet is scaled. If you wish to achieve the same with Aspose.Cells APIs, please set the PageSetup.FitToPagesTall & FitToPagesWide according to the application requirements. Aspose.Cells will automatically ignore any preferences set for Worksheet's zoom.
Note: I am working as Developer Evangelist at Aspose.
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---
abstract: 'Hong-Ou-Mandel interferometry allows one to detect the presence of entanglement in two-photon input states. The same result holds for two-particles input states which obey to Fermionic statistics. In the latter case however anti-bouncing introduces qualitative differences in the interferometer response. This effect is analyzed in a [*Gedankenexperiment*]{} where the particles entering the interferometer are assumed to belong to a one-parameter family of [*quons*]{} which continuously interpolate between the Bosonic and Fermionic statistics.'
author:
- Vittorio Giovannetti
title: 'Entanglement and statistics in Hong-Ou-Mandel interferometry'
---
[**Topic:**]{} Quantum measurements, Quantum optics and related technologies, Quantum entanglement and non-locality, New perspectives for Foundations of Quantum Mechanics from Quantum Information
Introduction
============
Interferometry is a widely used quantum optical technique for characterizing the non classical behaviors of light, like squeezing or entanglement (see for instance [@WALLS] and references therein). In particular the presence of entanglement in states of two multi-mode photons can be detected by means of Hong-Ou-Mandel (HOM) interferometry [@HOM; @ERDMANN]. Indeed, the visibility of the coincidence counts at the output ports of such interferometer allow to detect entanglement in the incoming two-photon states: the visibility of separable two-photon inputs is in fact upper bounded by half of the maximum achievable visibility (e.g. the visibility associated with the entangled [*twin-beam state*]{} emerging from a monochromatic pumped $\chi^{(2)}$ non-linear crystal). In the context of mesoscopic physics [@BEN] a Fermionic analog of the HOM interferometer has been recently proposed in Ref. [@NOSTRO]: here metallic leads play the role of photonic multi-mode optical paths and current-current correlations play the role of the coincidence counts. Notwithstanding the change in the input particle statistics (from Bosonic to Fermionic) and the corresponding transition from an underlying bouncing behavior to an underlying anti-bouncing behavior, it turns out that the HOM interferometer retains its full entanglement witnessing capability. As a matter of fact the output signals of the Fermionic HOM implementation [@NOSTRO] can be easily mapped into the output signals of the standard Bosonic implementation: the only difference being a different definition of the “regions” which are accessible or not accessible to separable inputs. Motivated by the recent interest in the relations between entanglement, statistics and interferometry [@BOSE; @NOSTRO; @LIM; @BEN; @LAVAGNO; @ZHANG; @HBT; @BURKARD] we present a simple theoretical model which permits to analyze them in a broader theoretical context. In particular, we discuss an hypothetical HOM set-up where the two particles entering the interferometer obey to generalized quantum statistics [@GREEN; @GREEN1]. This allow us to interpolate continuously between the Bosonic HOM interferometer and its Fermionic counterpart.
We start by briefly reviewing the standard Bosonic HOM interferometer. Then we introduce the [*quon*]{} algebra [@GREEN; @GREEN1] and discuss a HOM-[*Gedankenexperiment*]{} which operates on particles obeying to the corresponding deformed statistics.
Using Hong-Ou-Mandel interferometer to detect entanglement in multi-mode two-photon states {#s:quons}
==========================================================================================
The prototypical HOM set-up [@HOM] is sketched in Fig. \[figu1\]. Here two multi-mode photons enter the interferometer following, respectively, the optical paths associated with the input ports $A_1$ and $A_2$. Their (possibly entangled) state is described by the vector $$\begin{aligned}
|\Psi\rangle =\sum_{k_1,k_2} \Phi(k_1,k_2) a_1^\dag(k_1)a_2^\dag(k_2) |
\O \rangle
\label{input}\;,\end{aligned}$$ where $|\O\rangle$ is the vacuum and where $a_j(k)$ is the photonic annihilation operator associated with $k$-th mode of the port $A_j$ which obeys standard Bosonic commutation relations, i.e. $$\begin{aligned}
\left[a_j(k), a_{j^\prime}(k^\prime) \right] &=& 0 \label{prima} \\
\left[ a_j(k), a_{j^\prime}^\dag(k^\prime) \right]
&=& \delta_{jj^\prime} \; \delta_{kk^\prime} \label{seconda}\;.\end{aligned}$$ In these expressions $k$ labels the different frequencies of the modes propagating along the optical paths $A_{1,2}$ (for the sake of simplicity the entering signals are supposed to have definite polarization) and $\Phi(k_1,k_2)$ is the two-photon spectral amplitude which satisfies the normalization condition $$\begin{aligned}
\sum_{k1,k_2} |\Phi(k_1,k_2)|^2 =1 \;. \label{norma}\end{aligned}$$ Within the interferometer the modes of port $A_2$ undergo to phase-shifts transformations introduced through controllable delays (represented by the white box of Fig. \[figu1\]) which transform $a_2(k)$ into $a_2(k) e^{i\varphi_k}$. The two optical paths then interfere at the 50/50 beam-splitter BS where photo-coincidence $C_{12}$ are measured at the output ports $B_1$ and $B_2$. We will see that the presence of entanglement in the photon input state $|\Psi\rangle$ can be revealed by studying $C_{12}$ as a function of the controllable delays.
Assuming perfect detection efficiency and considering the limit of long detection times $T$ the average coincidence counts of the photo-detectors operating on $B_1$ and $B_2$ can be expressed as $$\begin{aligned}
C_{12} = \lim_{T\rightarrow \infty} \int_{0}^T d t_1
\int_{0}^T d t_2 \; \frac{
\langle \Psi | I_{B_1}(t_1) I_{B_2}(t_2)
|\Psi \rangle}{T^2} \;, \label{coinc} \end{aligned}$$ where $I_{B_{1,2}}(t)$ are the normalized output intensity operators defined by $$\begin{aligned}
I_{B_j}(t) \equiv \sum_{k_1} \sum_{k_2}
e^{i (\omega_{k_1}-\omega_{k_2})t}
\label{photonumber} b_j^\dag(k_1) b_j(k_2)
\;,\end{aligned}$$ with $b_j(k)$ being the Bosonic annihilation operator associated with the $k$-th outgoing mode of port $B_j$. Evaluating the integrals and the limit of Eq. (\[coinc\]) gives $$\begin{aligned}
C_{12} = \langle \Psi | \;N_1 N_2 \;|\Psi \rangle \;, \label{coinc1} \end{aligned}$$ where for $j=1,2$, $N_j$ is the total photon number operator of the $B_j$ output port, i.e. $$\begin{aligned}
N_j = \sum_k n_j(k) \;, \label{tot}\end{aligned}$$ with $n_j(k)= b_j^\dag (k) b_j(k)$ being the number operator of the $k$-th mode. Analogously the average output photo-counts at $B_1$ and $B_2$ yield $$\begin{aligned}
i_{j} \equiv \lim_{T\rightarrow \infty} \int_{0}^T d t \; \frac{
\langle \Psi | I_{B_i}(t)
|\Psi \rangle}{T} =
\langle \Psi | \; N_j \;
|\Psi \rangle \;. \label{corrente} \end{aligned}$$ The operators $b_j(k)$ and $a_j(k)$ are connected through the input-output relations of the HOM interferometer, i.e. $$\begin{aligned}
b_1(k) &=& \left[ a_1(k) + e^{i\varphi_k} a_2(k) \right] /\sqrt{2}
\nonumber
\\
b_2(k) &=& \left[ a_1(k) - e^{i\varphi_k} a_2(k) \right] /\sqrt{2}\;,
\label{inout}\end{aligned}$$ with $\varphi_k$ being the controllable phase-shift and with the coefficient $1/\sqrt{2}$ coming from the 50/50 beam-splitter transformation. Notice that Eq. (\[inout\]) couples only those modes of $A_1$ and $A_2$ which share the same value of $k$ and that $\varphi_k$ depends explicitly upon such index. \[In Refs. [@HOM; @ERDMANN] for instance it is $\varphi_k = \omega_k \tau$ with $\omega_k$ being the frequency of the $k$-th mode and with $\tau$ being a tunable delay.\] With the help of the above definitions one can show that the average photon number (\[corrente\]) at each of the two output ports equals $1$. On the other hand Eq. (\[coinc1\]) yields $$\begin{aligned}
C_{12} = \frac{1 - w}{2}\;, \label{BOSONI}\end{aligned}$$ with $$\begin{aligned}
w =
\sum_{k_1,k_2} \Phi^*(k_1,k_2) \Phi(k_2,k_1)
e^{i (\varphi_{k_1} - \varphi_{k_2})}\;\label{theg} \;.\end{aligned}$$ It is possible to verify that for a suitable choice of the delays $\varphi_k$ and the spectral function $\Phi(k_1,k_2)$, the real quantity $w$ can take any values in the interval $[-1,1]$. Correspondingly $C_{12}$ can take values over the interval $[0,1]$. In the twin-beam state case of Ref. [@HOM; @ERDMANN], for instance, the plot of $w$ with respect to the controllable delay $\tau$ shows the so called [*Mandel dip*]{}, i.e. it nullifies for $\tau=0$ and approaches $1$ for sufficiently large $\tau$. This implies that, by defining the visibility $V$ of $C_{12}$ as the difference between the maximum and minimum values it assumes when varying $\varphi_k$, the maximum achievable value of $V$ for the two-particles state $|\Psi\rangle$ is $1$.
On the other hand, consider what happens when $|\Psi\rangle$ factorizes with respect to $A_1$ and $A_2$. In this case the spectral amplitude has the form $\Phi(k_1,k_2) = \phi_1(k_1) \phi_2(k_2)$ and Eq. (\[theg\]) becomes $$\begin{aligned}
w = w_{\mbox{sep}}\equiv | \sum_{k} \phi_1^*(k) \phi_2(k)
e^{i\varphi_k}|^2 \geqslant 0 \;.\label{thegsep}\end{aligned}$$ Consequently, the coincidence counts $C_{12}$ of factorizable input states $|\Psi\rangle$ are limited into the interval $[0,1/2]$ and their maximum allowed visibility (i.e. $1/2$) is only one half of the maximum achievable value (see Fig. \[figu2\]). As discussed in Ref. [@NOSTRO] the above result generalize to any (non necessarily pure) two-photon input density matrices which are separable with respect to $A_1$ and $A_2$. Therefore we can use $C_{12}$ as an entanglement witness for two-photon input states. Indeed it is sufficient to repeat the coincidence measurements for different values of the controllable delay: by realizing a value of $C_{12}$ or a visibility $V$ which are strictly greater than $1/2$ one can conclude that the input state of the system was entangled.
Bosons vs. Fermions
-------------------
In Ref. [@NOSTRO] a Fermionic implementation of the HOM interferometer has been proposed. There, effective two-electron states analog to the two-photon input states of Eq. (\[input\]) originate from a solid-state entangler [@BEN] and propagate along metallic leads which take the place of the optical ports $A_1$, $A_2$, $B_1$ and $B_2$ of Fig. \[figu1\]. Moreover, the coincidence counts $C_{12}$ of Eq. (\[coinc\]) is replaced by the current-current correlator of the outgoing leads $B_1$ and $B_2$. We refer the reader to Ref. [@NOSTRO] for a detailed discussion of the physical assumptions underlying such a scheme. For the purposes of the present manuscript it is sufficient to observe that an effective but rigorous description of such interferometer is obtained from the Eqs. (\[input\]), (\[coinc1\]) and (\[corrente\]) by simply replacing the Bosonic commutation rules (\[prima\]) and (\[seconda\]) with their Fermionic counterpart, i.e. $$\begin{aligned}
\left\{ a_j(k), a_{j^\prime}(k^\prime) \right\} &=& 0 \label{primaf} \\
\left\{ a_j(k), a_{j^\prime}^\dag(k^\prime) \right\}
&=& \delta_{jj^\prime} \; \delta_{kk^\prime} \label{secondaf}\;,\end{aligned}$$ with $\{r,s\} = rs + sr$ being the anti-commutator of the operators $r$ and $s$. With this substitution the average output electron-numbers $i_{1,2}$ of Eq. (\[corrente\]) remain equal to $1$. However the expression (\[BOSONI\]) for the coincidence-counts $C_{12}$ is replaced by $$\begin{aligned}
C_{12} = \frac{1 + w}{2} \label{FERMIONI}\end{aligned}$$ with $w$ as in Eq. (\[theg\]). Since the dependence of $w$ upon the factorizable properties of the input states $|\Psi\rangle$ is the same as in the Bosonic case, one can still use the visibility $V$ as a signature of entanglement. Indeed also in the Fermionic case, entangled inputs can have visibility $V$ greater than $1/2$, while separable have always visibility $V$ smaller than or equal to $1/2$. In the case where the label $k$ refers to the spin degree of freedom of the incoming electron this effect was first noted in Ref. [@BURKARD].
The sign difference between Eqs. (\[BOSONI\]) and (\[FERMIONI\]) implies that separable states of Fermions are forced to have $C_{12}$ greater than or equal to $1/2$ while separable states of Bosons have $C_{12}$ smaller than or equal to $1/2$ (see Fig. \[figu2\]). This phenomenon can be interpreted in terms of the different bouncing and anti-bouncing attitudes of Bose and Fermi statistics. Indeed due to the exclusion principle one expects the coincidence counts $C_{12}$ of Fermions to be typically higher than the corresponding Bosonic coincidence counts. To make this a quantitative statement let us consider the average value of $C_{12}$ over all possible two-particle states of Eq. (\[input\]), i.e. over all the normalized two-particles spectral amplitudes $\Phi(k_1,k_2)$. A simple symmetric argument can be used to show $$\begin{aligned}
\overline{w} =
\int \; d \mu(\Phi)\; \left[
\sum_{k_1, k_2} \Phi(k_1,k_2)
\Phi^*(k_2,k_1) e^{i( \varphi_{k_1}-
\varphi_{k_2})} \right] = 1/M \label{thegaverage}\;,\end{aligned}$$ with $M$ being the number of the $k$-th modes. Therefore the average coincidence counts is $$\begin{aligned}
\overline{C_{12}} =
\left\{ \begin{array}{lll}
1/2 - 1/(2M) & & \mbox{Bosons}
\\
\\1/2 + 1/(2M) && \mbox{Fermions.}
\end{array}
\right. \label{cmedio}\end{aligned}$$ It is worth noticing that entangled input states are, to some extent, insensitive to the complementarity behavior of Eq. (\[cmedio\]): indeed, as shown in Fig. \[figu2\], there are no regions which are strictly forbidden to them by the particle statistics.
HOM interferometry with quons {#quons}
=============================
In this section a [*Gedankenexperiment*]{} is analyzed where the particles entering the HOM interferometer are assumed to be quons [@GREEN; @GREEN1]. These have been introduced by Greenberg as an example of continuous interpolation between Bose and Fermi algebras (see Refs. [@CHAI; @LAVAGNO] for alternative definitions). We will use them to describe the sharp transition from the left part to the right part of Fig. \[figu2\] in terms of a continuous deformation of the particle statistics.
For $q\in [-1,1]$ the quon algebra is obtained by replacing the relations (\[seconda\]) and (\[secondaf\]) with the identity $$\begin{aligned}
a_j(k) a_{j^\prime}^\dag(k^\prime) - q \;
a_{j^\prime}^\dag(k^\prime) a_j(k)
&=& \delta_{jj^\prime} \; \delta_{kk^\prime} \label{quon}\;,\end{aligned}$$ and by neglecting [@NOTA] the relations (\[prima\]) and (\[primaf\]). For $|q|<1$, Eq. (\[quon\]) can be used to define a valid non-relativistic field theory but poses serious problems in deriving a reasonable relativistic quantum field theory [@GREEN; @GREEN1]. A proper Fock-like representation can be derived upon enforcing the vacuum condition $$\begin{aligned}
a_j(k) |\O\rangle = 0\;,
\label{vacuum}\end{aligned}$$ for all $j$ and $k$ [@GREEN; @GREEN1]. In particular, it can be shown that for $|q|<1$ the squared norms of all vectors made by polynomials of $a_j^\dag(k)$ acting on the vacuum state $|\O\rangle$ are strictly positive. For $q=1$ and $q=-1$ instead the squared norms are never negative and nullify for those configurations which are, respectively, totally symmetric and totally antisymmetric under permutations. This allows us to recover the Bosonic and Fermionic statistics as limiting cases of Eq. (\[quon\]) without explicitly imposing the conditions (\[prima\]) and (\[primaf\]), respectively. Of particular interest is also the $q=0$ case whose statistics can be interpret [@GREEN] as quantum version of the Boltzmann statistics, based on a system of identical particles having infinite number of internal degree of freedom.
The possibility of defining a proper Fock-like structure for the quon algebra (\[quon\]) implies the existence of number operators $n_j(k)$ which satisfy the standard commutation relations $$\begin{aligned}
\left[ n_j(k), a_{j^{\prime}}(k^\prime) \right] = -\delta_{jj^\prime}
\; \delta_{kk^\prime} \; a_j(k)
\label{number}\;,\end{aligned}$$ and which reduce to $a_j^\dag(k) a_j(k)$ in the Bosonic and Fermionic limits [@GREEN; @GREEN1].
In the following we will adopt a pragmatic point of view assuming that the equations of the previous section yield a legitimate description of our quon HOM interferometer. This is in part justified by the fact that the input-output relations (\[inout\]) map the quon annihilation operators $a_j(k)$ into annihilation operators $b_j(k)$ which still satisfy the quon algebra (\[quon\]) and the vacuum condition (\[vacuum\]). The only technical problem of our approach comes from the fact that for $|q|<1$ Eq. (\[coinc1\]) is not necessarily a real quantity (it is a consequence of the fact that no commutation relation between $a_1(k_1)$ and $a_2(k_2)$ is defined [@NOTA]). Consequently we replace the product $N_1 N_2$ with its Hermitian part, i.e. $$\begin{aligned}
C_{12} \equiv \langle \Psi | \frac{ N_1 N_2 + N_2 N_1}{2} | \Psi\rangle =
\Re{e} \; \langle \Psi | N_1 N_2 | \Psi\rangle \;. \label{coincq}\end{aligned}$$ \[In this expression $N_{1,2}$ is defined through Eq. (\[tot\]) in terms of the number operators $n_j(k)$ of Eq. (\[number\])\]. The right hand side term of Eq. (\[coincq\]) can now be computed by inverting the transformation (\[inout\]) and expressing the input state $|\Psi\rangle$ of Eq. (\[input\]) in terms of the operators $b_{j}(k)$. With the help of the relations (\[number\]) and (\[quon\]) and using the vacuum condition (\[vacuum\]) one can then verify that the quon “coincidence counts” of the $q$-algebra are [@NOTA10], $$\begin{aligned}
C_{12}(q) =\frac{1- q \; w}{2} \label{coincq1}\end{aligned}$$ with $w$ as in Eq. (\[theg\]). Analogously one can verify that for normalized input states Eq. (\[norma\]) still holds and that the average number $i_{1,2}$ is equal to $1$ for all $q$. As expected, for $q=\pm 1$ Eq. (\[coincq1\]) reduces to the Bosonic and Fermionic case. More interestingly for $q=0$, $C_{12}(q)$ does not depend upon the two-particle input state $|\Psi\rangle$ and has constant value $1/2$. This is exactly what one would expect from a classical model of the interferometer confirming the Boltzmann interpretation [@GREEN] of the $q=0$ algebra.
Taking into account that for generic input state one has $w\in [-1,1]$ the following bounds can be derived: $$\begin{aligned}
\begin{array}{cll}
\frac{1-q}{2} \leqslant C_{12}(q) \leqslant \frac{1+q}{2} &
&\mbox{for $q\in [0,1]$} \\ \\
\frac{1+q}{2} \leqslant C_{12}(q) \leqslant \frac{1-q}{2} & &
\mbox{for $q\in [-1,0]$} \;.
\end{array} \label{allowed}\end{aligned}$$ On the other hand for factorizable amplitudes $\Phi(k_1,k_2)$, the function $w$ obeys Eq. (\[thegsep\]). Therefore for these states one has $$\begin{aligned}
\begin{array}{cll}
\frac{1-q}{2} \leqslant C_{12}(q) \leqslant \frac{1}{2} &
&\mbox{for $q\in [0,1]$} \\ \\
\frac{1}{2} \leqslant C_{12}(q) \leqslant \frac{1-q}{2} & &
\mbox{for $q\in [-1,0]$} \;.
\end{array}\label{allowed1}\end{aligned}$$ Finally, we can use Eq. (\[thegaverage\]) to compute the average value of value of $C_{12}$ with respect all possible two-particle input states $|\Psi\rangle$, i.e. $$\begin{aligned}
\overline{C}_{12}(q) = 1/2 -q/(2M) \label{cmedioq}\;.\end{aligned}$$ The above constraints and Eq. (\[cmedioq\]) have been plotted in Fig. \[figu3\]: they indicate that the transition from the Bosonic regime to the Fermionic regime is characterized by a critical point at $q=0$. Here the values $C_{12}> 1/2$ which for $q>0$ were pertinent to non factorizable input states $|\Psi\rangle$, become accessible to factorizable configurations. At the same time, however the values $C_{12}<1/2$ become inaccessible to them. Following the discussion of the previous section this effect can be interpreted as a continuous transition from a bouncing behavior to an anti-bouncing behavior with $q=0$ corresponding to the classical “neutral” point. Moreover, Eq. (\[cmedioq\]) provides an indirect confirmation of the interpretation [@GREEN] of the $q=0$ case in terms of the statistics a system of identical particles having infinite number of internal degree of freedom. In effect according to such expression the limit $M\rightarrow \infty$ and $q\rightarrow 0$ of the average coincidence counts $\overline{C}_{12}(q)$ are equivalent.
Conclusions
===========
We analyzed how different quantum statistics affect HOM interferometry. In particular we focused on the interferometer response to initially separable/entangled inputs. By introducing a family of $q$-deformed algebras we interpolated between the Bosonic and Fermionic regimes which were previously discussed in Ref. [@NOSTRO]. Our results indicate a progressive attenuation of the bouncing behavior when moving from the Bose statistics to the Fermi statistics a progressive attenuation of the bouncing behavior.
We thank Prof. Rosario Fazio and Dr. Diego Frustaglia for comments and criticisms. This work was in part supported by the Quantum Information research program of Centro di Ricerca Matematica Ennio De Giorgi of Scuola Normale Superiore.
[99]{} Walls, D.F., and Milburn, G.J., (1994), [*Quantum Optics*]{} (Springer: Berlin); Kobolov, M.I., (1999), Rev. Mod. Phys., [**71**]{}, 1539; Mandel, L., (1999), Rev. Mod. Phys., [**71**]{}, S274. Hong, C.K., Ou, Z.Y., and Mandel, L., (1987), Phys. Rev. Lett. [**59**]{}, 2044. Kwiat, P.G., Steinberg, A.M., and Chiao, R.Y., (1992), Phys. Rev. A [**45**]{}, 7729; Pittman, T.B., Strekalov, D.V., Migdall, A., Rubib, M.H., Sergienko, A.V., Shih, Y.H., (1996), Phys. Rev. Lett., [**77**]{}, 1917; Erdmann, R., Branning, D., Grice, W., Walmsley, I.A., (2000), Phys. Rev. A, [**62**]{}, 053810; Giovannetti, V., Maccone, L., Shapiro, J.H., and Wong, F.N.C., (2002), Phys. Rev. Lett., [**88**]{}, 183602; Atatüre, M., Di Giuseppe, G., Shaw, M.D., Sergienko, A.V., Saleh, B.E.A., Teich, M.C., (2002), Phys. Rev. A., [**65**]{}, 023808. Beenakker, C.W., Eprint cond-mat/0508488, to be published in [*Quantum Computers, Algortihms, and Chaos*]{}, International School of Physics Enrico Fermi, Vol. [**162**]{}, and references therein; Burkard, G., Handbook of Theoretical and Computational Nanotechnology. v.2 cond-mat/0409626; Büttiker, M., Phys. Rev. B [**46**]{}, 12485 (1992). Giovannetti, V., Frustaglia, D., Taddei, F., and Fazio, R., Eprint cond-mat/0606413. Lim, Y.L., Beige, A., (2005), New J. Phys., [**7**]{}, 155. Bose, S., Home., D., (2002), Phys. Rev. Lett., [**88**]{}, 050401. Lavagno, A., Swamy, P.N., (2000), Phys. Rev. E, [**61**]{}, 1218. Lavagno, A., Swamy, P.N., (2002), Phys. Rev. E [**65**]{}, 036101. Zhang, Q.H, Padula, S.S., (2004), Phys. Rev. C [**69**]{}, 024907. Samuelsson, P., Sukhorukov, E.V., Büttiker, M., Phys. Rev. Lett [**92**]{}, 026805 (2004). Burkard, G. and Loss, D., Phys. Rev. Lett. [**91**]{}, 087903 (2003). Greenberg, O.W., (1990), Phys. Rev. Lett. [**64**]{}, 705. Greenberg, O.W., (1993), Phys. Rev. D, [**43**]{}, 4111. Biedenharn, L.,(1989), J. Phys. A, [**22**]{},L873; Macfarlane, A., (1989), J. Phys. A, [**22**]{}, 4581; Chaichian, M., Gonzalez Felipe, R., and Montonen, C., (1993), J. Phys. A: Math. Gen., [**26**]{}, 4017. For $q\neq \pm 1$, no (deformed) commutation rules can be imposed among $a_j(k)$ and $a_{j^\prime}^\dag(k^\prime)$ (see Ref. [@GREEN] for details). It should be stressed that to derive Eq. (\[coincq1\]) the exact expression of the $n_j(k)$ in term of the annihilation operators is not required. Only the commutation relation (\[number\]) is needed.
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Dear Editor,
We thank Jain *et al*.\[[@CIT1]\] for their interest in our article.\[[@CIT2]\] We do agree that these lesions do improve with topical steroids. What is important here is the presence of additional systemic features like skin eruptions or involvement of a mucosa. In our patient, there were skin eruptions and painful buccal mucosal ulcers following a viral illness. Herpes simplex virus-induced erythema multiforme (EM) minor constitutes 15-60% of EM minor. The treatment is a combination of acyclovir and oral steroids.\[[@CIT3]\]
Treatment with oral steroids along with oral acyclovir is definitely justified in our case. In contrast, we do see cases of EM minor with only ocular involvement in the form of coin-shaped lesions which will definitely improve only with topical steroids just like the case described by Jain *et al*.\[[@CIT1]\] As we have described the confocal microscopy findings speculating an immunological mechanism in the development of these coin-shaped lesions on the cornea, these lesions will definitely respond to topical steroids.
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Q:
Are there any numbers so that $a*b\equiv 0 \pmod n$ but not $a\equiv 0 \pmod n$ and not$ b \equiv 0 (\pmod n) $?
Are there any numbers so that $a*b\equiv 0 \pmod n$ but not $a\equiv 0 \pmod n$ and not$ b \equiv 0 (\pmod n) $?
$n=\{2,3,4,6,7\}$
After trying some numbers I would say no, but is there any proof for that?
A:
Actually there are. For example, $2 \times 3 = 6 \equiv 0 $ (mod $6$)
In fact, for every composite (i.e. non-prime) number, this will be the case: if $n = ab$, then $ab \equiv 0 $ (mod $n$)
However, if $n$ is prime, then you're right that this cannot happen - since if $ab \equiv 0$ (mod $p$) for some prime $p$, then by definition, $ p | ab$, so since p is prime, $p|a$ or $p|b$ so we must have $a \equiv 0$ (mod $p$) or $b \equiv 0$ (mod $p$).
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Elective treatment of the neck for second primary tumors of the head and neck.
The aim of this study was to define the role of elective neck dissection in patients with a second N0 head and neck squamous cell carcinoma (HNSCC). We carried out a retrospective study in 74 patients with a second N0 HNSCC treated with an elective neck dissection. Thirteen patients (17.6%) had occult neck node metastases. The risk of occult neck nodes was low for patients with a second glottic tumor (0%), and for patients with non-glottic T1-T2 tumors who had received previous radiotherapy in the neck (5.3%). Patients with non-glottic locally advanced tumors (T3-T4) and non-glottic T1-T2 tumors who had not received previous radiotherapy in the neck had a risk of occult neck nodes of 28.1 and 33.3%, respectively. Elective neck dissection could be omitted in patients with glottic tumors and in patients with an early tumor (T1-T2) who had received previous radiotherapy in the neck.
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Shelley, I wanted to see if I could get the PAA in for Ava. I think she did a very good job on the
Transwestern meeting and I would like to recognize her for that. If you approve, then I will
send to Fran to fill out the appropriate information. Thanks. Lynn
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In establishments where cards are played, say for the purposes of gambling, a number of packs of cards are usually employed for each table where a card game, such as blackjack is played. The cards are normally shuffled by hand and then placed in a shoe or other device from which cards may be withdrawn by a dealer one at a time. Shuffling cards by hand is obviously time consuming and labour intensive and additionally often does not ensure full and even mixing of cards. Additionally a large number of packs of cards, often up to six packs or more, are used at each table on each day and those packs are then discarded at the end of the day. An apparatus has been provided in the past to facilitate shuffling of cards, however, such apparatus tends only to shuffle cards in the same way as achieved manually. Such apparatus is very expensive and has not proved particularly effective.
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High-grade carcinoma of the prostate: a comparison of current local therapies.
To determine the impact of either single or combined local therapeutic modalities for poorly differentiated (Gleason score 8 to 10) prostate cancer. Between 1987 and 1996, 156 patients were diagnosed with biopsy proven, poorly differentiated (Gleason score 8 to 10), clinically localized prostate cancer. Of these patients, 87 were treated with radical prostatectomy alone, 19 with radiotherapy, and 24 with both prostatectomy and postoperative radiotherapy. The median follow-up time was 74.6 months. The 5-year biochemical progression-free survival (PFS) for patients with a Gleason score of 8 to 10 was 65%, 30%, and 20% for patients treated with surgery plus postoperative radiotherapy, radiotherapy alone, and surgery alone, respectively (P <0.0001 between postoperative radiotherapy and all other groups, P = 0.6131 between surgery and radiotherapy). The 5-year clinical PFS was 80%, 60%, and 35% for patients treated with surgery plus postoperative radiotherapy, radiotherapy alone, and surgery alone (P <0.0001 between postoperative radiotherapy and all others, P = 0.1975 between surgery and radiotherapy). The independent prognosticators for biochemical failure included serum prostate-specific antigen level greater than 20 ng/mL and seminal vesicle invasion; only seminal vesicle invasion was prognostic for clinical failure. Patients with high-grade prostate cancer (Gleason score 8 to 10) have uniformly poor, but apparently similar, biochemical and clinical PFS rates when treated by either prostatectomy or radiotherapy alone. The addition of postoperative radiotherapy in the treatment of these patients may be associated with improved biochemical and clinical PFS compared with either modality alone.
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FCC Destroyed Media Ownership Report
Study found local ownership means more local news
A 2004 Federal Communications Commission study that showed locally owned television stations provide more local news than others was ordered destroyed by FCC officials, and only came to light this week when a copy was leaked to Sen. Barbara Boxer (D.-Calif.).
Three years ago, then-FCC chair Michael Powell launched a proceeding on the effects of local ownership on television news as part of his drive to further deregulate media and allow for even greater consolidation. But the report commissioned under Powell turned out to undermine his argument that consolidation has no ill effects on local news, and, according to former FCC lawyer Adam Candeub, senior managers ordered "every last piece" of the study destroyed (AP, 9/14/06). On September 12, Senator Boxer, armed with the leaked report, questioned current FCC Chair Kevin Martin about it at his renomination hearing.
According to the report, locally owned stations in fact deliver nearly six minutes more of total news and almost five-and-a-half more minutes of local news in a 30-minute newscast than stations with non-local owners. This adds up to 33 more hours of local news a year--a remarkable figure, and a damning one for big media's allies in the FCC, who are required to protect the public interest and to promote localism.
Former FCC Chairman Michael Powell...made many high-sounding pronouncements about the need for media policy to be rooted in empirical evidence. Powell also attempted to separate out the issue of media consolidation from localism, claiming that most of the millions of comments to the Commission stemmed from a concern about local content, not a concern about concentration of ownership into fewer hands.
Martin, who succeeded Powell in 2005 as chair, voted in 2003 for ownership rules that would have dramatically raised ownership caps. The rules were sharply contested by media activists and others, and a federal appeals court struck them down in 2004. Martin told Boxer he hadn't been aware of the report and has promised to keep "an open mind" on media consolidation as the FCC embarks once again on a review of its media ownership rules (Daily Variety, 9/13/06). The FCC has since posted the full report on its website.
Powell likewise denied any knowledge of the report or responsibility for its suppression (AP, 9/15/06).
Boxer has called on the FCC's inspector general to conduct a formal, independent investigation into the suppression of the study. As the FCC revisits its ownership rules once again, transparency and a true commitment to the public interest are vital.
ACTION: Contact the FCC and encourage the Inspector General to conduct an investigation into the suppression of the media ownership report.
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Q:
app.relaunch([options]) is not working in electron
I want to change userData path with path defined by user. So, I'm fetching the path from UI, storing it into a file. So that next time app launches, it changes the path.
I wanted to restart the app as soon as user has selected the path. I tried app.relaunch() function. But it didn't work, neither it returned error.
I used exact same example mentioned in documentation. http://electron.atom.io/docs/api/app/#apprelaunchoptions
A:
Calling app.relaunch() will not actually quit the app, you need to follow it by a call to app.quit() or app.exit().
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CS-T** We are approaching Lisbon’s airport runway 03, and now it is time to enjoy the superb views. Down there we have the historic and touristic Belem area, and two major monuments are visible. The Belem tower by the river Tagus, and the Jerónimos Monastery. To the right you have the tropical garden and to the left the Belem Cultural Centre (aka CCB). The Belenenses football stadium is also clearly visible, and in the back, though hidden by the haze is Sintra. This one goes to my good friend Geraldes.
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Namecoins coupon code
something to represent both of you, for both of you to wear, or you might be looking at something just for her. This traffic exchange is better than I first expected. Traffic Exchange: Bitcoin Blizzard A bitcoin faucet and traffic exchange. Please be aware your country may or may not charge you a customs fee to receive your package. This is a reliable, paying site. In this case, a crush can be anyone from a romantic interest in a celebrity. . Related Posts, bitcoin Video Crash Course, join over 94,000 students and know all you need to know about Bitcoin. EarnCrypto lets you earn big by completing offers surveys, playing games, or watching videos.
CoinAd, get free bitcoins for viewing websites for a few seconds. Many items are suitable gifts for Man Crush Monday, ranging from fashion clothing items to jewelry pieces. A piece of jewelry can reflect your affection and love. This rarely happens, but we want our customers to be prepared for possible extra expenses. Here, we have perfected the art of cutting coins and making all kinds of beautiful jewelry from them. The Best Jewelry to Give on Woman Crush Monday. You are at: Home reviews Comparisons wallets »KeepKey vs trezor Bitcoin Hardware Wallet Review. The customers are responsible for paying the fee. Jewelry pieces are one of the most popular gift options for Man Crush Monday. This faucet now adds a daily loyalty bonus to your claims, an extra 1 for every consecutive day you claim, up to a 100 bonus double your bitcoin! First we take our ideas to our designer, then decide on the final draft. (Real wallets dont have a 24-hour delay on withdrawals.) For a place to store these coins, and an exchange where you can trade them for Bitcoin or 100 other coins, sign up for a free Cryptsy account, too.
Namecoins #handmade #handmadewithlove #etsyshop # jewelrystore #c ustommade.Its quite impossible for the pictures to do them justice.My husband and I started cutting designs out of coins from around the world almost 30 years ago.It began as a hobby, but it wasn t long before we became.
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A wafer is a thin slice of semiconductor material, such as crystalline silicon, used to fabricate integrated circuits (“ICs”). Numerous ICs may be formed on a single wafer, and these ICs are separated from each other by way of a wafer singulation (or “dicing”) process. Prior to wafer singulation, the ICs on the wafer are tested as a quality control measure, with poorly performing ICs being identified for subsequent removal.
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Multi-pole rotating electromechanical devices, such as motors, generators, re-gen motors, alternators, brakes and magnetic bearings are comprised of rotors and electro-mechanical components. AC motors rotate by producing a rotating magnetic field pattern in the electro-mechanical component that causes the rotor to follow the rotation of this field pattern. As the frequency varies, the speed of the rotor varies. To increase the speed of the motor, the frequency of the input source must be increased.
High frequency motors manufactured with the proper materials can be very efficient. For certain applications, like electric or hybrid cars, highly efficient electric motors are desirable.
The construction of electro-mechanical components for high frequency electric motors and generators is problematic. Iron or steel components are quite common in electric motors and generators. However, at high frequencies, such as those greater than 400 Hz, conventional iron or steel components are no longer practical. The high frequency of the AC source increases the core losses of the iron or steel components, reducing the overall efficiency of the motor. Additionally, at very high frequencies, the component may become extremely hot, cannot be cooled by any reasonably acceptable means and may cause motor failure.
For construction of electro-mechanical components used in high frequency electric motors, ribbon made from soft magnetic material provides distinct advantages. Examples of soft magnetic ribbon materials would be either 1) conventional material typically defined as 0.008″ and thicker, non grain oriented with a typical Si content of 3%+/− ½% or 2) alternate soft materials that are 0.007″ or thinner with Si content of 3% to 7%, amorphous, or nanocrystalline alloys and other grain oriented or non grain oriented alloys. Some soft magnetic ribbon materials exhibit inherent characteristics that make their use in high frequency electro-mechanical rotating devices highly desirable. Some soft magnetic ribbons are easy to magnetize and demagnetize, which means an electro-mechanical component made with these metals would have low power loss, low temperature rise at high frequency, extremely fast magnetization and easy conversion of electrical to mechanical energy. An electro-mechanical component made of such an metal would generate less core losses and be able to operate at much higher frequencies, resulting in motors and generators of exceptional efficiency and power density.
Soft magnetic materials are commercially produced as ribbon or strip. A preferred example of a soft magnetic metal ribbon is Metglas®, which is an amorphous material, manufactured by Honeywell, Inc. Soft magnetic metal ribbons are very thin and of varying width. Manufacturing components of soft magnetic metal ribbon requires winding the soft magnetic ribbon into a shape and then heat processing the shape. Simple three dimensional shapes, such as toroids, can currently be constructed from soft magnetic metal ribbon.
However electro-mechanical components are often not simple three dimensional shapes. The electro-mechanical component can have numerous slots for accommodating motor coils in a generally toroidal structure.
Attempts to create complex three dimensional configurations from soft magnetic metal ribbon have heretofore been commercially unsuccessful. Various manufacturing techniques have been attempted by industry such as but not limited to: wire electrical discharge machining, electrochemical creep grinding, conventional electrical discharge machining, cutting, stamping, acid etching and fine blanking. None have proven satisfactory for reasons such as cost-effectiveness, manufacturing repeatability, or process cycle time.
This inability to fabricate complex three dimensional shapes from soft magnetic ribbon has been the significant impediment to producing high efficiency axial flux motors and generators. A method to produce electro-mechanical components from soft magnetic ribbon in a cost effective, end use functional, high volume capable method that will also provide substantial design flexibility for end use requirements is highly desirable.
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FEMEN Topless Protesters
Ukrainian Topless Protesters: What's Their Message?
There were many sheer looks yesterday at Nina Ricci's Spring 2014 show in Paris, but two uninvited women showed even more skin on the catwalk. Two topless women from Ukraine's FEMEN protest group ran on stage before one was punched by a model and they were escorted out by security. Shouting "fashion fascism" and apparently protesting the assumption that Ukrainian women are sex slaves, the pair managed to disrupt the show momentarily.
FEMEN, founded in 2008, protests sex tourism, fashion, and even environmental causes. On Friday, a group of topless members armed with flares stormed a boat on the Seine in Paris in support of Greenpeace. And earlier this year, they reportedly protested "anorexic" models outside the Versace show in Paris. The group's website defines FEMEN as a "famous organization of topless women activists, who defend with their breast sexual and social equality in the world." While some may question the sensibility of going nude to protest sexual exploitation, FEMEN claims "the magic of the body" gets people interested and gives them "the courage of the act make you want to riot."
High-profile fashion brands aren't FEMEN's only targets. In April, a member of the group flashed Russian President Vladimir Putin when he was in Hanover, Germany, with Chancellor Angela Merkel. The demonstration was in support of the feminist punk band Pussy Riot, whose members are being detained in Russia. Putin didn't seem to mind, giving the women thumbs-ups. He also added later, "It is better to be dressed if one wants to discuss political matters." That might be true, but it won't get you as much attention.
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Family of seven forced to stay
in their incomplete house 01/04/2001 SUM
AFTER waiting for seven years, Yahya Abu and his family finally moved into
their low-cost home in Taman Dalma, Semenyih, Kajang, Selangor, last week.
The only thing stopping the 40-year-old general worker from calling it his
"dream house" is that it is not completed as work on the project had stopped
in 1995!
493 abandoned projects worth
RM15 bil 01/04/2001 SUM
ABANDONED housing projects are a bane to everyone. A blot on the landscape,
a constant reminder of lost investments for buyers, extra work for the Housing
and Local Government Ministry and a black mark in the developer's book.
Squatters in their own house01/04/2001 SUM
AFTER waiting for seven years, Yahya Abu and his family finally moved into
their low-cost home in Taman Dalma, Semenyih, Kajang, Selangor, last week.
The only thing stopping the 40-year-old general worker from calling it his
"dream house" is that it is not completed as work on the project had stopped
in 1995! Without basic amenities such as water and electricity supply, Yahya
has to resort to tapping electricity from the developer's site office opposite
his house.Family of seven forced to stay in their incomplete house With the
help of his five children, he uses containers to get some clean water from
a factory about a kilometre away. They do this twice a day. For washing, Yahya
and his family use rain water collected in a manhole.
Developers should be more careful
selecting contractors 02/04/2001 NST
I WOULD be grateful if you could publish my letter for the benefit of landowners
and developers who may indiscriminately appoint contractors for their housing
projects. After having read your report "A new Puchong township in the pipeline"
(NST, March 15), I would like to put in a word of advice to all landowners
and developers to be more careful when selecting their contractors.
The higher cost of low-cost
03/04/2001 MM-CLAS
THE issue of low-cost housing has always been a vexatious one. The original
price-tag of RM25,000 for a low-cost house was revised to RM42,000 in 1999
with the Cabinet's approval in the hope that it would trigger private sector
developers into building more low and medium low- cost houses. Alas, this
was not to be. Developers barely reached the 20 per cent mark of the target
set by the authorities, preferring instead to build higher- cost units.
Completed but still waiting
for approval 03/04/2001 NST
Purchasers of about 550 units in two blocks of the Venice Hill Condominium
blocks of the Venice Hill Condominium and Golf Resort in Ulu Langat, Selangor,
are in a quandary as the project that was launched in the mid-90s and is virtually
completed still has not obtained the required building plan approvals
New law needed to protect house
buyers: Ahmad Zahid 05/04/2001 BT
A NEW law is needed to protect the interest of buyers in case housing projects
are abandoned, said a government backbencher. Datuk Ahmad Zahid Hamidi (BN-Bagan
Datoh) said the move would be seen as the Government coming to the defence
of the people
Unsold properties reached RM9.8b
in 2000 06/04/2001 BT
UNSOLD properties in the residential, industrial and commercial sectors in
the country amounted to RM9.83 billion at the end of last year, up 11.6 per
cent from the first half of 2000, according to a Finance Ministry report.
Deputy Finance Minister Datuk Chan Kong Choy expects the unsold properties
situation to ease within the next few years.
Reviving Sentul
Raya project 07/04/2001 NST-CITY
THE multi-billion ringgit Sentul Raya project, which was halted three years
ago during the economic slowdown, is expected to be continued in the middle
of this year. However, there are several changes in the project which the
new developer, YTL Corporation Berhad, and property owners KTM Berhad, will
make with approval from the housebuyers.
Abdullah: Punish
errant developers 07/04/2001 NST
KUALA LUMPUR, Fri. - Developers who fail to complete their projects should
be penalised as the failure will cause unnecessary hardship to others, Deputy
Prime Minister Datuk Seri Abdullah Ahmad Badawi said today. For instance,
he said, house buyers would be in the lurch if developers delayed or failed
to complete projects. The situation would be worse if the projects were abandoned.
Check them out before approval
07/04/2001 NST
THE Government has advised local authorities and financial institutions to
scrutinise feasibility studies for projects before granting approval to build
or give out loans to developers. Deputy Minister of Finance Datuk Chan Kong
Choy said the Government found the property overhang situation worrying and
appealed to the organisations to exercise caution when deciding to give approvals
or loans for projects.
Planning laws must meet the
needs 07/04/2001 NST
TOWARDS the end of last year, some residents of Damansara Heights in Kuala
Lumpur were quite distressed when news began circulating about a proposed
development in their midst. If you are not familiar with the place or its
history, let me tell you right away that when the area was first developed
over 30 years ago, it was meant for the construction of bungalows only - except
for a limited stretch of land reserved for shops and offices. It was for this
reason that people with money bought property here.
We don't have the money, says
developer 08/04/2001 SUM
TAMAN Dalma developer Visioneast Development Sdn Bhd said it could not continue
with the project due to financial constraints. A senior official, met at the
company's office in Taman Melawati, Selangor, last Thursday, said the project
was launched in 1991 and the company had budgeted that each of the 106 units
would cost RM14,000 to build.
We just want to move in'
08/04/2001 SUM
WE hope the banks can help us. This was the appeal from the 20-member task
force comprising buyers of the abandoned Taman Dalma project in Semenyih,
Kajang, Selangor, in the hope of solving their six-year-old headache.
No plans to amend law to control
allocation of houses 10/04/2001 The Star
THE Housing and Local Government Ministry has no intention to draft a new
law or amend the current legislation to regulate the allocation of low-cost
housing. Minister Datuk Seri Ong Ka Ting said the state governments were in
charge of renting out or allocating low-cost units to whoever was eligible.
Condo owner wants defects rectified
11/04/2001 MM
SIX months after CHIANG's family moved into their RM303,000 Menara Duta 11
condominium in Jalan Segambut Dalam, they were saddled with problems of leaking
in the balcony and bedroom. That was in 1998. After several appeals, the developer,
Vital Best Sdn Bhd, finally repaired the balcony last February since it was
on common property.
Ka Ting: 324 abandoned housing
projects revived11/04/2001 The Star
Dewan Rakyat A TOTAL of 324 housing projects which were abandoned since the
mid-1980s have been revived by the Government. Housing and Local Government
Minister Datuk Seri Ong Ka Ting said the projects involved the construction
of some 59,616 houses and 43,448 buyers. "At the same time, 19 projects have
been taken over by other developers and we have targeted another 56 which
have the potential to be revived. "We are currently carrying out feasibility
studies on these projects, which involve some 21,182 houses and 13,325 buyers,
with the co-operation of Syarikat Perumahan Negara,'' he told reporters at
the Parliament lobby here yesterday. However, the ministry, he added, had
also identified 115 projects which could no longer be revived due to flagging
interest from consumers or where the developers had wound up operations.
Builder offers to open joint FD account with buyers12/4/2001 The Star By Derrick Vinesh
A developer has proposed to open a joint fixed deposit account with its house
buyers for a sinking fund to promote transparency in its business transactions.
Eternal Holdings Sdn Bhd managing director Ong Tai Chin said his company was
among the first to adopt the approach, noting that most developers opened
their own bank accounts for the fund.
Let condo owners call the shots
13/04/2001 NST
I OWN and live in a condominium and from my experience there are many reasons
why I should not. Ownership - this word usually means you are the lord and
master and you make the decisions. Not so for condominiums. Your hands are
bound by the developer because unless you have a strata title you do not have
control as the maintenance of the common property is in the hands of the developer.
Working out the `perfect' SPA
13/04/2001 NST-PROP
THE first response was quite expected. Sham, who has more than 10 years' experience
handling property transactions on behalf of purchasers as well as developer
clients, said that a complete panacea, an all-encompassing solution that can
resolve all problems presently faced by purchasers, cannot be formulated or
created overnight. Since the SPA does not stand alone but must remain part
and parcel of the Housing Developers Act and the Regulations made thereunder,
the entire existing legal infrastructure must also be completely revamped.
Facilities not provided
13/04/2001 MM
LIVING in condominiums may be stressful, as residents of Tiara Duta condominiums
in Ampang found out. The main problems are incomplete clubhouse, no parking
bays for visitors, lifts often not functioning, high maintenance fees for
unsatisfactory service rendered.
Proposed amendments submitted
13/04/2001 The Star
KUALA LUMPUR: The proposed amendments to the Housing Developers Act have been
submitted to the Attorney-General's office.
Sell with moral responsibility
13/04/2001 MM-CLAS
MINISTER of Housing and Local Government Datuk Seri Ong Ka Ting urged developers
to sell their properties with the highest moral responsibility. "Take care
of the buyers and protect their interests," said Ong who also added that developers
need to be conscious of the fact that in some cases, the life's savings of
purchasers are involved.
Rehda: Let buyers form pro tem
panels 13/04/2001 The Star
THE Housing and Local Government Ministry should allow house buyers to form
pro tem management corporations soon after obtaining Certificate of Fitness
(CF) for their apartment units.
Crimson Land completes project
nine months ahead 14/04/2001 NST
AT a time when property purchasers live in uncertainty of whether their houses
will be delivered on time, developer Crimson Land Bhd has inspired market
confidence by completing its D'aman Crimson project in Petaling Jaya nine
months ahead of schedule. Crimson Land sales and marketing director Datuk
Tan Seng Chee said owners of the 1,068 units would be given vacant possession
of their houses by May this year.
Selangor MB gets memo on Balakong
flats issue 14/04/2001 NST-CITY
THE Selangor Government has been urged to investigate allegations of irregularities
in the allocation of low-cost homes to some residents from the KTM Berhad
longhouse in Balakong. About 50 people, representing 600 affected families,
turned up at the State administrative complex in Shah Alam last Thursday to
hand over a memorandum on the issue to Selangor Menteri Besar Datuk Dr Mohd
Khir Toyo, appealing to him to look into the matter.
Businesswoman cries foul over
being sold a RM580,000 show unit 15/04/2001 NSUNT
PENANG, Sat. - A 29-year-old businesswoman is crying foul over a developer
selling her a bungalow which was only a show unit built with a one-year permit.
Teoh Boay Lan alleged that the developer did not submit any building plans
to the Seberang Prai Municipal Council and, as a result of it, the completed
unit could not be issued with a Certificate of Occupation.
RM15b of business if housing
projects revived 16/04/2001 BT
THE country can generate about RM15 billion worth of economic activities if
all the 493 abandoned housing projects are revived and rehabilitated, said
chairman of Syarikat Perumahan Negara (SPN) Datuk Ahmad Zahid Hamidi. Based
on a study synergising various aspects of the housing industry, related areas
such as financing, supply of building materials, workforce, architecture and
landscaping could easily produce some RM11 billion worth of business while
another RM4 billion will be in terms of its supporting activities for the
small and medium-sized industries.
Homes fit to crack up
17/04/2001 MM
A GROUP of residents in Bukit Sentosa, Rawang, wants the authorities to ensure
that their cracking houses will not collapse. This is because they have lost
faith in the Hulu Selangor District Council that issued their double-storey
terrace houses with certificates of fitness last year when the cracks had
already appeared.
Court orders
appointment of receiver and manager 17/4/2001 The Star
A High Court yesterday ordered, by consent, the appointment of a receiver
and manager to continue with the completion of a stalled medium-cost apartment
project in Jalan Lumba Kuda.
Housebuyers act to revive
stalled project 17/04/2001The Star By Choong Kwee Kim
Purchasers of units at the stalled Majestic Heights project are executing
plans to salvage the project themselves and are not banking on the rescue
by a new 'white knight' developer.
New law to protect high-rise
buyers 18/04/2001The Star
SUBANG JAYA: High-rise residential unit owners and developers will jointly
manage buildings until the strata titles are issued, under a new law being
planned by the Government.
No new CF for major repairs done18/04/2001The Star
IPOH: Buildings which have been issued with a certificate of fitness need
not get another after major repairs are done to it, state Local Government
Committee chairman Datuk Chang Ko Youn said. H
Amendments to Housing Act to
protect housebuyers 18/04/2001 NST
AMENDMENTS to the Housing Developers Act would greatly improve the housing
industry and offer added protection to home buyers, said the secretary- general
of the Housing and Local Government Ministry, Datuk Khalid Husin. He said
the main amendments being proposed to address the shortcomings of the existing
Act would include increasing the paid-up capital of licensed developers and
the appointment of a deputy controller to improve efficiency and speed up
the issuance of development licences.
Ministry submits
draft of new law to Cabinet 18/04/2001 BT
THE Housing and Local Government Ministry has completed the draft of the new
Common Properties and Building's Management and Maintenance Bill and will
submit it to the Cabinet soon before tabling it in the Parliament. Minister
Datuk Seri Ong Ka ting said at present, the law is being studied by the Attorney-General's
chamber to ensure there would be no overlapping of jurisdictions by the involved
parties.
Low-cost housing
blues 18/04/2001 NST
A POOR take-up rate was recorded in new low-cost housing projects, particularly
for stratified units, that were launched during the fourth quarter of last
year. According to the Residential Property Stock Report launched recently
by the Ministry of Finance's Valuation and Property Services Department (VSPD),
only 46 per cent of the 1,047 low-cost houses (or non-stratified units) and
three per cent of the 1,451 low-cost flats launched during this period were
sold.
Sec-gen on how to solve property
overhang18/04/2001The Star
The prevailing property market overhang reflects a major weakness in town
and country planning, Housing and Local Government Ministry secretary-general
Datuk Khalid Husin said.
Condo
buyer sues builder over vacant possession19/04/2001 The Star By K. Kasturi Dewi
A geophysicist, who is suing a developer for failing to deliver vacant possession
of his condominium unit in Penang, claimed he was incurring expenses travelling
from Singapore while waiting for his property to be handed over to him.
Buildings ready but still no
CF 19/04/2001 MM
AFTER four long years, the shop and office buildings project in Westport Tech-Zone,
Pulau Indah Klang, was finally completed last year. However, until now, the
buyers are unable to shift into their new office or rent the units out as
there is no Certificate of Fitness from the local authority.
Piling reinforced
19/04/2001 MM
THE developer of a housing project in Bukit Sentosa, Rawang, has started remedial
works to strengthen the soil which had caused 12 houses there to crack. Talam
Corporation Berhad general manager of customer service, Michael Khor, said
work by its subsidiary, Nobel Rights Sdn Bhd, involved additional piling to
strengthen the soil on which the houses were built.
Revival of abandoned projects
can spur growth 20/04/2001 NST-PROP
THE crucial role that the property market plays in keeping the wheels of the
nation's economy moving cannot be overstated. This was clearly expressed by
Syarikat Perumahan Negara (SPN) chairman Datuk Ahmad Zahid Hamidi who early
this week said that the country can generate about RM15 billion worth of economic
activities if all the 493 abandoned housing projects are revived and rehabilitated.
Residents meet council for
solution 20/04/2001 MM
DISAPPOINTMENT was written on the faces of the 48 people who arrived in two
buses at the Subang Jaya Municipal Council yesterday to meet its president,
Ahmad Fuad Ismail, over their housing woes. Due to their failure to set an
appointment for a meeting with Fuad, the residents from Taman Sungai Besi
Indah in Seri Kembangan had to settle for a meeting with deputy president
Arpah Abdul Razak.
Housebuyers guide on website
20/04/2001 The Star
PENANG: Housebuyers who face various problems such as housing delays or defects
can now visit a new website to find out more about their rights. House Buyers
Association (HBA) secretary-general Chang Kim Loong said yesterday the HBA
website would serve as an "A to Z'' guide for purchasers.
Owners, developer reach settlement
20/04/2001 The Star
KUALA LUMPUR, Thurs. - Thirty-two condominium owners, who sued housing developer
Endah Raya Realty Sdn Bhd and two others for breach of contract over the Shang
Village condominium project in Kelana Jaya, today entered into a settlement.
The terms of the settlement agreement were not disclosed. The agreement was
brought before High Court judge Datuk Azmel Ma'amor in his chambers
Residents meet council for
solution 20/04/2001 MM
DISAPPOINTMENT was written on the faces of the 48 people who arrived in two
buses at the Subang Jaya Municipal Council yesterday to meet its president,
Ahmad Fuad Ismail, over their housing woes. Due to their failure to set an
appointment for a meeting with Fuad, the residents from Taman Sungai Besi
Indah in Seri Kembangan had to settle for a meeting with deputy president
Arpah Abdul Razak.
Build first, then sell policy
mooted for houses20/04/2001 Malaysia Kini
The government has been urged to implement a 'Build first then sell' concept
- where developers can sell their properties only after they are fully-built-
to overcome numerous complaints by disgruntled home buyers relating to abandoned
housing projects.
Condo purchasers
to lodge report against developer 21/04/2001 NST
NEARLY 25 purchasers of the Venice Hill Condominium and Golf Resort's Tower
10 units have unanimously agreed to lodge a police report next week against
the developer, Venice Hill Resort Living Sdn Bhd (VHRL), for criminal breach
of trust. VHRL is a member of the Li Foong Group of Companies.
`Submit Form 7F'
warning 21/04/2001 NST
KUALA LUMPUR, Fri. - Housing developers have been given a final warning to
inform the Housing and Local Government Ministry of the progress of projects
and financial standing or risk a fine or jail sentence.
Forcing flat-owners to pay their
dues23/04/2001 The Star
While the government has encouraged the construction of flats and other high-rise
units to meet the housing shortage, the sad fact is that Malaysians are still
not used to living in such accommodation.
Developers `no' to holding
back 20pc not payment 26/04/2001 NST
DEVELOPERS have deemed inequitable the suggestion that the last 20 per cent
of the purchase price for houses be held back to contra claims for late delivery
without resorting to court action. They were responding to a call made by
Consumers Association of Penang president S.M. Mohamed Idris.
Developers told to dismantle
cranes at abandoned buildings 26/04/2001 NST
SEREMBAN, Wed. - The State Government has ordered developers of abandoned
projects to dismantle construction cranes at the site of uncompleted buildings.
Menteri Besar Tan Sri Mohamad Isa Abdul Samad said today the cranes posed
a danger to the public.
Pitfalls of buying a model
house 27/04/2001 NST
THERE is a strong urge bordering on an irresistible compulsion for people
with money and no patience to buy an existing model house, also called a show
house, built by a housing developer on the occasion of the launch of a new
project, or a new phase of a project. Buying a show unit means having the
advantage of seeing what you purchased, unlike throwing down a hefty deposit
on a house which has yet to be built - and not knowing for certain whether
it will indeed be built and completed on the agreed date.
Flats residents `fighting'
over parking lots 27/04/2001 MM
IT is a war-zone at BAHARI's residence at Flat L35 Taman Pandan Jaya, Ampang.
He says almost all the residents are "fighting" for the limited parking lots
and some even go to the extent of building a permanent garage with their vehicle
registration number on it.
Two decrepit apartment blocks
in Johor Baru to be demolished 30/04/2001 NST
JOHOR BARU, Sun. - Two of Johor Baru's shabbiest landmarks - the Bukit Cagar
and Lumba Kuda public housing flats - will be demolished within five years
to make way for development. "The apartment blocks are too decrepit to upgrade
and are out of step with the times," said Menteri Besar Datuk Abdul Ghani
Othman
Landslide threat to expensive
home 30/04/2001 MM
THE RM500,000 semi-detached house which Alvin Lai bought about 15 months ago
in Taman Sungei Besi Indah, is set to crumble. Unless, something is done to
stop the erosion.
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Q:
How to convert this query to CakePHP 3
Here is my vicidial_log table call_date column look like
+---------------------+
| call_date |
+---------------------+
| 2016-02-06 03:35:48 |
| 2016-02-06 03:42:38 |
| 2016-02-06 07:38:09 |
| 2016-02-06 07:39:11 |
| 2016-02-06 07:41:09 |
| 2016-02-06 07:42:21 |
| 2016-01-06 07:44:19 |
| 2016-01-06 07:45:28 |
| 2016-01-06 07:47:08 |
| ................... |
+---------------------+
I want to group by call_date without time.
SQL QUERY
SELECT DATE(call_date) AS call_date FROM vicidial_log GROUP BY DATE(call_date);
OUTPUT
+---------------------+
| call_date |
+---------------------+
| 2016-02-06 |
| 2016-01-06 |
| ................... |
+---------------------+
How can I convert this query to CakePHP 3?
A:
From model :
$query = $this->find();
$date = $query->func()->DATE([
'call_date' => 'identifier'
]);
$query->select(['call_date'=>$date])
->group(['call_date'=>$date]); /* or simply ->group($date) */
From Controller :
$query = $this->your_table_name->find();
$date = $query->func()->DATE([
'call_date' => 'identifier'
]);
$query->select(['call_date'=>$date])
->group(['call_date'=>$date]); /* or simply ->group($date) */
For results :
$query->toArray();
/* or */
$query->all();
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Kyle Greig’s on fire – USL defences are terrified
It’ll be an emotional homecoming for Kyle Greig when he returns to Taft Stadium on Saturday.
Greig spent two years with OKC Energy before joining the Whitecaps this offseason, building a lot of relationships there with the coaching staff, players, fans and the local community.
With MLS as his clear goal, Greig felt that Vancouver provided a better opportunity to put himself in the shop window. To impress Carl Robinson and the rest of the ‘Caps coaching staff and earn a shot with Vancouver’s first team.
To do that he knew he needed to perform on the pitch right away and he’s certainly done that, banging in 10 goals in 13 appearances for WFC2 this season and averaging a goal every 101 minutes of play. There is no doubt the ‘Caps are taking notice, but Greig knows he has to keep it going to make his dreams a reality.
“It was a big goal of mine to start very, very well on an individual basis,” Greig admitted to AFTN when we caught up with him at training this week. “I’m happy with where I’m at, but I don’t want to have a second half of the season that’s slow, so I want to keep this momentum going.”
Greig returns to Oklahoma City in excellent form, and rested after missing last week’s win over Portland through yellow card accumulation. He admits it will “be a little bit weird” to be changing in the away team’s locker room, but heads back to face to his old club for the first time with a strikerate far higher than the 16 goals he got for the Energy in his 51 appearances there.
WFC2 head coach Alan Koch has been delighted with Greig’s initial success in Vancouver and credited his captain for playing such a big part in the club’s excellent start to the season.
“You can see him in training, he’s very, very confident,” Koch said of Greig. “His finishing is great in training, so it’s not surprising that he carries it over and scored in the games because you can see he looks very good. Football’s about confidence. When you train well, you play well, and when you to have success in the games, like he’s had, you continue to feel good about yourself and you carry that over into the games.
“It was frustrating to be missing him last week. We’re glad to have him back this week, particularly for him. It’s obviously a huge game going back to playing against his old team. He’s a huge part of our team and he’s a big part of why we’ve had success so far.”
Greig was part of an OKC side that had an excellent season last year, narrowly losing out to LA Galaxy II in the Western Conference final. He knows what it takes to be successful in the league and is confident this young WFC2 side has what it takes to make a run at this year’s USL Championship.
“I think it’s huge that we come to this halfway point and we’re top of the league,” Greig told us. “Some teams have games in hand on us, but at the same time they know they have to win to get to where we’re at. If we have the confidence that we can win these games and get points on the road, get points at home, then I think it’ll put us in a good position at the end of the season.”
Saturday’s game with OKC will mark the halfway point of the season for the Whitecaps. They head in to it atop of the Western Conference standings, four points clear of Sacramento Republic, with 28 points from their first 14 matches.
If the ‘Caps leave Oklahoma with a win, they will have amassed one more point in their first half of the 2016 season than they did all of last year. It’s been a remarkable turnaround for the team.
“When we were climbing the table, some of the guys were saying ‘I think we have almost as many wins as we had last year’, kind of joking around a little bit,” Greig said. “But it does show that this team’s come a long way since last year. There’s a lot of guys that were here last year and there’s a lot of new faces that help build this young team.”
The OKC game will be the first of four straight games on the road for WFC2 over the next month. A tough schedule, but not a daunting one for a team that has picked up four wins and 14 points (half their overall total) away from home so far this season.
The team is confident of producing the goods no matter where they have to play.
“I definitely think we can win each game that is presented in front of us,” Greig feels. “It’s obviously tough on the road and it’s going to be tough in a place like Oklahoma City, where they have a lot of fans and it’s hot and the pitch is narrow. We’re going in thinking we can get three points, but any points will be great for us.”
There’s no doubting that Greig will be heavily marked upon his return to face the Energy. More focus on him will likely leave more room to maneuver for the likes of Daniel Haber, Alphonoso Davies and others.
OKC head coach Jimmy Nielsen knows Greig well, having brought him to the club to begin with back in 2014, and is fully aware of the threat he poses his side on Saturday.
“Kyle Greig is a big presence in the box, and has scored a lot of goals this season,” Nielsen told AFTN in a phone interview this week. “We had the pleasure to have Kyle our first two season’s here. Unbelievable guy and a great player. He really deserves all the success he’s had so far with the Whitecaps. I’m really happy on his behalf.”
And as for Greig’s MLS aspirations, Nielsen, a man with four season’s experience in the league with Sporting KC, has no doubts that the ‘Caps striker has what it takes to make it at the top level of the game here.
“I think he can go a long way,” Nielsen told us. “And yes, why not MLS? He’s big, he’s fast, strong. He’s got a good nose for goals. If he gets on the right path then, yeah. Why not?”
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Introduction {#s1}
============
*Legionella pneumophila* is an environmental bacterium ubiquitously found in freshwater, where it is associated with biofilm communities (Lau and Ashbolt, [@B70]; Hilbi et al., [@B54]). Protozoan predators like amoebae are part of these communities and feed on bacteria residing within these biofilms. *L. pneumophila* has developed a way to survive and replicate within these free-living protozoa by forming a unique compartment called the *Legionella*-containing vacuole (LCV). The LCV is a pathogen vacuole, wherein *L. pneumophila* dodges lysosomal degradation by acquiring components of early and late endosomes, mitochondria, the endoplasmic reticulum and ribosomes (Isberg et al., [@B60]; Urwyler et al., [@B111]; Hilbi and Haas, [@B53]). To establish this intracellular niche, the bacterial Icm/Dot type IV secretion system (T4SS) is essential, as it translocates around 300 different "effector" proteins into the host cell, many of which target central eukaryotic pathways like endocytic, secretory or retrograde vesicle trafficking by exploiting small GTPases, phosphoinositide lipids and other host factors (Hubber and Roy, [@B59]; Finsel et al., [@B33]; Haneburger and Hilbi, [@B49]; Rothmeier et al., [@B92]; Hoffmann et al., [@B55]). Besides its natural protozoan hosts, *L. pneumophila* also replicates within human alveolar macrophages and epithelial cells, thus causing a severe pneumonia called Legionnaires\' disease. Most processes involved in survival in protozoa or macrophages are very similar and appear to be evolutionarily conserved (Gao et al., [@B37]; Greub and Raoult, [@B44]; Hoffmann et al., [@B56]). In addition to biofilm and protozoan niches, *Legionella* spp. are also naturally found in physically more challenging habitats, such as extremely acidic environments, antarctic freshwater lakes and water sources with temperatures over 60°C (Hilbi et al., [@B54]). Accordingly, these facultative intracellular bacteria are an example of a microorganism colonizing many different environmental niches.
To survive within its extra- and intracellular niches, *L. pneumophila* employs a biphasic life cycle, where it alternates between two different forms in response to environmental and metabolic stimuli (Molofsky and Swanson, [@B77]). In its transmissive form the pathogen is motile, resistant to environmental stress like nutrient starvation and infectious to host cells. In its replicative form the bacteria lack these traits but are able to replicate intracellularly (Rowbotham, [@B93]; Brüggemann et al., [@B11]). Further manifestations of *L. pneumophila* differentiation include a mature intracellular form (MIF) that develops late during infection (Garduno et al., [@B39]). MIFs are motile, metabolically inert, highly infectious and loaded with cytoplasmic inclusions of poly-3-hydroxybutyrate. Moreover, under harsh conditions, *L. pneumophila* appears to adopt a viable but non-culturable (VBNC) state (Steinert et al., [@B102]; Garcia et al., [@B38]; Al-Bana et al., [@B2]). To ensure bacterial survival in different environments, the biphasic life cycle of *L. pneumophila* is strictly regulated. Consequently, *L. pneumophila* employs a multitude of regulatory systems devoted to the control of gene expression, including transcriptional regulators and two-component systems (Molofsky and Swanson, [@B77]).
As *L. pneumophila* survives in various environmental niches, it is likely that the bacterium exploits numerous different carbon and energy sources. Furthermore, the intracellular milieu might represent a richly set table for pathogens, as eukaryotic host cells contain many different nutrients, which are potentially accessible to intracellular pathogens (Eisenreich et al., [@B24]; Rohmer et al., [@B91]; Abu Kwaik and Bumann, [@B1]). An intriguing aspect of intracellular metabolism is its compartmentalization into processes that occur within the host cytoplasm, the LCV lumen or the bacteria (Figure [1](#F1){ref-type="fig"}).
{#F1}
Early metabolic studies suggested that amino acids are the major if not only source of carbon and energy for *L. pneumophila* (Pine et al., [@B80]; Tesh and Miller, [@B104]; Tesh et al., [@B106]). However, the subsequent availability of genome sequences, transcriptome, proteome and metabolism data indicated that *L. pneumophila* possess much broader metabolic capacities (Cazalet et al., [@B15]; Chien et al., [@B18]; Urwyler et al., [@B112]; Eylert et al., [@B28]; Faucher et al., [@B30]; Hoffmann et al., [@B55]; Schunder et al., [@B98]). In this review we will summarize the metabolic capacities of *L. pneumophila* regarding amino acid and carbohydrate degradation. Moreover, we will highlight further nutrient requirements of the bacteria and assess the regulation of their life cycle by metabolites.
Amino acid metabolism
=====================
Initial studies of the nutrient requirements of *L. pneumophila* in chemically defined minimal media showed a preference for amino acids as main source of carbon and energy (Pine et al., [@B80]; Ristroph et al., [@B89]; Tesh and Miller, [@B104]; Tesh et al., [@B106]). A preference for amino acid utilization is also illustrated in the genome sequence of *L. pneumophila*, where around 12 classes of ATP binding cassette transporters, amino acid permeases and proteases can be found (Cazalet et al., [@B15]; Chien et al., [@B18]). Furthermore, genes involved in synthesis and transport of amino acids are highly induced during growth inside macrophages (Faucher et al., [@B30]). *L. pneumophila* employs transport systems to take up and utilize amino acids (Sauer et al., [@B95]), but also exploits host cell transporters (Wieland et al., [@B116]) and host proteolytic processes (Price et al., [@B85]).
*L. pneumophila* is an obligate aerobe organism and auxotroph for several amino acids including cysteine, arginine, isoleucine, leucine, threonine, valine, and methionine. The observed auxotrophy corresponds to the notion that cysteine biosynthetic genes and other anabolic genes are absent in the genomes of *L. pneumophila* (Cazalet et al., [@B15]; Chien et al., [@B18]; Glöckner et al., [@B43]; D\'Auria et al., [@B21]; Schroeder et al., [@B97]) and *L. longbeachae* (Cazalet et al., [@B14]; Kozak et al., [@B69]). Compared to chemically defined media, the complex ACES-buffered yeast extract (AYE) broth routinely used to grow *L. pneumophila* contains several additional amino acids: alanine, asparagine, glutamine and glycine. The common solid growth medium for *Legionella* species is buffered charcoal-yeast extract (BCYE) agar, supplemented with L-cysteine and ferric pyrophosphate. *L. pneumophila* growth depends on excess cysteine in the medium (Feeley et al., [@B31]; George et al., [@B41]). Yet, the amount of cysteine added to the BCYE medium is much higher than what is required to support growth. The major part of cysteine in the *Legionella* growth medium is rapidly oxidized to cystine and becomes unavailable to the bacteria, as *L. pneumophila* is not able to utilize this compound (Ewann and Hoffman, [@B27]). The remaining concentration of cysteine is around 0.5 mM, which is enough to support *Legionella* growth. Furthermore, using radio-labeled cysteine and mutant strains, it was found that cysteine is not only imported by specific transporters but also consumed during *L. pneumophila* growth (Ewann and Hoffman, [@B27]).
*L. pneumophila* is also auxotroph for arginine, as the bacteria lack enzymes that allow synthesis of arginine from glutamate. However, the bacteria produce arginine in chemically defined medium supplemented with ornithine or citrulline, which are precursors of arginine emerging in the later steps of the synthesis from glutamate (Tesh and Miller, [@B105]; Hovel-Miner et al., [@B57]). Furthermore, *L. pneumophila* mutants lacking the arginine repressor ArgR fail to replicate within host cells. ArgR might sense the availability of arginine within the host. This leads to the expression of genes (many of them not involved in arginine metabolism), which are required for intracellular growth (Hovel-Miner et al., [@B57]).
The identification of the phagosomal transporter A (PhtA) revealed a major role of threonine not only for replication but also for differentiation of *L. pneumophila* (Sauer et al., [@B95]) (Figure [1](#F1){ref-type="fig"}). A mutant strain lacking *phtA* does not grow in a chemically defined medium, but is rescued by excess tryptone or dipeptides containing threonine, indicating that PhtA is not the only threonine uptake system. Intriguingly, *phtA* mutant bacteria are defective for intracellular replication in macrophages due to their inability to differentiate from the transmissive to the replicative state. Analogously, PhtJ was identified as a valine transporter also required for differentiation and replication within macrophages (Sauer et al., [@B95]; Chen et al., [@B17]). These findings highlight the role of the Pht transporters as means for *L. pneumophila* to scavenge amino acids from host cells.
Further evidence for the importance of the Pht transporter family for nutrient acquisition of *L. pneumophila* was obtained by investigating the *phtC-phtD* locus (Chen et al., [@B17]). The *phtC* and *phtD* genes are paralogs in an operon containing genes involved in nucleotide metabolism. The transporter genes are required for successful replication within macrophages and survival of thymidine deprivation. Expression of *phtC* and *phtD* in *E. coli* bestowed pyrimidine transport activity upon strains lacking all known nucleoside transporters, identifying PhtC and PhtD as thymidine transporters (Fonseca et al., [@B34]).
To take up and utilize amino acids, *L. pneumophila* does not only produce many own systems, but also exploits host metabolic functions (Figure [1](#F1){ref-type="fig"}). The eukaryotic neutral amino acid transporter SLC1A5 was found to be upregulated in *L. pneumophila*-infected cells, and blocking the transporter with the competitive inhibitor BCH (2-amino-2-bornonane-carboxylic acid) or depletion by RNA interference impaired intracellular growth of *L. pneumophila* (Wieland et al., [@B116]). This study demonstrated the requirement of a single host cell transporter for intracellular replication and also indicated that SLC1A5 may be recruited to the LCV, thus enabling *L. pneumophila* to import amino acids from the cytoplasm into the LCV lumen. Other host-cell transporters might be utilized in a similar manner. Notably, similar to *L. pneumophila*, *Francisella tularensis* modulates the expression of SLC1A5 upon infection of THP-1 human monocytes and is also impaired for intracellular replication when this transporter is downregulated (Barel et al., [@B10]).
*L. pneumophila* uses the Icm/Dot T4SS to translocate effector proteins across the LCV membrane to interfere with central host cell processes (Figure [1](#F1){ref-type="fig"}). The Icm/Dot substrate AnkB subverts amino acid metabolism and protein degradation by hijacking the host cell ubiquitination machinery and the proteasome to create nutrients for bacterial growth (Al-Khodor et al., [@B4]). AnkB harbors several eukaryotic domains: an F-box domain that allows interaction with the host SCF1 ubiquitin ligase complex, two ANK domains, which mediate protein-protein interactions in eukaryotes and a CaaX motif that is modified by farnesylation (Price et al., [@B83], [@B82],[@B84]; Ensminger and Isberg, [@B26]; Ivanov et al., [@B61]; Lomma et al., [@B73]). Farnesylation of AnkB leads to localization of the effector to the LCV membrane, and intracellular replication of *L. pneumophila* fails when farnesylation is blocked. Anchoring of the effector to the LCV membrane recruits polyubiquitinated host cell proteins, which are degraded by the host proteasome generating a pool of amino acids utilized for intracellular bacterial replication (Price et al., [@B85]).
Isotopolog profiling is a powerful approach to study metabolic pathways. The method is based on the incorporation of carbon isotopes from stable isotope-labeled precursors such as \[U-^13^C~3~\]serine or \[U-^13^C~6~\]glucose. To elucidate the metabolic pathways and fluxes used, key metabolites such as protein-derived amino acids or storage compounds are then analyzed for the presence of labeled carbon atoms (Zamboni et al., [@B118]). Metabolomic flux analysis and isotopolog profiling have recently provided detailed insights into the metabolism of the pathogenic bacteria *Listeria monocytogenes* (Gillmaier et al., [@B42]) or *Streptococcus pneumonia* (Hartel et al., [@B51]), and the metabolic responses of infected host cells to the pathogens have also been investigated (Eisenreich et al., [@B25]).
Upon growth of *L. pneumophila* in AYE medium supplemented with \[U-^13^C~3~\]serine, incorporation of the ^13^C-label indicated that the amino acid was not only used for protein biosynthesis but also to synthesize other amino acids and poly-3-hydroxybutyrate (Eylert et al., [@B28]). Thus, in agreement with earlier studies (Pine et al., [@B80]; George et al., [@B41]; Ristroph et al., [@B89]) serine can serve as a major carbon source during growth of *L. pneumophila* in broth. Yet, no ^13^C-label was detected in isoleucine, leucine, phenylalanine, tyrosine, histidine, proline or valine, confirming the auxotrophy of *L. pneumophila* regarding these amino acids (Eylert et al., [@B28]). Finally, isotopolog profiling also revealed that *L. pneumophila* growing intracellularly in *Acanthamoeba castellanii* previously fed with \[U-^13^C~6~\]glucose utilizes amoebae-derived amino acids (e.g., phenylalanine, tyrosine) for protein biosynthesis (Schunder et al., [@B98]).
Carbohydrate and polysaccharide metabolism
==========================================
While amino acids seem to represent the preferred carbon source of *L. pneumophila*, the bacteria can also metabolize carbohydrates, other small organic compounds and complex nutrients (Figure [1](#F1){ref-type="fig"}). Early studies using ^14^C-radio-labeled substrates indicated that glucose, α-ketoglutarate, pyruvate, glycerol and acetate are metabolized by *L. pneumophila*, yet only some of these compounds stimulated extracellular bacterial growth under the conditions used (Pine et al., [@B80]; Weiss et al., [@B115]; Tesh et al., [@B106]). Moreover, during infection of macrophages *L. pneumophila* genes required for glycerol catabolism---namely *lpg1414* and *glpD*---were highly upregulated compared to growth in rich broth (Faucher et al., [@B30]). Therefore, glycerol likely plays a role during intracellular growth of *L. pneumophila*, similar to other intracellular bacteria such as *L. monocytogenes* (Eylert et al., [@B29]; Joseph et al., [@B65]) and *Salmonella enterica* (Steeb et al., [@B101]).
Glucose was not found to stimulate growth of *Legionella* spp.; however, the genomes of *L. pneumophila* (Cazalet et al., [@B15]; Chien et al., [@B18]; Glöckner et al., [@B43]; D\'Auria et al., [@B21]; Schroeder et al., [@B97]) as well as *L. longbeachae* (Cazalet et al., [@B14]; Kozak et al., [@B69]) encode complete pathways required for metabolism of carbohydrates, including the Emden-Meyerhof-Parnas (EMP) pathway, the Entner-Doudoroff (ED) pathway, as well as an incomplete pentose phosphate (PP) pathway. In support of the notion that carbohydrate metabolism is crucial during infection, genes associated with the ED pathway, as well as a glucokinase and a glucoamylase, were upregulated upon intracellular growth of *L. pneumophila* in *A. castellanii* (Brüggemann et al., [@B11]). Another intracellular pathogen that depends on sugar assimilation via the ED pathway during intracellular growth is *S. enterica*. Yet, in this case the parallel exploitation of several different host nutrients enhances bacterial virulence (Steeb et al., [@B101]).
Using isotopolog profiling, it was recently shown that *L. pneumophila* indeed catabolizes glucose via the ED pathway (Eylert et al., [@B28]). Upon growth in a chemically defined medium containing \[U-^13^C~6~\]glucose, followed by analysis of the isotopolog pattern by mass spectrometry and NMR spectroscopy, the ^13^C-label was recovered with high efficiency in alanine and also in poly-3-hydroxybutyrate. In contrast, an *L. pneumophila* mutant lacking the glucose-6-phosphate dehydrogenase gene (*zwf*), the first gene of an operon comprising the genes of the ED pathway (*zwf*-*pgl*-*edd*-*glk*-*eda*-*ywtG*), did not incorporate label from glucose and was outcompeted by the wild-type strain in co-infection experiments using *A. castellanii* (Eylert et al., [@B28]). In line with these observations, *L. pneumophila* lacking other components of the ED pathway, either glucokinase (*glk*), phosphogluconate dehydratase (*edd*), 2-keto-3-deoxy-phosphogluconate aldolase (*eda*) or the putative sugar transporter (*ywtG*), was no longer able to metabolize glucose and was defective for growth in *Acanthamoeba culbertsoni* or mammalian cells (Harada et al., [@B50]). Together, these findings strongly support the notion that the ED pathway is essential for glucose metabolism and intracellular growth of *L. pneumophila*. The results also implicate that under the conditions prevailing within LCVs in host cells *L. pneumophila* does not solely grow on amino acids as carbon and energy sources, but rather, carbohydrates are also utilized (at least as co-metabolites). Yet, the relative contribution of amino acids and carbohydrates to intracellular growth is difficult to assess, and many carbohydrates do not support extracellular growth as sole source of carbon and energy.
The transporters promoting the uptake of sugars have not been studied in molecular detail at present. The gene *ywtG* (lpg0421) is conserved among *L. pneumophila* and *L. longbeachae*, and annotated as a putative D-xylose (galactose, arabinose)-proton symporter (Cazalet et al., [@B15], [@B14]). However, arabinose appears to be barely taken up by *L. pneumophila* (excluding genetic approaches based on the arabinose promoter, P*~bad~*). Moreover, glucose-1-phosphate is metabolized much faster than glucose-6-phosphate or glucose, suggesting that the former compound is transported efficiently into the cells (Weiss et al., [@B115]).
In addition to simple carbohydrates and small organic compounds, polymeric compounds also likely serve as carbon sources for *L. pneumophila*. The exogenous supply of polyamines during infection moderately favored intracellular replication of *L. pneumophila* (Nasrallah et al., [@B78]). Moreover, similar to other bacteria (Khosravi-Darani et al., [@B67]), *L. pneumophila* might use the intracellular "energy reserve" poly-3-hydroxybutyrate as an endogenous source of carbon and energy, which is synthesized via pyruvate and acetyl-coenzyme A (James et al., [@B62]; Eylert et al., [@B28]). Further support for the notion that *Legionella* spp. degrade complex polysaccharides stems from the genome sequences. *L. longbeachae* harbors a number of genes likely involved in cellulose degradation (Cazalet et al., [@B14]), and *L. pneumophila* contains genes putatively involved in the degradation of cellulose, chitin, starch and glycogen (Cazalet et al., [@B15]).
The Lsp type II secretion system (T2SS) is essential for intracellular growth of *L. pneumophila* in amoebae and macrophages (Hales and Shuman, [@B45]; Liles et al., [@B71]) (Figure [1](#F1){ref-type="fig"}). Proteome studies on the type II "secretome" of *L. pneumophila* revealed that the bacteria secrete a chitinase (ChiA), as well as an endoglucanase, which metabolizes carboxymethyl cellulose (Debroy et al., [@B22]). An endoglucanase (CelA) was indeed found to degrade cellulose (Pearce and Cianciotto, [@B79]), and a eukaryotic-like glycoamylase (GamA) degraded carboxymethyl cellulose, glycogen and starch (Herrmann et al., [@B52]). Yet, neither CelA nor GamA was required for growth of *L. pneumophila* in amoebae. In summary, insights from genomics, transcriptomics, metabolomics, as well as biochemical experiments indicate that *L. pneumophila* utilizes simple and also complex carbohydrates as important sources of carbon and energy during extra- and intracellular growth.
Micronutrient requirements
==========================
Iron is essential for growth of most if not all bacteria, as it is a co-factor for many enzymes of the central metabolism as part of prosthetic groups like heme or iron-sulfur clusters (Ratledge and Dover, [@B87]). Moreover, the availability of iron is especially important for pathogens, as iron limitation plays an important role in host defense against infections. For *L. pneumophila* iron represents an essential nutrient and has to be supplemented in high concentrations to growth media (Reeves et al., [@B88]; Ewann and Hoffman, [@B27]). The major iron-containing protein of *L. pneumophila* is aconitase of the tricarboxylic acid cycle (Mengaud and Horwitz, [@B75]). *L. pneumophila* grown under iron-limited conditions showed reduced virulence and was impaired for survival in host cells (James et al., [@B63]). Furthermore, host cells treated with iron chelators did not support growth of *L. pneumophila*, presumably due to iron limitation, as the addition of iron as iron-transferrin or ferric iron-nitrilotriacetate reversed growth inhibition (Gebran et al., [@B40]; Byrd and Horwitz, [@B12]; Viswanathan et al., [@B114]). Notably, patients with iron overload or smokers are at increased risk for Legionnaires\' disease, probably because their lungs contain increased levels of iron (Fields et al., [@B32]; Vikram and Bia, [@B113]).
Iron exists in equilibrium between a ferrous (Fe^2+^) and a ferric (Fe^3+^) form, depending mostly on the pH and availability of oxygen (Williams, [@B117]). In *L. pneumophila*, many systems are devoted to iron metabolism and involved in iron reduction, complexation and transport (Figure [1](#F1){ref-type="fig"}). Iron reductase enzymes may promote iron assimilation in the periplasm and cytoplasm (Johnson et al., [@B64]; Poch and Johnson, [@B81]). Iron reduction is also catalyzed by the secreted compound homogentisic acid (HGA) and its polymerized derivative HGA-melanin (Chatfield and Cianciotto, [@B16]; Zheng et al., [@B119]). HGA is a product of the phenylalanine and tyrosine catabolism of *L. pneumophila* and was identified as the brown pigment secreted by *L. pneumophila*, which is produced from oxidative polymerization of HGA to HGA-melanin (Steinert et al., [@B103]). HGA and HGA-melanin stimulate growth of *L. pneumophila* under iron-limiting conditions, enhance the uptake of iron and can release ferrous iron from transferrin and ferritin, two major protein iron chelators of mammalian cells (Zheng et al., [@B119]).
*L. pneumophila* chelates and transports iron with the secreted high-affinity iron siderophore legiobactin (Liles et al., [@B72]; Starkenburg et al., [@B100]). The gene *lbtA*, which has homology with siderophore synthetases, and *lbtB* that encodes a homolog of a multidrug efflux pump, were identified as key players in the synthesis of legiobactin (Allard et al., [@B6]). Moreover, an *L. pneumophila lbtA* mutant strain showed reduced ability to infect lungs of A/J mice, demonstrating the importance of legiobactin *in vivo* (Allard et al., [@B5]). Iron is also transported in *L. pneumophila* via the *FeoAB* system (Robey and Cianciotto, [@B90]). The *L. pneumophila feoAB* operon bears homology to the *E. coli* system, a well-characterized ATP-driven ferrous iron transporter. An *L. pneumophila feoB* mutant was outcompeted by wild-type bacteria during infection of A/J mice, highlighting an important role of the transporter *in vivo*.
Finally, in addition to iron, the extracellular growth of *L. pneumophila* is also stimulated by calcium, magnesium and zinc. Calcium and magnesium might also play a role in biofilm formation, as the two metal ions enhance the adherence of *Legionella* to surfaces (Reeves et al., [@B88]; Koubar et al., [@B68]).
Metabolic regulation of differentiation and virulence
=====================================================
The biphasic life cycle of *L. pneumophila* is regulated by a variety of environmental and metabolic stimuli (Molofsky and Swanson, [@B77]). As long as nutrients are not limiting, the post-transcriptional regulator CsrA suppresses transmission traits and promotes replication (Molofsky and Swanson, [@B76]). Given the importance of amino acids as carbon source for *L. pneumophila*, it is not surprising that these compounds are also main regulatory factors of the phenotypic switch (Byrne and Swanson, [@B13]; Sauer et al., [@B95]). Amino acid starvation or otherwise nutrient-limiting conditions trigger the shift from the replicative/non-motile to the virulent/motile form of *L. pneumophila*, which is mediated through the second messenger guanosine 3′,5′-bispyrophosphate (ppGpp) (Hammer and Swanson, [@B47]; Dalebroux et al., [@B20]) (Figure [2](#F2){ref-type="fig"}). The "alarmone" ppGpp is synthesized by the synthase RelA as part of the "stringent response" that senses the accumulation of uncharged tRNAs at the ribosome. A second stringent response enzyme called SpoT also synthesizes ppGpp (Dalebroux et al., [@B19]). However, rather than sensing amino acid shortage, SpoT monitors fatty acid biosynthesis by interacting with the acyl-carrier protein ACP (Edwards et al., [@B23]). In addition, SpoT hydrolyzes ppGpp during exponential growth to ensure that transmissive traits are not expressed during replication.
{#F2}
The alternative sigma factor RpoS (σ^38^/σ^S^) represents the pivotal transcriptional regulator of the *L. pneumophila* life cycle (Hales and Shuman, [@B46]; Bachman and Swanson, [@B8]; Zusman et al., [@B121]). An *L. pneumophila rpoS* mutant is not affected regarding extracellular growth in broth and retains significant stress resistance, but is not able to replicate in amoebae. This severe defect in intracellular replication is not due to impaired Icm/Dot function or *icm*/*dot* gene expression, but because of major transcriptional changes affecting basic cellular processes and other central regulatory networks (Hovel-Miner et al., [@B58]). The transcription of more than 70 genes required for central metabolism, 40 of these associated with amino acid metabolism, was negatively regulated in the *rpoS* mutant. Furthermore, small regulatory RNAs (*rsmY* and *rsmZ*) (Rasis and Segal, [@B86]; Sahr et al., [@B94]), two component systems (CpxRA, PmrAB), the transcriptional regulator ArgR and the quorum sensing response regulator LqsR (Tiaden et al., [@B110]) are regulated by RpoS (Bachman and Swanson, [@B9]; Hovel-Miner et al., [@B58]) (Figure [2](#F2){ref-type="fig"}).
At least three two component systems and one quorum sensing system influence the virulence of *L. pneumophila:* CpxRA (Gal-Mor and Segal, [@B35]; Altman and Segal, [@B7]), PmrAB (Zusman et al., [@B120]; Al-Khodor et al., [@B3]; Rasis and Segal, [@B86]), LetAS (GacAS) (Hammer et al., [@B48]; Gal-Mor and Segal, [@B36]; Lynch et al., [@B74]) and the *Legionella* quorum sensing (*lqs*) gene cluster (Tiaden et al., [@B109]) (Figure [2](#F2){ref-type="fig"}). The *lqs* system of *L. pneumophila* comprises the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT (Kessler et al., [@B66]), and the response regulator LqsR (Tiaden et al., [@B110]). LqsA produces the compound LAI-1 (*Legionella* autoinducer-1, 3-hydroxypentadecane-4-one) (Spirig et al., [@B99]), which presumably binds to the cognate sensor kinases. The kinase-mediated phosphorylation signal converges on LqsR (Schell et al., [@B96]), which among many other processes also controls the switch from the stationary phase to the replicative phase (Tiaden et al., [@B110]). Lqs-regulated processes include pathogen-phagocyte interactions, production of extracellular filaments, natural competence for DNA uptake and the expression of a 133 kb genomic "fitness island" (Tiaden and Hilbi, [@B107]). Furthermore, transcriptome analysis of *L. pneumophila* strains lacking *lqsR*, *lqsS* or *lqsT* or the entire *lqs* cluster indicates that the Lqs system also regulates a number of metabolic pathways (Tiaden et al., [@B110], [@B108]; Kessler et al., [@B66]).
Conclusions and perspectives
============================
The amoebae-resistant bacterium *L. pneumophila* colonizes a variety of extra- and intracellular niches in the environment. Upon reaching the human lung, *L. pneumophila* grows in mammalian macrophages and possibly also in epithelial cells. Accordingly, the bacteria are equipped to utilize a broad range of compounds as carbon and energy sources. In addition to amino acids, which initially have been regarded as the main if not sole nutrients, carbohydrates have recently been shown to be catabolized by extra- and intracellularly growing *L. pneumophila*. Novel technological approaches such as isotopolog profiling allow analyzing metabolic fluxes with unprecedented resolution and sensitivity. Transcriptome and genome studies indicate that a number of other compounds, including complex polysaccharides, are also metabolized by *L. pneumophila*. Further studies will unravel the manifold and robust metabolic pathways that the bacteria employ to thrive in diverse environmental niches. Importantly, future investigations will also shed light on the intricate relationship between the physiology and pathogenesis of *L. pneumophila*, and thus might contribute to control Legionnaires\' disease.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Ina Haneburger and Bernhard Steiner for critically reading the manuscript. Research in our laboratory was funded by the Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, the Deutsche Forschungsgemeinschaft (DFG; SPP 1316, SPP 1617) and the Swiss National Science Foundation (31003A-125369).
[^1]: Edited by: Wolfgang Eisenreich, Technische Universität München, Germany
[^2]: Reviewed by: Marina Santic\', University of Rijeka, Croatia; Joseph Vogel, Washington University School of Medicine, USA
[^3]: This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.
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Hey I dont see to much about the Hawks at all over here in NH, but I was wondering something...
Did Wilson play that well in the rpeeseason or was Flynn that BAD in the preseason wilson got the job?? 9I mean you guys went out and paid big bucks get Flynn so I am just wondering how Wilson was able win the job before game 1 even happened.)
Flynn definately wasn't underwhelming IMO. He seemed to be a very capable game managing "dink n dunk", "take whats there" type of QB, which is not a bad thing. He seemed to be able to take care of the ball and hit the open guy for a decent gain, but that's all he looked like. He looked like Kyle Orton or Matt Cassel, who aren't bad quarterbacks by any stretch, but they aren't "special". After the last few years, I'd have been happy as anything to have that kind of QB.
But Russell Wilson looked like he had the potential to be so much more than that, and still does IMO. But he's a rookie QB who's going to go through the rookie QB learning curve, which is where we find ourselves. Barring anything horrible, I can see Rusty having a pretty damn good career, full of playoff runs and pro-bowls.
FREE PEHAWK
Sac>CANI love Sac with all my heart, and wish I were half as handsome as he.^I know this is here, but leaving it b/c YOLO.
CANHawk wrote:Flynn definately wasn't underwhelming IMO. He seemed to be a very capable game managing "dink n dunk", "take whats there" type of QB, which is not a bad thing. He seemed to be able to take care of the ball and hit the open guy for a decent gain, but that's all he looked like. He looked like Kyle Orton or Matt Cassel, who aren't bad quarterbacks by any stretch, but they aren't "special". After the last few years, I'd have been happy as anything to have that kind of QB.
But Russell Wilson looked like he had the potential to be so much more than that, and still does IMO. But he's a rookie QB who's going to go through the rookie QB learning curve, which is where we find ourselves. Barring anything horrible, I can see Rusty having a pretty damn good career, full of playoff runs and pro-bowls.
Read this explanation and all but ignore the others... It was quite the polarizing issue so everyone tends to have a biased opinion. Also, Flynns contract wasn't that big, especially after the first year. He'll make about 5 mill next year and then 6 the year after if we keep him through the next 2, so he could be an interesting trade possibility. That is only if Russell Wilson holds onto the job, plays well, AND we can get another backup quarterback who won't be huge drop off from Flynn.
Russel outplayed Flynn in the preseason and showed the potential to be special. In addition the coaches and players said his work ethic, film study and leadership qualities tipped the scales in his favor. During camp we heard the praise about how he's the first here, last to leave and is out-studying everyone in the film room.
I tend to agree that Flynn's future is based on Wilson's progress, but while Flynn is higher paid...The FO only guaranteed that money for two years, so from a business standpoint he is a value and we did not over spend for him even as a back-up IMO.
I ask about the trade possibility because a team say Like Buffalo could give up as much as a 1st rounder to get Flynn (or at least a 2nd rounder) and while the hawks are vastly improved every team could use another high 2nd round pick or even earlier first round pick.
CANHawk wrote:Flynn definately wasn't underwhelming IMO. He seemed to be a very capable game managing "dink n dunk", "take whats there" type of QB, which is not a bad thing. He seemed to be able to take care of the ball and hit the open guy for a decent gain, but that's all he looked like. He looked like Kyle Orton or Matt Cassel, who aren't bad quarterbacks by any stretch, but they aren't "special".
Canadians must have a different definition for "underwhelming" because you just hit it on the head with your description of Flynn.
PatsFanNH wrote:lol ok, do you see the Hawks trying to trade one of these two QB's in the offseason? (be silly have such a high salary for a back up QB.)
It'll be hard to trade Flynn, IMO. There wasn't much of a market this off-season for the unproven quarterback and next year's might just be worse considering how many teams are seemingly "set" with their veteran/young quarterbacks.
Flynn's contract is $5.25 million next year with $2 mil guaranteed. Too high to trade for as a backup and probably too high for teams looking for, at best, a two-way quarterback competition. Teams looking for an upgrade would do better looking to the draft.
I could see Flynn staying through 2013, but if Wilson is still the starter by then, Flynn is gone. It's excusable to pay that now (or next year) to backup a 1st year or 2nd year young player, but not for a 3rd year starter (where Flynn is set to make $6.25m).
"People who don't punch their ponies in the face make me sick." - Louis C.K.
Flynn did in the preseason what Wilson's doing in the regular season (ie. Moving the ball into FG range and then stalling in the Red Zone or having to punt on every drive.)
In the preseason though, Wilson was scoring on almost every drive (at first against 2nd team Defenses, then in games 3 and 4 of the preseason, he did it against 1st team defenses) and looked phenomenal with a bajillion gallons of potential.
Therefore, it stands to reason then that if Flynn was behind center in the regular season, he would either be doing the same as Wilson or worse.
CANHawk wrote:Flynn definately wasn't underwhelming IMO. He seemed to be a very capable game managing "dink n dunk", "take whats there" type of QB, which is not a bad thing. He seemed to be able to take care of the ball and hit the open guy for a decent gain, but that's all he looked like. He looked like Kyle Orton or Matt Cassel, who aren't bad quarterbacks by any stretch, but they aren't "special".
Canadians must have a different definition for "underwhelming" because you just hit it on the head with your description of Flynn.
Maybe Flynn was just "whelming" and not over or under.
I think a word people have used in this thread applies. Wilson looked special, like he had a playmaking element to his game that just didn't seem to be there with Flynn. I recall back in the preseason someone saying that Flynn played capably and efficiently, but that he was uninspiring. That summed up my views on it, too.
Seahawk Sailor wrote:To sum it up for Pats fans: would you rather go with the rookie who looks like he could be a Tom Brady, or the veteran backup with three starts that looks like he could be a Drew Bledsoe?
LOL BB went with Bledsoe for a year so Brady could learn the offense and get comfortable lol.. and Bledsoe was good for like 4 years..
Like everyone has said, Flynn looked fine and capable, but Wilson just took the Seahawk Nation by storm and was effin dominant in the preseason. Hopefully after last weeks game the reigns will come off a little bit more, but I doubt it. I think because it IS the Pats, and what the forecast is calling for that the Seahawks are gonna try and turn this into a slobberknocker, last time they forced a game like that was when they demoralized the Cowboys. I don't ask this to be a douche, I ask because I really just don't know, but can the Pats play a grind it out beat the holy hell out of another team and take a beating game?
Last edited by Throwdown on Thu Oct 11, 2012 11:56 am, edited 1 time in total.
Seahawk Sailor wrote:To sum it up for Pats fans: would you rather go with the rookie who looks like he could be a Tom Brady, or the veteran backup with three starts that looks like he could be a Drew Bledsoe?
LOL BB went with Bledsoe for a year so Brady could learn the offense and get comfortable lol.. and Bledsoe was good for like 4 years..
You say this as though Bledsoe was only a temporary placeholder for Brady. IIRC, Bledsoe was paid a ridiculous contract the year they drafted Brady (something like 10 years/100 m.) I doubt even BB knew what they had in Brady that first season.
Edit: Did some research, Bledsoe was re-signed a year after they drafted Brady.
"People who don't punch their ponies in the face make me sick." - Louis C.K.
Flynn's salary is not a big deal given Wilson basically here on a premium. The way I see it is The Seahawks are spending (let's round) say 3 million dollars at the QB position in 2012, it doesn't matter how it's broken up...that 3 million still has to be spent. It's not on PC to pay these guys, he just needs to put who he believes is the best on the field. Besides, the owners rich b#!!!
That being said, Wilson lit it up in the off season and won the job straight up. Personally, I feel the decision took a little too long and is hampering is performance so far. His chemistry with the receivers has been lack luster...but!....showing definite signs of improvement...can't wait to see what will happen when the offense is on the same page! It's going to be awesome.
PatsFanNH wrote:I ask about the trade possibility because a team say Like Buffalo could give up as much as a 1st rounder to get Flynn (or at least a 2nd rounder) and while the hawks are vastly improved every team could use another high 2nd round pick or even earlier first round pick.
PatsFanNH wrote:I ask about the trade possibility because a team say Like Buffalo could give up as much as a 1st rounder to get Flynn (or at least a 2nd rounder) and while the hawks are vastly improved every team could use another high 2nd round pick or even earlier first round pick.
We already gave them TJack
Which is why the posts about us "not being able to trade Flynn" are ridiculous. I bet I read 1,000 posts about how it would be impossible to trade TJ since he was making $4 million. Well guess what, we got something out of him. If Flynn gets any playing time this year (whether through injury or because we are kicking the crap out of someone), his trade value could go up substantially. Besides, if the rookie QBs have proven anything this year it is that it is difficult to come in and start as QB. RW is tied for the best win loss record for all rookie QBs in the past 20 years, and he has been assisted by a phenomenal D and a great rushing game. Different teams will be in the market this year, different QBs will get hurt or bomb out, Flynn will be worth something guaranteed.
Throwdown wrote:Like everyone has said, Flynn looked fine and capable, but Wilson just took the Seahawk Nation by storm and was effin dominant in the preseason. Hopefully after last weeks game the reigns will come off a little bit more, but I doubt it. I think because it IS the Pats, and what the forecast is calling for that the Seahawks are gonna try and turn this into a slobberknocker, last time they forced a game like that was when they demoralized the Cowboys. I don't ask this to be a douche, I ask because I really just don't know, but can the Pats play a grind it out beat the holy hell out of another team and take a beating game?
1st Snow doesnt stop the Pats passing game and if anything slippery conditions are BETTER for theReceiver as he knows where he is going while the CB could lose their balance. So the Pats will pass alot still.
As for the slobberknocker game, I THINk they can do it now with these running backs especially since teams cant key on the running game even in bad weather.
PatsFanNH wrote:lol ok, do you see the Hawks trying to trade one of these two QB's in the offseason? (be silly have such a high salary for a back up QB.)
The Seahawks look at it as salary paid to a position...so they are spending X amount on the QB position, it doesn't matter to them if the starter or the backup is making more of the pot than the other.
They have the luxury to do that because Wilson is making 3rd round rookie money. His salary combined with Flynn's is STILL less than what a lot of teams pay their starting QB.
If Wilson were to get really good and be worthy of a pay raise in an extension, by that time Flynn will be nearing the end of his contract.
In week 17, after we beat the 9ers and seal the division and the #1 spot in the NFC, he'll get to throw for a franchise record 500 yards and 7 TDs against the Rams ....exactly like he did to Detroit when he was in Green Bay. (putting up monster digits in a meaningless game)
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243
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6
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9
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54
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5
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0
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3
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3
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7
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3
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4
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280
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21
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4
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39
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107
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22
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2
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4
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22
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6
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32
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65
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2
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42
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3
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3
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49
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36
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11
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4
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60
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387
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2
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11
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20
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5
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28
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51
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3
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14
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15
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4
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3
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2
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49
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3
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2
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2
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30
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5
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24
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7
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3
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315
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41
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48
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5
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5
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46
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2
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230
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21
What is the cube root of 6507
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Hydrogen could save regional railways
November 29, 2013
by Andreas Hoffrichter, The Conversation
Small, but not for long. Credit: niversity of Birmingham
There is increasing talk of electrification of the UK's railway network. But electrification is an expensive business, requiring much new hardware including masts, wiring, substations and so on. Such an investment can be justified for heavily used lines such as urban metro systems and inter-city routes, but not on less used lines such as regional routes. Railways will continue to need an energy supply system that does not rely on electrification for the foreseeable future.
This currently takes the form of diesel power. But in addition to environmental concerns over emissions, the price of diesel is likely to continue to increase. A point will be reached in the future where it is no longer economically viable or environmentally acceptable to use diesel, and hydrogen appears to be an attractive alternative.
Like electricity, hydrogen can be produced from many different primary sources including natural gas, coal and petroleum. It can also be produced using electricity to split water into its elements. Then there are several "green" production options being developed including the anaerobic digestion of waste material and the direct use of heat from solar energy. But unlike electricity, hydrogen can be stored and transported in discrete quantities in order to power trains, much like diesel or natural gas.
To release the energy stored in the hydrogen, one option is to use a fuel cell. In this device, hydrogen is combined with oxygen from the air to produce electricity, water and heat. There are no harmful emissions or exhaust fumes, and the technology is quiet, vibration-free and low maintenance – all attractive features for railway applications. Fuel cells are usually combined with batteries which help meet the peak power demand during hard acceleration, and to provide a store for energy recovered during braking.
A recent study by the University of Birmingham showed it is possible to store enough hydrogen onboard a modern train (a lightweight commuter train in this case) to provide a similar range to diesel power. In contrast to battery only systems, re-fuelling takes a similar amount of time to diesel, and only a limited number of re-fuelling points would need to be provided. Commercially, the cost of hydrogen is comparable to that for diesel, and the inherent ability of a hybrid system to store and reuse braking energy helps reduce overall consumption.
To show the viability of the technology, the UK's first hydrogen-powered locomotive, called the Hydrogen Pioneer, was developed and built using off-the-shelf technology. The locomotive uses a 1.1kW fuel-cell stack to charge four lead-acid batteries that provide the combined power for acceleration and store energy when braking. The 320kg-locomotive can haul wagons and passengers up to 4,000kg.
This video is not supported by your browser at this time.
Elsewhere in the world, hydrogen-powered railway vehicles have been constructed in Spain, the United States, China, and Japan. These have mostly been prototypes, but the first commercial operation of hydrogen power has been for mining locomotives, with a fleet now operating at a platinum mine in South Africa. In 2014, hydrogen-powered trams will start commercial operation in Aruba, with hydrogen generated solely from renewable sources.
It is possible that hydrogen-powered trains will see service on the UK network within the next few years, most likely on lower-speed applications such as trams or shunting locomotives. But hydrogen has the potential to play a larger role, powering those parts of the network where electrification does not make sense.
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16 comments
"But in addition to environmental concerns over emissions, the price of diesel is likely to continue to increase."
"Like electricity, hydrogen can be produced from many different primary sources including natural gas, coal and petroleum."
How do people with no knowledge of physics or energy conversion get published?
Prove to me that converting natural gas into H2 can be more cost, pollution and energy efficient than burning it directly in an engine. How can green science deserve any respect at all when it is represented by articles like this???
Prove to me that converting natural gas into H2 can be more cost, pollution and energy efficient than burning it directly in an engine.
Since they're not claiming it does - why should they need to prove it?
Even so I could see that sometimes there are considerations about global vs. local pollution. E.g. for trains operating largely within population centers it is sometimes better that they don't pollute locally - even if the combined global pollution by producing their fuel elsewhere is greater.
Local railroads need to plan economically with a long time horizon. The price of diesel can only go up, making such plans dicey (and quickly push them into the 'uneconomical' region).
With the amount of income of people using railroads stagnating/dropping they cannot hope to match this price increase with increased revenue in the long run. railroad owners need a fuel that will remain stable in price (or become cheaper as technology develops). Hydrogen could just be that fuel.
"Since they're not claiming it does - why should they need to prove it?" So if they are not claiming that converting viable fuels into hydrogen has any advantages at all, what exactly is the point of the article? Natural Gas is a superb fuel and converting it to H2 for transportation use is just plain stupid.
"railroad owners need a fuel that will remain stable in price (or become cheaper as technology develops). Hydrogen could just be that fuel."
There is absolutely no proof that H2 derived from fossil fuels could ever be more efficient than using the fuel directly.
There is absolutely no proof that H2 derived from fossil fuels could ever be more efficient than using the fuel directly.
The hydrogen from fossil fuels idea is debunked immediately by pointing out that methane (natural gas) can be used directly in a solid oxide fuel cell without conversion to hydrogen, because such fuel cells are internally reforming. They can even burn gasified diesel as long as there's no sulfur in it.
The output is just water and CO2 - no particulate matter or NOx or HCs - so AA's complaint about pollution and air quality doesn't apply.
The requirement for raw hydrogen is only a limitation of PEM fuel cells which pass the hydrogen ion through the membrane, whereas the SOFC works the opposite way by passing oxygen ions to burn whatever it is on the other side.
And methane stores twice the energy at half the pressure in a volume compared to hydrogen.
Like electricity, hydrogen can be produced from many different primary sources including natural gas, coal and petroleum. It can also be produced using electricity to split water into its elements.
All that applies to methane as well.
Then there are several "green" production options being developed including the anaerobic digestion of waste material
Anaerobic digestion of waste into methane is 99% efficient, into hydrogen just 5-10% efficient. The hydrogen digestion also releases byproduct organic solvents into the water, which actually makes it more polluted.
I too have troubles understanding what's the big deal with hydrogen? It's inferior to just about every other option you can imagine in almost every way imaginable. It takes the most space, it's the most flammable, it's the most difficult to contain, most expensive to obtain...
Look Anti, I read the article and unless there is an energy, pollution, and cost, effective way to produce H2 the whole article is a pipe dream.
Which is easily answered because you always have to ask yourself: cost effective compared to what?
If that which you compare it to (fossil fuels) is a finite resource then you have your answer. A finite resource can only become more expensive with time as it gets used up. Hydrogen is a (virtually) infinite resource so its cost is constant or will only drop with better technology. In such a setup there MUST come a point where it's cost effective.
If you look at the mounting cost due to environmental changes caused by fossil fuels (not to speak of the health costs) then that point has already long been passed. We're currently just deluding ourselves by kicking that particular can down the road.
People are still buying the hydrogen economy crap? Really? Let's see ... difficult to produce, difficult to transport and store, easy to use. Well, it has 1 of 3 worked out. Too bad the other two turn every corner fuel stop into a bomb just waiting for idiots to arrive.
"Hydrogen is a (virtually) infinite resource so its cost is constant or will only drop with better technology. In such a setup there MUST come a point where it's cost effective."
Look this article is about converting perfectly good fossil fuels into H2 which is a ludicrous use of precious resources.
If man ever develops a viable source of cheap renewable energy H2 could become a useful general purpose fuel after the containment and distribution issues are solved. As another poster pointed out, methane probably makes more sense as a fuel.
Look this article is about converting perfectly good fossil fuels into H2
No it is not. That is only mentioned because it is a possibility among many - for completeness sake.
Nowhere in the article does it say that this is the only or preferred way to do it. The article does not express a preference at all, but expressly stresses that there are green ways to do it. So if you want to interpret anything at all then they are preferring that.
Hydrogen is a (virtually) infinite resource so its cost is constant or will only drop with better technology. In such a setup there MUST come a point where it's cost effective.
No it isn't. Hydrogen isn't a resource since all of it has to be made. There's no hydrogen mines or hydrogen wells that spout infinite amounts of hydrogen - all of it has to be manufactured using -other resources-
What you're claiming is pure nonsense. It's like saying size AA alkaline batteries are a resource and it's just a matter of time before they become cost effective to drive your car on.
Of course with infinite resources we could make AA batteries, or hydrogen, so cheap to use that you could load your trunk full and drive into the sunset, but the point is that it would still be just a massive waste of effort.
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Saracens shine and Marcelo Bosch will make them even stronger
Saracens’ decision to add Argentina centre Marcelo Bosch to their powerful squad has proved very timely after Brad Barritt underwent ankle surgery that will keep him out of rugby until the New Year.
Bosch, who has been playing at Biarritz, has been linked with Saries for some time and Standard Sport understands the deal has now been finalised although the 21-cap back will not be available until the Rugby Championship concludes next month.
The Pumas round off the tournament against Australia in Rosario on October 5 but Sarries are aware Bosch could be needed for Argentina’s autumn programme, which starts against England at Twickenham on November 9 and is followed by matches against Wales and Italy.
Barritt has been the rock on which Saracens and England have built their defences and his loss will be felt by club and country as he will miss the November Tests with Australia, Argentina and New Zealand.
The centre first damaged his ankle during the Heineken Cup semi-final loss to Toulon in April and although he recovered in time for the Lions tour the ankle became a major issue again after he helped Sarries defeat London Irish last Saturday.
Given Barritt’s new lay-off, the signing of Bosch has taken on more importance as the 29-year-old is an experienced utility back and a long-range goal kicker.
Without Barritt, Sarries were still able to overwhelm Gloucester 44-12 at Allianz Park yesterday with the five-try victory taking them to the top of the Premiership. They were helped by the sending off of Gloucester prop Nick Wood after just 73 seconds for stamping on Jacques Burger’s head.
Wood apologised to Burger and the Sarries flanker said: “There are no hard feelings and you do silly things in rugby. I am sure he feels really bad about it. It’s tough when a team go a man down and we stuck to our game plan, particularly in the second half.”
Burger spent nearly two years battling to overcome a serious knee injury and although he was man of the match yesterday he believes he can get even better.
With Scotland captain Kelly Brown and highly rated young England flanker Will Fraser both set to return from injury, Burger knows he must make a big impression before the established rotation policy kicks in.
He said: “It will take some time for me to be 100 per cent in terms of decision making and I am just happy to be playing rugby again. I know that other guys are coming back into the squad.
“We have a healthy competition going in the back row at the club and you have to make the most of your chance with our rotation.”
London Irish were indebted to England wing Marland Yarde for their 20-18 win at Worcester with the 21-year-old running in two tries.
Irish director of rugby, Brian Smith, said: “Marland Yarde’s finishing is lethal and he is as good as there is in the game.
“He was finding his feet last season but now he’s the real deal. His biggest strength is his willingness to learn but I believe there’s still 25 per cent improvement to come.”
London Wasps lost 30-26 at Exeter while Harlequins were beaten 13-6 by Northampton at the Twickenham Stoop.
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The present invention relates to sheet feeding and, in particular, to the feeding of sheets, such as paper sheets, one-at-a-time from a stack and detecting double sheets.
It is known to feed sheets sequentially, i.e., one-at-a-time from a vertical stack, e.g., in sheet-printing and sheet-coating machines. For example, U.S. Pat. Nos. 7,125,014 and 7,207,558 disclose a sheet feeding apparatus employing suction belts which grip the upper surface of a top sheet in the stack and advance the sheet. The disclosures of those patents are incorporated by reference herein.
It is, of course, desirable to prevent the feeding of double sheets, defined herein as arising when a second sheet adheres itself to the underside of the sheet above it in the stack, e.g., due to static friction. Such double sheet feeding is undesirable, especially in the case where paper sheets are fed to a sheet-coating apparatuses in which the coatings are cured by passing the coated sheets beneath a heater which emits intense heat. In the case of double sheets being fed, the extra bottom sheet can become dislodged from the top sheet and possibly immobilized beneath the heater, whereupon overheating of the immobilized sheet can produce a fire.
Efforts to sense the feeding of double sheets are known, such as disclosed in U.S. Pat. No. 4,420,747 in which sheets are passed successively through the nip of a roller pair, a lower one of the rollers being driven about a fixed axis, and a top one of the rollers being freewheeling and vertically movable. The passing of a sheet through the nip causes the top roller to be displaced upwardly. That roller displacement is sensed by a transmitter which sends a signal to electric evaluator circuits. When double sheets pass through the nip, the greater thickness of the double sheets produces an increase in the roller displacement, which is sensed by the evaluator circuits, and an appropriate warning signal is produced. The disclosure of this patent is incorporated by reference herein.
Despite the precaution heretofore taken in the art to prevent the feeding of double sheets, room for improvement remains. For example, in the case of the roller pair disclosed in afore-mentioned U.S. Pat. No. 4,420,747, it will be appreciated that expensive rollers of high-precision manufacture and positioning are required in order to be able to reliably detect the minute difference in sheet thickness between a single sheet and double sheets, especially when very thin paper sheets are being fed. The reason is that if rollers of imprecise positioning or shape, e.g., of eccentric or out-of-round shape, are used, displacements of the freewheeling roller can occur just because of such imprecise positioning or shape. Since the difference in roller displacement between the feeding of single sheets versus double sheets is minute, the creation of such false displacements can produce unreliable results.
It would be desirable to provide a sheet feeding system which minimizes the chances for double sheets to be fed from a stack, and/or maximizes the chances for the feeding of double sheets to be detected along a feed path.
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A qualitative research synthesis exploring professional touch in healthcare practice using the threshold concept framework.
Touch is an integral part of human life. Consequently, touching and being touched are also fundamental to healthcare practice. Despite a significant literature on touch, it is rarely conceptualized or discussed in terms of the student journey from layperson to practitioner. We chose to explore professional touch using the threshold concepts framework (TCF), which provides a theoretical model for exploring the way in which learners encounter, engage with and understand fundamental concepts in a discipline. This qualitative research synthesis (QRS) describes the use of the TCF to identify key issues involved in developing and using professional touch. Through a cross-professional analysis and synthesis of recent international literature, we aimed to identify key characteristics of the transitional journey for professional touch. Three orders of analysis were applied, employing a methodology described by Major and Savin-Baden (An introduction to qualitative research synthesis: managing the information explosion in social science research, Routledge, London, 2010). Following identification of threshold characteristics in the overall sample of articles, second order analysis revealed the nuances of professional touch associated with the characteristics. The final synthesis led to identification of five themes: touch as dialogue; being changed by touch; multiple boundaries of touch; multiple meanings of touch and influences on touch. Whilst providing support for some assertions within the literature, this QRS also offers new insights into the complexity of professional touch. Given the paucity of explicit learning and reflection around professional touch in training programmes of health professionals, the TCF reveals ways in which professional preparation might be improved to promote understanding of the role and impact of touch in practice.
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Introduction {#s1}
============
Notch system is highly conserved from invertebrates to mammals and regulates cell fate decisions during the development of various organs ([@bib5]; [@bib23]). This system is composed of four Notch receptors (Notch1--4) and four Notch ligands, Delta-like (Dll) 1, Dll4, Jagged (Jag) 1, and Jag2 in mammals; the interaction between these receptors and ligands induces the proteolysis of the Notch receptor, resulting in the translocation of Notch intracellular domain (NICD) into the nucleus, where the binding of NICD with transcription regulators such as Rbpj and MamL1 activates several target gene transcription. It is well known that Notch system plays pivotal roles in the development of various mammalian tissues, including neuron/glia, intestine, lung, pancreas, and so on. Interestingly, the interaction between Notch receptors and ligands is different in each tissue.
The Notch system plays essential roles in T lymphopoiesis in the thymus. Loss-of-function experiments using Notch1-deficient hematopoietic progenitor cells (HPCs) and Dll4-deficient thymic epithelium in vivo clearly demonstrated that the Notch1-Dll4 interaction is pivotal for T lymphopoiesis in the thymus ([@bib32]; [@bib18]; [@bib20]). It has been long believed that three-dimensional distinctive structure of the thymus was necessary for T lymphopoiesis. However, it has been found that T lymphopoiesis can be recapitulated in an in vitro monolayer-culture system, in which HPCs bear the exogenous active fragment of Notch1 or are cultured on Notch ligand-expressing stromal cells ([@bib15]; [@bib33]). In this culture system, Dll1, as well as Dll4, can promote HSCs to differentiate into T lineage cells; however, the efficiency of Dll1 to promote HSC differentiation into T cells is lower than that of Dll4 ([@bib4]; [@bib27]).
As Dll1 expression is not clearly detected in the mammalian thymus ([@bib12]), and the Dll1-deficient thymic epithelium remained intact ([@bib16]), this in vitro result seems not be worth paying attention to. However, from the evolutionary point of view, cartilaginous fish possesses only Dll1 but not Dll4, suggesting that Dll1 is the only Notch ligand which contributes to T lymphopoiesis in the thymus of cartilaginous fish ([@bib3]). As cartilaginous fish lack the development of some major T cell sub-populations in the thymus ([@bib39]), the acquisition of Dll4 seems to be associated with the explosive evolution of T cell-mediated immune system. Furthermore, it has been reported that in some processes, such as the development of arterial vascular endothelium and presomitic mesoderm, Dll1 is not fully substituted by Dll4 ([@bib30]; [@bib38]). Taken together, the physiological function of Dll1 and Dll4 is different. However, the molecular basis underlying this difference, and the preferential contribution of Dll4 to T lymphopoiesis is not fully understood.
Dll1 and Dll4 are type I cell-surface proteins that are composed of several distinct domains; MNNL (module at the N-terminus of Notch ligands), DSL (Delta/Serrate/Lag-2), and 8 EGF-like repeats ([@bib23]). A study that used recombinant soluble Notch1 and Dll proteins revealed that the N-terminus of Dll1/4 spanning from MNNL to the 3^rd^ EGF-like repeat is enough to compare the simple affinity to Notch1, which was 10-fold higher for Dll4 than for Dll1 ([@bib2]). Especially, the 1^st^ and 2^nd^ EGF-like repeat of Dll1 are known to form a distinct secondary structure. The DOS motif is common among Dll1, Jagged1, Jagged2---but not in Dll4---which were reported to contribute to binding with Notch receptors ([@bib23]; [@bib22]). Therefore, it is assumed that Dll4 binds to Notch1 with stronger affinity than Dll1 and in a different manner from other Notch ligands, as far as the truncated and soluble form of Notch receptors and ligands are concerned.
The high-resolution structure of Notch1-Dll4 was revealed using a high-affinity mutant of Dll4, showing that Dll4 binds to Notch1 via two domains, MNNL and DSL ([@bib25]). However, the contribution of the MNNL domain to the difference between Dll1 and Dll4 with the swapping strategy has not been reported. Recently, it has been shown that functional differences between Dll1 and Dll4, observed in vivo, resides in the extracellular domains ([@bib38]). Moreover, these ligands differ in their ability to activate different Notch receptors, such as Notch1 and Notch2, in vitro, which is due to their N-terminal region that encompasses MNNL to the 3^rd^ EGF-like repeat. However, their difference cannot simply be accounted for by interfacial residues in the MNNL-DSL region ([@bib38]), which has been observed using high-resolution structural information ([@bib25]); moreover, the part(s) of the domain, which have significant functions in the interaction with Notch1, remain to be known.
In this study, we confirmed the superior function of Dll4 compared to Dll1 as a transmembrane Notch ligand; Dll1 could not promote T cell development in the BM and Dll4 could deliver Notch1-mediated signaling more prominently than Dll1 in vitro. We constructed several domain-swapping mutants of Dll1/Dll4 and estimated the ability of each domain to induce T lymphopoiesis and Notch1-mediated signaling in vitro. Among the domains previously reported, we revealed that the MNNL domain of Dll4 contributes to the stable binding to Notch1, presumably via flexible loop structure with disulfide bond including several interfacial residues within the domain. In contrast, that of Dll1 contains a similar loop structure with limited range of motion. These results suggested that Dll1 and Dll4 differently bind with Notch1 due to the different dependency on their domain structures.
Results {#s2}
=======
Ectopic expression of Dll4 but not Dll1 induces T cell development in the bone marrow {#s2-1}
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We established conditional transgenic mice in which one copy of *Dll1* or *Dll4* gene was transcribed by CAG promoter after a Cre-dependent gene depletion of floxed *GFP* cDNA with the translational termination codon at *Rosa26* locus (hereafter referred to as iD1 for Dll1 and iD4 for Dll4 Tg mice, respectively; [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}). The expression of GFP could be observed in CD45^+^ hematopoietic and PDGFRα^+^ mesenchymal cells in the bone marrow (BM) ([Figure 1A](#fig1){ref-type="fig"}), although the expression of GFP in both cell lineages of iD1 mice were obviously higher than those of iD4 mice, which seemed to be due to the difference of sequences between the inserted *Dll1* and *Dll4* cDNA ([Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}). These mice were bred with *Rosa^CreER^* mice, and administered tamoxifen to systemically induce ectopic expression of Dll1 or Dll4. Up on the exogenous expression of Dll1 or Dll4 in the BM, substantial reduction in abundance of B220^+^CD19^+^ B-lineage cells was observed ([Figure 1B and C](#fig1){ref-type="fig"}), suggesting that Dll1 or Dll4-mediated Notch signaling occurred in hematopoietic progenitor cells (HPCs), and abrogated B cell development in the BM. However, ectopic appearance of Thy1^+^CD4^+^CD8^+^ immature T cells was detected only in the BM with exogenous Dll4 ([Figure 1B and C](#fig1){ref-type="fig"}), even though the expression level appeared to be lower. These results indicated that Dll4 can stimulate Notch signaling into HPCs more efficiently than Dll1, and that the amount of Notch signaling necessary for the inhibition of B cell development is lower than that required for the induction of T cell development in the BM.
{ref-type="fig"}.](elife-50979-fig1){#fig1}
Dll4 promotes the growth and development of Thy1^+^CD25^+^ T-lineage cells on OP9 stromal cells from hematopoietic progenitor cells more efficiently than Dll1 {#s2-2}
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To evaluate the potency of Dll1 and Dll4 at inducing T-lineage cells from HPCs on OP9 stromal cells ([@bib32]; [@bib17]) in vitro, we established the OP9 transfectants that expressed either Dll1 or Dll4 on the surface in response to doxycycline removal (Dox, Tet-off system). The expression of these proteins on the cell surface was quantitatively detected by intracellular staining with an isotype control or by an anti-HA mAb that recognizes the C-terminus HA epitope of both Dll1 and Dll4 in a dose-dependent manner ([Figure 2A](#fig2){ref-type="fig"}, left panels). This system enabled the direct comparison of the potencies of Dll1 and Dll4, and confirmed that Dll4 induced T-lineage cells on OP9 cells more efficiently than Dll1 did from HPCs as shown previously ([@bib4]; [@bib27]). In every comparable situation, for similar induction of Thy1^+^CD25^+^ T-lineage cells, lower surface expression of Dll4 was required ([Figure 2A](#fig2){ref-type="fig"}). Moreover, by using serial dilution of Dox, we determined the EC~50~ values of Dll1 or Dll4 expression in OP9 cells for the induction of CD25 during the initiation of T cell differentiation ([Figure 2B](#fig2){ref-type="fig"}). Based on the results from three independent experiments, we estimated the EC~50~ values at 16710 ± 1820 and 4217 ± 1594 dMFI for Dll1 and Dll4 ([Figure 2---source data 2](#fig2sdata2){ref-type="supplementary-material"}), respectively, and demonstrated that Dll4 in OP9 cells exhibited higher ability to induce T-lineage cells than Dll1.
{ref-type="fig"}.\
Figure 2---source data 2.Measurement of the induction efficiencies for the CD25^+^ T-lineage cells of serial expression of Dll4 or Dll1 as EC~50~ as shown in [Figure 2B](#fig2){ref-type="fig"}.\
Figure 2---source data 3.Raw data used to generate [Figure 2---source data 2](#fig2sdata2){ref-type="supplementary-material"}.](elife-50979-fig2){#fig2}
DOS motif present in the 1^st^/2^nd^ EGF-like repeat (EGF1-2) of extracellular regions of Dll1, but not in those of Dll4, represents a unique structure and modifies the function of both Dll1 and Dll4 {#s2-3}
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To investigate the molecular basis for the functional difference between Dll1 and Dll4, we generated several swapping chimeras of the DSL and the 1^st^ and 2^nd^ EGF-like repeats (EGF1-2, depicted E1-2 in [Figure 3](#fig3){ref-type="fig"}) containing DOS motif in Dll1 ([Figure 3A](#fig3){ref-type="fig"}) in order to determine whether the EGF1-2 of Dll4 that lacks the DOS motif will allow it to function better with Notch1. To evaluate that, we prepared the cell lines producing soluble or transmembrane Notch1/2 proteins with or without glycosyl modification by Lfng or Mfng, glycosyltransferase, because the fringe-modified Notch increases the affinity towards the Dll family members ([Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}). The glycosyl modification of Notch receptors by Lfng or Mfng is well known to be effective in enhancing their affinities towards Dll but reduced their affinities to the Jag family members ([@bib11]; [@bib13]). Dll1-based chimeras with Dll4-derived EGF1-2, named D1-D4E1-2 and D1-D4DSL/E1-2 ([Figure 3A](#fig3){ref-type="fig"}), lost their ability to bind to Notch receptors ([Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}). Next, the activity of signal transduction via Notch1 was examined by luciferase reporter assay (TP-1 reporter, driven by 6xRbpj binding sites) with or without Lfng. Expression levels of NotchL were monitored by the expression of GFP in this experiment and almost all transfectants were comparable. ([Figure 3---figure supplement 2](#fig3s2){ref-type="fig"}). While the high levels of NotchL expression used in this experiment made it less sensitive to the difference between Dll1 and Dll4, the effects of the domain-swapping chimeras were strongly evident. These chimeras exhibited reduced activity to transduce Notch1-mediated signaling in the reporter assay ([Figure 3B](#fig3){ref-type="fig"}). Importantly, reduced or eliminated activity for signal transduction by D1-D4DSL and D1-D4DSL/E1-2 was seen even though these constructs were expressed even more highly than Dll1. As only D1-D4E1-2 transfectant showed the lower expression of GFP ([Figure 3---figure supplement 2](#fig3s2){ref-type="fig"}), its weak activity in the reporter assay might be due to the insufficient expression. However, its binding efficiency with Notch1/2 ([Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}) and signal transduction by D1-D4DSL/E1-2 in the reporter assay were also low. Thus, it was likely that the DOS motif present in EGF1-2 of Dll1 is necessary for the function of Dll1, which was consistent with the previous report ([@bib24]). Moreover, these swapping mutants did not promote T cell differentiation in vitro ([Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}). Conversely, Dll4-based chimeras with Dll1-derived EGF1-2, D4-D1E1-2, and D4-D1DSL/E1-2 enhanced their activities, binding to Notch1 ([Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}), signal transduction via Notch1 ([Figure 3B](#fig3){ref-type="fig"}) and induction of T cell differentiation ([Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}). These results indicated that the DOS motif also contributes to enhancing the activity of Dll4. Similar results were obtained with other swapping mutants containing one amino acid substitution---P^260^ of Dll1 to N, which was conserved in the DOS motif, or vice versa---D1-P260N and D4-N258P (data not shown), as shown in our previous study ([@bib24]). Moreover, the swapping chimeras of DSL domains neither efficiently bind to Notch receptors nor induce Notch signaling ([Figure 3B](#fig3){ref-type="fig"} and [Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}), indicating their specificity to exert full activity. These results suggested that DOS motif, shared by NotchLs except Dll4, is crucial for the functionality of Dll1 and even capable of enhancing that of Dll4 in chimera. Thus, the superiority of Dll4 cannot be explained by the difference in DSL and EGF1-2 domains, which are known to be critical for binding to Notch receptors.
{ref-type="fig"}). (**B**) The swapping chimeras of DSL domain and/or the 1^st^/2^nd^ EGF-like repeats transduce Notch signaling. Stable transfectants expressing murine Notch1 and control vector (Notch1, open column) or Notch1 and Lfng (Lfng-N1, filled column) were transiently transfected with a TP1-luciferase reporter plasmid, pGa981-6, and a pRL-TK plasmid for internal control. Cells were harvested at 24 hr after transfection, and co-cultured for an additional 40 hr with the transfectants expressing the DSL/E1-2 swapping chimeras. The relative induction of luciferase activity in each sample (mean ± SD, n = 3; \*\*\*, p\<0.001; unpaired Student's *t*-test) was calculated and described as fold activation against the control (value from the culture with mock transfectant not expressing Notch ligand). Data represents three independent experiments. (**C**) Monoclonal antibody originally established by us, HRJ1-5, broadly reacted with murine Jag1, Jag2, and Dll1, but not with Dll4. The transfectants of BM-derived mesenchymal cell line, originally not expressing any Notch ligands, established in our lab, expressing murine Jag1, Jag2, Dll1, or Dll4, were stained with HRJ1-5, and analyzed by flow cytometry. Open histograms indicate HRJ1-5 staining and filled histograms indicate staining with control hamster IgG. (**D**) Reactivity of HRJ1-5 with Dll chimeras. Each transfectant shown in the panel was stained with HRJ1-5 and analyzed as in C.\
Figure 3---source data 1.Raw data (Fold activation) of luciferase activity used to generate the graph in [Figure 3B](#fig3){ref-type="fig"}.](elife-50979-fig3){#fig3}
Further, we have established a hamster-derived mAb, HRJ1-5, by immunization with purified rat Jag1 protein, which was broadly cross-reactive with murine and human Dll1, Jag1, and Jag2, but not to Dll4 ([Figure 3C](#fig3){ref-type="fig"} and data not shown). To identify the epitope recognized by HRJ1-5, we validated the reactivity of this mAb to the chimeras ([Figure 3D](#fig3){ref-type="fig"}). HRJ1-5 clearly bound to mutants with Dll1-derived EGF1-2 with DOS motif, indicating that the DOS motif contributes to the maintenance of the unique structure recognized by the antibody, which is critical for their function as NotchL.
The MNNL domain of Dll4 is necessary for its function and is related to the superiority of Dll4 over Dll1 {#s2-4}
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Previous reports have indicated that the truncated proteins of Dll family members from the N-terminus to the 3^rd^ EGF-like repeat in the extracellular region are sufficient to trigger the Notch signaling ([@bib2]; [@bib24]), and that Dll4 directly binds to Notch1 via its MNNL and DSL domains ([@bib25]). We next examined the significance of MNNL domain of Dll4 using other swapping chimera constructs ([Figure 4A](#fig4){ref-type="fig"}), which included HA tag for comparing protein expression levels. This shows that the chimeras are expressed to similar absolute levels per cell. Furthermore, swapping the MNNL domain in Dll1 does not affect expression of its DOS motif as detected by HRJ1-5, although swapping the MNNL domain of Dll4 does appear to reduce its reactivity with an antibody against the Dll4 DSL domain ([Figure 4---figure supplement 1](#fig4s1){ref-type="fig"}). Interestingly, Dll4-based chimera with Dll1-derived MNNL domain (D4-D1MNNL) reduced its binding to soluble Notch1 and Notch2, while Dll1-based chimera with Dll4-derived MNNL region (D1-D4MNNL) showed comparable activity to that of Dll1 ([Figure 4B](#fig4){ref-type="fig"}). Similarly, only D4-MNNL lost its activity to induce Notch1-mediated signaling with or without Lfng in the luciferase reporter assay ([Figure 4C](#fig4){ref-type="fig"}). Moreover, D4-D1MNNL failed to inhibit B cell development and support T cell differentiations in vitro ([Figure 4D](#fig4){ref-type="fig"}). These results demonstrated that the MNNL domain of Dll4 is necessary for its function as NotchL, and that there might be some difference(s) in MNNL domain of Dll1 and Dll4, which is responsible for the superiority of Dll4 over Dll1 for T cell induction.
{ref-type="fig"}). (**B**) Binding activity of the MNNL swapping chimeras with soluble Notch1 and Notch2. Soluble Notch receptors comprised the N-terminus to the 15^th^ EGF repeats and the Fc domain of human IgG1. The transfectants of BM-derived mesenchymal cell line expressing Dll1 (D1), Dll4 (D4), or their MNNL swapping chimeras (D4-D1MNNL and D1-D4MNNL) were incubated with soluble Notch1 (sN1) or Notch2 (sN2), and their binding was detected by anti-human IgG Ab as described in the Materials and methods. Soluble Notchs were produced by CHO cells expressing murine Lfng (red line), Mfng (blue line), or vector control (green line), and used in this experiment. (**C**) The MNNL swapping chimeras transduce Notch signaling. Reporter assay was carried out as shown in [Figure 3B](#fig3){ref-type="fig"}. The relative induction of luciferase activity in each sample (mean ± SD, n = 3; \*\*, p\<0.01; N.S.: not significant; unpaired Student's *t*-test) was calculated. Data represents three independent experiments. (**D**) Induction of T-lineage cells by MNNL swapping chimeras in in vitro. Fetal liver-derived lineage markers-negative c-kit-positive hematopoietic progenitors were cultured on the OP9 cells expressing Dll1, Dll4, or their MNNL chimeras for 13 days. After the cultures, live cells were stained with CD19, Thy1, CD4, and CD8, and analyzed by flow cytometry. Numbers in the dot-plot represent the relative percentages for each corresponding fraction or quadrant in Thy1^+^CD19^-^ fraction (red square).\
Figure 4---source data 1.Raw data (Fold activation) of luciferase activity used to generate the graph in [Figure 4C](#fig4){ref-type="fig"}.](elife-50979-fig4){#fig4}
Molecular dynamic simulation enables different characterization of structural dynamics in MNNL domain {#s2-5}
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In order to determine the significance of the MNNL domain of Dll4 in forming a complex with Notch1, we constructed Dll/Notch1 complexes (Dll4/Notch1, depicted in [Figure 5A](#fig5){ref-type="fig"} and D4-D1MNNL/Notch1) and performed molecular dynamic simulations using these complexes. Consistent with our in vitro and in vivo experiments, the interaction energy of Dll4/Notch1 ([Figure 5B](#fig5){ref-type="fig"}, black line) was lower than that of D4-D1MNNL/Notch1 ([Figure 5B](#fig5){ref-type="fig"}, red line), suggesting that the former interaction was more stable. Then, we focused our attention on the differences in the amino acid sequence of the loop structure with disulfide bond (C-C loop, dotted circle, [Figure 5A and D](#fig5){ref-type="fig"}) present in the MNNL domain, between Dll1 and Dll4 ([Figure 5C](#fig5){ref-type="fig"}). This is because the C-C loop of Dll1 contains a characteristic proline-rich amino acid region (PEPP-region, underline, [Figure 5C](#fig5){ref-type="fig"}) that restricts the movement of the C-C loop in the MNNL domain ([Figure 5D](#fig5){ref-type="fig"}), probably due to the conformational rigidity created by the presence of several proline residues (magenta sticks, lower panels in [Figure 5D](#fig5){ref-type="fig"}). This loop structure also includes the residues (^64^His and ^65^Phe, bold green, [Figure 5C](#fig5){ref-type="fig"}) that comprise the binding surface of MNNL domain of Dll4 which interacts with Notch1 ([@bib25]). As shown in [Figure 5E](#fig5){ref-type="fig"}, it was revealed that the movement of the C-C loop in the MNNL domain of Dll1 (red line) is completely restricted in comparison with that of Dll4 (black line). This difference was shown in the movies (Dll4, [Figure 5---video 1](#fig5video1){ref-type="video"}; Dll1, [Figure 5---video 2](#fig5video2){ref-type="video"}). Therefore, we presumed that the restricted movement of the C-C loop in the MNNL domain of Dll1 prevents the inducible binding with Notch1. However, in the MNNL domain of Dll4, the flexible movement of the C-C loop is retained, which enables Dll4 to interact with Notch1 via the key residues ([Figure 5C and D](#fig5){ref-type="fig"}, [Figure 5---video 1](#fig5video1){ref-type="video"}, [Figure 5---video 2](#fig5video2){ref-type="video"} and [Figure 5---figure supplement 1](#fig5s1){ref-type="fig"}).
{#fig5}
Furthermore, we constructed a Dll4-based mutant with a Dll1-derived PEPP-region (Dll4-PP, [Figure 5C](#fig5){ref-type="fig"}) to examine the contribution of the C-C loop in the MNNL domain to the complex formation with Notch1. The interaction energy of Dll4-PP/Notch1 and the movement of the C-C loop in the MNNL domain of Dll4-PP ([Figure 5B and E](#fig5){ref-type="fig"}, blue lines) were nearly identical to those of D4-D1MNNL or Dll1 ([Figure 5B and E](#fig5){ref-type="fig"}, red lines). This difference was also found in the movie ([Figure 5---video 3](#fig5video3){ref-type="video"}). Thus, these results revealed that the flexible movement of the C-C loop in the MNNL domain of Dll4 plays an important role in complex formation with Notch1.
The loop structure in MNNL domain of Dll4 with a wide range of motion contributes to efficient signal transduction {#s2-6}
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To confirm the significance of the dynamic motion of the C-C loop in MNNL domain, we compared the Notch binding and activating abilities of Dll4-PP---which retains two unique proline residues---with those of Dll4 ([Figure 5C](#fig5){ref-type="fig"}). Consistent with the simulation analysis, Dll4-PP showed weaker ability to bind to soluble Notch receptors ([Figure 6A](#fig6){ref-type="fig"}) and to transduce Notch signaling in the reporter experiment ([Figure 6B](#fig6){ref-type="fig"}), which were similar to those observed on using the Dll4-based chimera with Dll1-derived MNNL domain, D4-D1MNNL ([Figure 4B--D](#fig4){ref-type="fig"}). The strong reduction in functional activity of Dll4-PP was seen despite virtually unchanged levels of protein expression ([Figure 6---figure supplement 1](#fig6s1){ref-type="fig"}). These results suggested that the C-C loop in MNNL domain of Dll4 with a wide range of motion uniquely contributes to the triggering of Notch signaling.
{ref-type="fig"}. (**B**) Inducing activity of Notch signaling in vitro was examined by the co-cultures of NotchL and Notch1/Lfng transfectants as shown in [Figure 3B](#fig3){ref-type="fig"}. The fold activation against the control was calculated from each sample (mean ± SD, n = 3; \*\*, p\<0.01; N.S.: not significant; unpaired Student's *t*-test). Data represents three independent experiments. Expression of NotchLs were monitored by the GFP expression, the intracellular staining with anti-HA mAb and the surface staining with HRJ1-5 or anti-Dll4 mAbs ([Figure 6---figure supplement 1](#fig6s1){ref-type="fig"}).\
Figure 6---source data 1.Raw data (Fold activation) of luciferase activity used to generate the graph in [Figure 6B](#fig6){ref-type="fig"}.](elife-50979-fig6){#fig6}
Discussion {#s3}
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In this study, we focused on the difference between Dll1 and Dll4, and revealed that Dll family members bind to Notch and trigger the signaling differently based on the structural features: DSL plus DOS motif for Dll1 and DSL plus MNNL for Dll4. The MNNL of Dll1 loses the ability to move widely with its rigidity and the EGF1-2 of Dll4 lacks the DOS motif. It has been well known that the DSL domain is highly conserved from invertebrates to mammals and is essential to maintain the structure and function of NotchLs ([@bib37]; [@bib7]; [@bib6]; [@bib19]). Moreover, the additional domain, MNNL in Dll4 and EGF1-2 in Dll1, is further required for the stable interaction with Notch. We showed here that Dll1 and Dll4 differently use the neighboring domains of DSL. However, these results were mainly obtained in vitro and might be seen under conditions in the middle of the dynamic range between Notch and NotchL, where the additional domains seem to be essential. Therefore, our conclusions need to be verified in physiological condition.
Molecular basis of the interaction between Notch1 and Dll4 was provided by the high-resolution structural analysis and showed that several interfacial residues within the MNNL and DSL domains of Dll4 directly interact with Notch1 ([@bib25]). However, it was reported that the Dll4-based mutant containing an exchange of the MNNL from 65^th^ Phe (Dll4) to Tyr (Dll1) ([Figure 5C](#fig5){ref-type="fig"}, bold green), and of DSL with several contact residues from Dll1, did not alter its selectivity toward Notch1 characteristic of Dll4. Therefore, it was suggested that residues outside of the MNNL-DSL contact interface of Dll4 contribute to the functional characteristics of Dll4 ([@bib38]). Alternatively, in this study, we demonstrate that, when approaching Notch1, the MNNL of Dll1 does not behave as that of Dll4 and that Dll1 efficiently triggers Notch1 depending on the DOS motif present in EGF1-2 of Dll1 but not Dll4. Thus, the unique approach of Dll family members is based on the domains used differently in the binding to Notch1, but not on the distinctive amino acid residues.
We observed the difference between ectopic Dll1 and Dll4 in the ability to induce and suppress T- and B-lineage cells, respectively, in the BM, where exogenous Dll proteins were expressed, at least, in both hematopoietic and mesenchymal cell lineages. Previous studies with retrovirus-mediated transduction of Dll1 or Dll4 into hematopoietic cells and transfer into lethally irradiated mice suggested the superiority of Dll4 over Dll1; however, these experiments did not ensure the sufficient expression of Dll1 to function ([@bib8]). In our established mouse model, exogenous Dll1, as well as Dll4, clearly suppressed B cell development in the BM; however, it was not enough for the appearance of CD4, CD8-bearing immature T cells. These results suggested that Dll4 in BM transduces the Notch signaling into HPCs for T cell development more efficiently than Dll1 and confirmed that there is a different requirement regarding the threshold of Notch signaling for the suppression of B cell and the induction of T cell lineages in vivo, as previously shown in vitro ([@bib21]). We have previously shown that on the monolayer culture of HPCs, Gata3 is necessary only for the induction of the T-lineage cells, but not for the suppression of B-lineage cells by Notch signaling, suggesting the presence of different mechanisms downstream of Notch signaling ([@bib18]). The quantitative difference in Notch signaling seems to be converted into the distinctive cellular events at the hematopoietic progenitor stage.
Although it has been suggested that Dll4 induces Notch1-mediated signaling more efficiently than Dll1, its quantitative evaluation was only conducted by their affinities using soluble forms of both Dll and Notch1 proteins as truncated parts of the extracellular domains and demonstrated that Dll4 binds with more than 10-fold higher affinity than Dll1 ([@bib2]). As NotchL triggers Notch signaling in the neighboring cells only as a transmembrane protein and not in its truncated soluble form, alterations in its levels should be quantitatively evaluated only as a transmembrane protein. In this study, we aimed to compare full-length Dll proteins as transmembrane form by measuring their serially diluted expression on stromal cells and by their ability to induce T-lineage cells. Further, we evaluated their biological effect on HPCs. Dll4 present on stromal cells supported T cell differentiation 3--6-fold more efficiently than Dll1, which seemed to be less when compared to the difference in their affinities to Notch1. HPCs express both Notch1 and Notch2 on their surface, and the latter contributes to the induction of T-lineage cells only on interacting with Dll1 ([@bib4]; [@bib10]), indicating that the total signaling induced by Dll1 was mediated by Notch1 as well as Notch2, while that induced by Dll4 was through Notch1 alone. We realized that the functional difference in the ability to induce T-lineage cells should be assessed under this situation.
It is obvious that the determination of T cell fate is regulated only by the *trans*-activation of Notch signaling between NotchL and Dll4 on thymic epithelium (environment), and Notch1 on thymic immigrants (progenitors). In contrast, Notch-NotchL interaction seems to facilitate not only *trans*-activation but also *cis*-inhibition during the development of various tissue, because Notch signaling occurs between two equivalent progenitors in which Notch and NotchL simultaneously function, according to the lateral inhibition theory ([@bib9]; [@bib36]). In fact, functional difference between Dll1 and Dll4 is also observed during somitogenesis and Dll4 cannot rescue the defect of Dll1, which is explained by the difference between their action through the *cis*-inhibition ([@bib30]). This situation is not suitable due to its complexity and makes it difficult to understand the correlation between molecular events and biological responses. In this study, we showed their functional and structural difference for the T cell induction in which Notch-NotchL interaction only occurs between the cells with *trans*-activation, which reflected their affinities.
The MNNL domain is known to be essential for triggering Notch signaling in the immobilized short fragment ([@bib2]; [@bib24]; [@bib35]). The MNNL domain is conserved between the two NotchL families, and missense mutations were found in Alagille syndrome ([@bib6]). Moreover, Jag1 exhibited structural similarity to the C2 domain of protein kinase C, which bears phospholipid-binding properties in a calcium-dependent manner and is also necessary for efficient Notch activation ([@bib6]). Recent study reported that the MNNL of Dll4 binds directly to Notch1 ([@bib25]), while the similarity to PKC is not detected in those of Dll ligands ([@bib25]; [@bib19]), raising the question of how MNNL contributes to the binding to Notch. Here, we showed that the MNNL domain of Dll4---that contains the loop structure with disulfide bond (C-C loop) with a wide range of motion---was critical for binding to the Notch receptor and activation of the Notch pathway, a process that results in the promotion of T cell development in vitro. The Phe^65^ and His^64^ residues in the C-C loop partly comprise the interface that binds to Notch1, and directly interacts with the O-Fuc moiety on Thr^466^ and Leu^468^/Ile^477^ residues in the EGF12 domain of Notch1 ([@bib25]). The wide motion ensured by the flexible C-C loop structure seems to contribute to a more effective interaction with Notch1 EGF12. The actual signal transduction as assessed by luciferase reporter assay, was more affected by the substitution and insertion of unique proline residues in MNNL domain of Dll4, compared to the simple binding to Notch. This reduced signal transduction suggests that the motion of the C-C loop serves to generate the pulling force for the transendocytosis of the Notch extracellular domain. ([@bib40]; [@bib29]). The Dll4 mutant with Dll1-derived MNNL was less effective than that with two proline residues, suggesting that other part(s) of MNNL play a role in the triggering of Notch signaling.
It has been recently shown that in addition to MNNL and DSL domains, the 3^rd^ EGF-like repeat is essential for the distinctive function of Dll ligands ([@bib38]). We have confirmed that the 3^rd^ EGF-like repeat of Dll4, but not of Dll1, plays an important role in signal transduction (unpublished data). In contrast to Dll1, which approaches the Notch receptor via the DSL domain and the 1^st^ and 2^nd^ EGF-like repeat with the DOS motif, Dll4 seems to access Notch via the MNNL and DSL domains, supported by the 3^rd^ EGF-like repeat. The structural cooperation among these regions should be further investigated for the precise understanding of Notch-NotchL interaction.
Materials and methods {#s4}
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Plasmids and constructs {#s4-1}
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We cloned cDNAs encoding the swapping mutants of Dll1 and Dll4 as below: D1-D4DSL, *Dll1* cDNA was digested by SalI/ScaI, removing the DSL region, and ligated with synthetic Dll4-derived cDNA encoding DSL domain flanked by 5' SalI and 3' ScaI sites; *D4-D1DSL*, *Dll4* cDNA was digested by AflII/ScaI, removing the DSL region, and ligated with synthetic Dll1-derived cDNA encoding DSL domain flanked by 5' AflII and 3' ScaI sites; *D1-D4E1-2*, *Dll4* cDNA was digested by ScaI/Bst1107I, removing the 1^st^/2^nd^ EGF region, and ligated with synthetic Dll4-derived cDNA encoding the 1^st^/2^nd^ EGF region flanked by 5' ScaI and 3' Bst1107I sites; *D4-D1E1-2*, *Dll4* cDNA was digested by ScaI/BglII, removing the 1^st^/2^nd^ EGF region, and ligated with synthetic Dll1-derived the 1^st^/2^nd^ EGF region flanked by 5' ScaI and 3' BglII sites; *D1-D4MNNL*, *Dll1* cDNA was digested by EcoRI (5' terminal as cloning site) and ScaI, removing MNNL and DSL regions, and ligated with EcoRI/ScaI fragment from D4-D1DSL encoding Dll4-derived MNNL and Dll1-derived DSL domains; *D4-D1MNNL*, *Dll4* cDNA was digested by EcoRI and ScaI, removing MNNL and DSL regions, and ligated with EcoRI/ScaI fragment from D1-D4DSL encoding Dll1-derived MNNL and Dll4-derived DSL domains. All cDNAs and variants for NotchLs were cloned into the MIGR1 retrovirus vector ([@bib31]).
Establishment of BM-derived mesenchymal cells expressing NotchLs {#s4-2}
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For establishment of BM-derived mesenchymal cell lines (VF) expressing intact NotchLs or their variants, retroviruses encoding NotchLs and mutants were obtained after transfection into the Plat-E ecotropic packaging cell line as described previously ([@bib1]). Retrovirus-infected cells were collected 48 hr after the infection and analyzed for GFP expression by flow cytometry. GFP-positive cells were obtained by sorting, giving rise to a \>99% pure population. Expression of Notch ligands or chimeric molecules on the cell surface was detected by staining with anti-Dll1, anti-Dll4 mAbs or HRJ1-5 mAb recognizing the DOS motif at the E1-2 region as described previously ([@bib1]; [@bib28]), or by intracellular staining with anti-HA mAb (Cell Signaling Technology, Danvers, MA). An isotype control of biotinylated hamster IgG and rabbit IgG were purchased from BD Biosciences (Tokyo, Japan) and Cell Signaling Technology, respectively. For serial induction of Dll1 and Dll4, lentivirus encoding those constructed in Retro-XTM Tet-Off Advanced Inducible Expression System (Takara Bio Inc, Shiga, Japan) were prepared and infected into OP9 stromal cells. These OP9 transfectants were treated with Dox (0.03 \~ 0.1 ng/ml) to suppress the full expression of Dll molecules, and their amounts of the expression were quantitatively determined by flow cytometry with the intracellular anti-HA staining.
Co-culture assay with stromal cells {#s4-3}
-----------------------------------
In vitro cultures of lineage marker-negative c-kit-positive fetal liver (FL) cells with stromal cells were described previously ([@bib15]; [@bib1]). Briefly, lineage markers- (Lin; TER119, CD19, Mac-1, Gr-1) negative, c-kit-positive cells were isolated from E15.5 embryos and plated at 1 × 10^4^ cells on a monolayer of OP9 for T cell induction into CD25^+^ DN3 stage with serial expression of Dll for 7 days in the presence of 5 ng/ml murine IL7 (Peprotech, London, UK) and 5 ng/ml human Flt3L (Peprotech) in 24-well culture dishes or 5 × 10^2^ cells on OP9 expressing Dll or its swapping mutants for T cell induction into DP stage for 13 days in the presence of 1 ng/ml IL7 and 5 ng/ml Flt3L. After cultures, growing cells were collected and analyzed for surface markers for 7 days (Lineage markers: Gr1, CD11b, CD11c, DX5 and ST2; CD45, Thy1.2, CD19, CD44 and CD25) or 13 days (CD45, CD19, Thy1.2, CD4 and CD8) cultures by flow cytometry. OP9 cell line was obtained from Dr. Yokoyama who published the report using this cell line ([@bib41]). We have confirmed the micoplasma-free status before the experiments.
Antibodies and flow cytometry {#s4-4}
-----------------------------
FITC- and APC-CD19, CD11b, GR-1, FITC-TER119, FITC-CD44, PE-Dll1, Dll4, APC-c-kit, CD11c, B220, ST2, PE/Cy7-CD4, APC/Cy7-Thy1.2 and CD19 mAbs were all purchased from BioLegend (San Diego, CA). PE-CD19, PerCP/Cy5.5-CD25, APC-CD8, DX5, PDGFRα and PE/Cy7-CD45 mAbs were purchased from Thermo Fisher Scientific (Tokyo, Japan). Anti-NotchLs mAb, HRJ1-5, was established by immunization of Armenian hamsters with a recombinant rat Jagged1-human Fc chimeric protein (34) (R and D Systems Inc, Minneapolis, MN). Expression of NotchLs assessed by HRJ1-5 was detected by biotinylated goat anti-hamster IgG Ab (Thermo Fisher Scientific) and PE-conjugated streptavidin (BD Bioscience). Intracellular staining of HA-labeled Dll1 or Dll4 was detected by rabbit anti-HA mAb (Cell Signaling Technology) and DyLight649-conjugated donkey anti-rabbit IgG Ab (BioLegend). Expression of cell surface markers was analyzed by flow cytometry with a FACSVerse (BD Biosciences).
Establishment and analysis of conditional dll transgenic mice {#s4-5}
-------------------------------------------------------------
The basal targeting vector for *Rosa26* locus, ES cells with Rosa26 acceptor (IDG26.10--3) and the expression vector of PhiC31 integrase were kindly provided by Dr. R. Kühn ([@bib14]) (GSF National Research Center, Munich, Germany). Murine Dll1 or *Dll4* cDNA was cloned into EcoRI site of the modified targeting vector with CAG promoter and floxed GFP cassette, and introduced into ES cells bearing Rosa26 acceptor allele with the expression vector of PhiC31 integrase, followed by selection with G418. Resistant colonies were isolated and analyzed for cassette exchange by PCR with P1 and P2 or P3 primers ([Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}) and by Southern blotting of BamHI-digested genomic DNA using *GFP* cDNA fragment as probe. Almost all clones showed only 8.5 kb band, indicating correct cassette exchange. Positive ES clones containing one copy of targeting construct were injected into C57BL/6 blastocysts. Resulting chimeras were bred to C57BL/6 or RosaCreER mice ([@bib34]) and offspring were checked for germline transmission as Cre-dependent inducible Dll1 (iD1) or Dll4 (iD4) transgenic mice.
The expression of GFP in BM cells obtained from RosaCreER^+^ iD1 and iD4 mice were analyzed with staining of CD45 and PDGFRα by flow cytometry. These mice were daily given tamoxifen (2 mg, ip; Sigma-Aldrich Japan, Tokyo, Japan) fourth times. Then, four weeks later, cells obtained from the BM were analyzed by flow cytometry. These Tg mice showed differences in the expression level of GFP. The knock-in strategy ensured the insertion of one copy of the GOI into the identical locus, and we were able to observe the same level of GFP expression in every ES cell clone obtained. Thus, we hypothesized that during or after the developmental processes of murine embryos, this locus may be regulated via some cis-element(s) included in the cDNAs, especially the 5' UTR (\~300 kb in Dll4,\~50 bp in Dll1) sequences.
All mouse experiments were approved by the Animal Experimentation Committee (Tokai University, Isehara, Japan).
Binding activity of NotchLs to soluble Notch1 and Notch2 {#s4-6}
--------------------------------------------------------
Expression vectors (pTracerCMV, Thermo Fisher Scientific) encoding murine Notch1- and Notch2-human IgG1 chimeric proteins were provided by Dr. S. Chiba (Tsukuba Univ., Japan), and the protein was collected from culture supernatant of stable transfectants of CHO cells as shown previously ([@bib35]). All transfectants of NotchLs were incubated with the above supernatant for 30 min at RT, then treated with PE anti-human IgG Ab (Rockland), and analyzed by flow cytometry to detect the binding with soluble Notch proteins.
Transient reporter assay {#s4-7}
------------------------
Reporter assays were carried out by the transient transfection of reporter plasmids TP1-luciferase (pGa981-6, including six copies of RBPJk binding sites, constructed by Dr. L. Strobl [@bib26]) and pRL-TK (Promega, Tokyo, Japan) into N1/3T3, which was established as a stable transfectant with pTracer-CMV (Invitrogen, Carlsbad, CA) encoding mouse Notch1. Each reporter plasmid (50:1) was cotransfected into 5 × 10^4^ cells in 24-well plates by a liposome-based method (Transfast, Promega) according to the manufacturer's instructions. Following 24 hr culture after transfection, target cells were detached by trypsinization and co-cultured with stimulators (5 × 10^4^, VF cells expressing NotchLs) for 40 hr. Cell lysates from the mixtures of two kinds of cells were then used for the luciferase assay. Lfng-N1/3T3 was generated by the infection of retroviruses encoding Lfng ([@bib1]).
In silico analysis {#s4-8}
------------------
The construction of the three-dimensional structure of Dll/Nothc1 complexes were performed with MOE, version 2016.08 (CCG Inc, Montreal, Canada) based on the Brookhaven Protein Databank 4XLW (Dll4 and Dll4-PP) and 4XBM (Dll1). Molecular mechanics calculations for these complexes were performed to prepare the initial structures for the molecular dynamics (MD) simulations using the Amber99 force field in MOE. MD simulations for these complexes were performed using the program AMBER 14 (<http://ambermd.org/>) with the AMBER force field and the modified TIP3P every 2.0 fs. The non-bonded interaction energy in terms of electrostatic and Van der Waal's between Dll/Notch1 complexes were calculated by NAMD Energy plug-in in VMD. The root-mean-squared deviation (RMSD) of the C-C loop in the MNNL domain of Dll (Dll1, Dll4, Dll4-PP) were analyzed by CPPTRAJ in AMBER 14.
Funding Information
===================
This paper was supported by the following grants:
- http://dx.doi.org/10.13039/501100001691Japan Society for the Promotion of Science 16K08848 to Katsuto Hozumi.
- http://dx.doi.org/10.13039/501100001700Ministry of Education, Culture, Sports, Science, and Technology 22021040 to Katsuto Hozumi.
We thank Drs. R Kühn, S Chiba for providing the basal targeting vector for *Rosa26* locus, ES cells with Rosa26 acceptor and the expression vector of PhiC31 integrase, and soluble Notch1/Notch2 constructs; Dr. N Abe, Mr. S Ochiai for technical assistance and Drs. M Ito and N Hirayama for valuable discussion and comments for this manuscripts. We would like to thank Editage for English language editing.
Additional information {#s5}
======================
No competing interests declared.
Formal analysis, Investigation, Visualization.
Formal analysis, Investigation, Visualization.
Data curation, Software, Validation, Methodology.
Resources.
Resources, Supervision.
Conceptualization, Validation, Methodology.
Conceptualization, Validation.
Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration.
Animal experimentation: All animal experiments were performed under protocols approved by the Animal Experimentation Committee of Tokai University (Approval No.: 165015, 171002, 182026, 193040), which is further monitored by the Animal Experimentation Evaluation Committee of Tokai University with researcher for Humanities/Sociology and external expert.
Additional files {#s6}
================
###### Key resources table.
Data availability {#s7}
=================
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, 4 and 6.
The following previously published datasets were used:
KershawNJChurchNLGriffinMDWLuoCSAdamsTEBurgessAW2015X-ray crystal structure of Notch ligand Delta-like 1RCSB Protein Data Bank4XBM
LucaVCJudeKMPierceNWNachuryMVFischerSGarciaKC2015Complex of Notch1 (EGF11-13) bound to Delta-like 4 (N-EGF2)RCSB Protein Data Bank4XLW
10.7554/eLife.50979.sa1
Decision letter
Kurosaki
Tomohiro
Reviewing Editor
Osaka University
Japan
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
**Acceptance summary:**
This paper focuses on the structural differences between Δ-like 1 (DLL1) and Δ-like 4 (DLL4) as ligands for Notch1 in T-cell development. The authors found that ectopic expression of DLL4 but not DLL1 induced T cell development in the bone marrow. Furthermore, they demonstrated that the MNNL domain of DLL4 is necessary for the Notch signaling and that the flexible movement of the C-C loop in the MNNL domain of DLL4 was important for binding and function. These findings are valuable and novel in regard to molecular selectivity. In the revised manuscript, authors provided a good overview of the previous work and explained the unique points of the current study.
**Decision letter after peer review:**
Thank you for submitting your article \"Δ-like 1 and Δ-like 4 differently require their extracellular domains for triggering Notch signaling\" for consideration by *eLife*. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Satyajit Rath as the Senior Editor.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
Summary:
This paper from a pioneer in the field of Notch regulation of T-cell development focuses on the structural differences between DLL1 and DLL4 as ligands for Notch1. They first found that thymus immature T cells only appeared in the BM with exogenous DLL4 more efficiently DLL1. Second, more importantly, they demonstrated that the MNNL domain of DLL4 is necessary for the Notch signaling and that the flexible movement of the C-C loop in the MNNL domain of DLL4 was important for binding and function. These findings are valuable and novel in regard to molecular selectivity.
Essential revisions:
There are three points.
First, in regard to writing, authors did not fairly refer similar Tveriakhina\'s paper. Authors should write more explicitly with this paper\'s results, and more importantly, they should clearly mention which points are still not unanswered, which has been tackled by this manuscript.
Second, thymus in vivo results are not strongest part of this manuscript. The experiments in Figure 1 supports Figure 2, but the differences in expression level between the DLL4 expressing transgene and the DLL1 expressing transgene, in the hematopietic cells and stromal cells, make the results difficult to interpret. Authors do not need to remove these results, but they should discuss the results more carefully in light of this expression difference.
Finally, it is very important to quantitate the expression levels of the different chimeras in the main in vitro experiments, Figure 3 and Figure 4. The binding to Notch1 by a cell is hard to interpret unless we know how strongly each of the different chimeras is expressed on that cell.
\[Editors\' note: further revisions were suggested prior to acceptance, as described below.\]
Thank you for resubmitting your work entitled \"Δ-like 1 and Δ-like 4 differently require their extracellular domains for triggering Notch signaling in mice\" for further consideration by *eLife*. Your revised article has been evaluated by Satyajit Rath (Senior Editor) and a Reviewing Editor.
The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:
As seen in the below reviewers\' comments, reviewers request no more additional experiments, but do request authors to carefully re-write the manuscript, particularly from the following points.
1\) Emphasize the unique points of this study, compared with previous reports (for instance, the results of Tveriakhina et al).
2\) Describe somewhere that there could be more contribution of additional domains to DII4 vs Dll1 affinity for Notch1 that would be seen under conditions more in the middle of the dynamic range.
3\) It is not always clear to the readers what alternative hypotheses can be ruled out at each point.
4\) Emphasize the implications of the new supplementary data.
5\) It is also not clear in the text with distinguishing between effects that are clear and effects that are not.
Reviewer 1:
I can see that the revised version has dealt with some points raised by reviewers, and the manuscript has been improved clearly. However, their explanation was not adequately clarified their advantage. Advance of this manuscript would be a role of E3 domain in comparison with the Tveriakhina\'s work, and the main point is that the reduced number of Pro residues in a C-C loop of the MNNL domain explain the advantage of DLL4 as ligands for Notch1. It will be more understandable as a clear advantage if the authors emphasize the specific points about the previously non-covered part of the DLL4 function.
Reviewer 2:
The authors have definitely improved the manuscript, and their addition of the new supplementary figures showing measurements of expression levels is particularly welcome. The work is quite interesting and suggests There is still a concern about the way Figure 3 was done and the way it was interpreted. However, this is not the most important figure in the paper, and on balance, I believe that the paper\'s remaining issues can be dealt with by changes in the writing. These issues are: (1) in the Discussion, still not fully explaining how the results in this paper specifically relate to the results reported by Tveriakhina et al; (2) concern about trying to compare affinities of binding under conditions that are apparently saturating; (3) needing more guidance for readers to understand how the newly included experiments successfully rule out some alternative explanations while favoring the explanations of the authors.
1\) The Discussion has apparently not been updated since the previous submission, but it seemed important to include some kind of summary of how far the results of Tveriakhina et al. foreshadowed this paper, and how far this paper went beyond that previous report.
2\) I thank the authors for explaining that they were trying to achieve such high levels of overexpression of the Notch ligands that they would saturate the Notch receptors in Figures 3 and 4. However, this is not a standard way to measure affinity differences, because the binding difference that could be due to affinity is overwhelmed by the high level of ligand. This is a problem with the way the experiments in Figures 3-4 are interpreted in terms of \"affinity\". In fact, the authors see no difference between wildtype Dll1 and Dll4 in the experiments of Figures 3B and 4C in the presence of Lfng, despite the fact that they have previously shown very different affinities in Figure 2. Therefore it is not clear why this data is highlighted as a way to find out what parts of the protein account for the stronger binding.
It is surprising but fortunate for the authors that the main effects seen in the chimeras are to reduce binding, even from this extremely saturated level, when the Dll1-Dll4-DSL-E1,2 chimeras are used. In fact, stronger evidence for a true binding affinity difference is seen under different conditions (soluble Notch or soluble Δ) as shown in Figure 3---figure supplement 1, panels A and B. This deserves much more emphasis and should be pointed out as a separate finding on its own. But as a caveat, please also acknowledge somewhere that there could be more contribution of additional domains to Dll4 vs. Dll1 affinity for Notch1 that would be seen under conditions more in the middle of the dynamic range.
3\) In the central experiments describing functional changes of the chimeras compared to wildtype, it is not always clear to the reader what alternative hypotheses can be ruled out at each point. Unfortunately, although there is valuable new evidence to support the authors\' arguments in the new supplementary figure panels, very often these panels are never mentioned in the text.
4\) There are still some problems in the text with distinguishing between effects that are clear and effects that are not. Figure 3 and its supplements remain a little unsatisfying because there is no independent way to compare the levels of the Notch ligand proteins expressed among all the constructs in this experiment. (In this regard, Figure 4 and Figure 6, where the HA tag is used, are much more convincing.) However, it is still possible to help guide the reader through Figure 3. For example, one construct, D1-D4-E1-2, is poorly expressed at the construct reporter level and there is no evidence presented in the paper that this particular chimeric protein is stably expressed at all (Figure 3, Figure 3---figure supplement 1, Figure 3---figure supplement 2). In a case like this, it is possible that the lack of activity is due to lack of stable protein. However, the D1-D4DSL-E1-2 construct is at least expressed well transcriptionally (Figure 3---figure supplement 2), and this construct is equally nonfunctional. It would help the reader if the phenotype of D1-D4DSL-E1-2 were highlighted more, to emphasize the loss of function where the evidence is stronger. More important is the fact that the swap of D1-E1-2 into Dll4 actually enhances its activity instead of diminishing it a very surprising result. Here also the binding evidence and expression evidence are more convincing, but again need to be emphasized more.
10.7554/eLife.50979.sa2
Author response
> Essential revisions:
>
> There are three points.
>
> First, in regard to writing, authors did not fairly refer similar Tveriakhina\'s paper. Authors should write more explicitly with this paper\'s results, and more importantly, they should clearly mention which points are still not unanswered, which has been tackled by this manuscript.
In accordance with your comments, we have introduced an explanation addressing the findings and unanswered points in Tveriakhina\'s paper in the Introduction. Moreover, we have added a further clarified our findings in the following paragraph, which are in contrast to the results reported by Tveriakhina et al.
*Second, thymus* in vivo *results are not strongest part of this manuscript. The experiments in Figure 1 supports Figure 2, but the differences in expression level between the DLL4 expressing transgene and the DLL1 expressing transgene, in the hematopietic cells and stromal cells, make the results difficult to interpret. Authors do not need to remove these results, but they should discuss the results more carefully in light of this expression difference.*
We have deliberated as to why the Dll1/4 Tg mice demonstrated the differences in expression levels. However, a definite explanation has not been reached. The knock-in strategy ensures the insertion of one copy of GOI into the identical locus, and we observed the same intensity of GFP expression in every ES cell clone obtained (please refer to Materials and methods). Thus, we postulate that during or after the developmental processes in murine embryos this locus was regulated via some cis-element(s) included in the cDNAs, especially 5' UTR (\~300 kb in Dll4, \~50bp in Dll1) sequences. This estimation has been added to the revised Material and methods section.
In this study, we aimed to confirm the superiority of Dll4 over Dll1 for the induction of T lymphopoiesis. Using these Tg mice, we successfully emphasized their differences in vivo. We have suitably revised the explanation in the Results section.
*Finally, it is very important to quantitate the expression levels of the different chimeras in the main* in vitro *experiments, Figure 3 and Figure 4. The binding to Notch1 by a cell is hard to interpret unless we know how strongly each of the different chimeras is expressed on that cell.*
We prepared every NotchL-transfectant by the repeated infection with retrovirus encoding NotchL chimeras and ensured their overexpression, monitored by the expression of GFP. In the revised manuscript, we have provided additional supplemental figures (for Figures 3, 4 and 6; introduced in each Figure legend) with their comparable fluorescence intensities. Some transfectants demonstrated lower expression of GFP; however, the protein levels of NotchLs (discuss later, monitored by intracellular HA staining) were comparable. Hence, we hypothesize that there seems to be a GFP threshold level, which ensures the saturating expression of the NotchL protein.
In case of transfectants shown in Figures 4 and 6, critical to obtain our experimental conclusion, we evaluated their expression using the intracellular staining with anti-HA mAb, assessing the total amount of the NotchL, and by the surface staining with anti-Dll4 mAb recognizing mainly the DSL region of Dll4 or with HRJ1-5 mAb recognizing the DOS motif at E1-2 region, detecting the surface expression of NotchL (Figure 4---figure supplement 1 and Figure 6---figure supplement 1). The NotchL expression in all transfectants was comparable between each Dll1/4 and Dll1/4-based chimeras; however, D4-D1MNNL demonstrated a lower expression than Dll4 as observed by anti-Dll4 mAb staining. As their protein levels evaluated by HA-stating were comparable, the affinity to anti-Dll4 mAb seemed to be affected by the replacement of the MNNL region. However, as the expression level was identical between Dll4 and D4-PP, we believe that our conclusion is accurate.
\[Editors\' note: further revisions were suggested prior to acceptance, as described below.\]
> The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:
>
> As seen in the below reviewers\' comments, reviewers request no more additional experiments, but do request authors to carefully re-write the manuscript, particularly from the following points.
>
> 1\) Emphasize the unique points of this study, compared with previous reports (for instance, the results of Tveriakhina et al).
We add the new second paragraph in Discussion section explaining the unique points of this study compared with previous papers, especially Tveriakhina\'s paper.
> 2\) Describe somewhere that there could be more contribution of additional domains to DII4 vs Dll1 affinity for Notch1 that would be seen under conditions more in the middle of the dynamic range.
We mention the limit of our experiment in the first paragraph in Discussion section as reviewer 2 indicated.
> 3\) It is not always clear to the readers what alternative hypotheses can be ruled out at each point.
According to the suggestion of reviewer 2, we declare the implication of the reporter assay in Results section. In addition, we remove the word "affinity" in the explanation of our results, because it is difficult to evaluate in these experiments as indicated by reviewer 2.
> 4\) Emphasize the implications of the new supplementary data.
We emphasize the implication of the supplemental figures of Figures 3, 4 and 6 as reviewer 2 suggested.
> 5\) It is also not clear in the text with distinguishing between effects that are clear and effects that are not.
We add the explanation of Figure 3---figure supplement 2 in Results section as reviewer 2 suggested.
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Follow me on Twitter
From an execution by rifle in Somalia and the 2nd Asian Youth Games in China to an alleged nerve gas attack in Syria and the world’s largest alphorn orchestra, TIME presents the best pictures of the week.
While not sleeping this morning, for some reason or another I was reflecting and counted all of my closest male friends and ended up with a number north of fifteen. One of them I only met a year ago. Another I’ve still never met in person.
Watching HGTV with my 5 year old grandson and they are showing people who are looking for their second (vacation) home in Hawaii. Me: “I am so jealous of these people, Iwish I had a second home.” Grandson: ” But you do have two homes already. Your first house (which my son and his family rent, including my grandson) and this house (where my husband and I live), sooo…….
From violent protests in Egypt and the start of the holy month of Ramadan to a driverless oil-tanker train explosion in Quebec and the running of the bulls in Spain, TIME presents the best pictures of the week.
My daughter Journey has recently become a fan of the Disney Channel. She just turned 7, she has officially outgrown Nick Jr. and since Nick seems to show SpongeBob all day, she has moved over to the Disney channel. At first, she was pretty much only watching Phineas and Ferb. Eventually, she started watching the rest of the line up including Good Luck Charlie, Austin and Ally, Shake It Up and Jessie.
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Hermitian connection
In mathematics, a Hermitian connection is a connection on a Hermitian vector bundle over a smooth manifold which is compatible with the Hermitian metric
on , meaning that
for all smooth vector fields and all smooth sections of .
If is a complex manifold, and the Hermitian vector bundle on is equipped with a holomorphic structure, then there is a unique Hermitian connection whose (0, 1)-part coincides with the Dolbeault operator on associated to the holomorphic structure.
This is called the Chern connection on . The curvature of the Chern connection is a (1, 1)-form. For details, see Hermitian metrics on a holomorphic vector bundle.
In particular, if the base manifold is Kähler and the vector bundle is its tangent bundle, then the Chern connection coincides with the Levi-Civita connection of the associated Riemannian metric.
References
Shiing-Shen Chern, Complex Manifolds Without Potential Theory.
Shoshichi Kobayashi, Differential geometry of complex vector bundles. Publications of the Mathematical Society of Japan, 15. Princeton University Press, Princeton, NJ, 1987. xii+305 pp. .
Category:Complex manifolds
Category:Structures on manifolds
Category:Riemannian geometry
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Up-to-the-minute advice, information, resources, and, on occasion, commentary on federal and New Jersey state income taxes, and the various New Jersey property tax rebate programs, and insights and observations on tax policy and professional tax practice, by 40-year veteran tax professional Robert D Flach.
The distinction had
meaning for me in the will of a recently deceased uncle.
* Another “blast
from the past” from GETTING YOUR FINANCIAL DUCKS IN A ROW, this one by Sterling
Raskie, discusses the “alphabet soup” of financial planners (as I discuss the
“Alphabet Soup” of tax preparation at FINDING A TAX PROFESSIONAL).The post is titled “A Note About Designations”.
You will note that
Sterling correctly says (highlight is mine) “CPAs may be qualified to
prepare tax returns” and not that they are
qualified to prepare tax returns.That
“may” is very important to note.Thanks
to Sterling for getting it right.
“Jersey Shore TV star Mike “The Situation”
Sorrentino and his brother Marc were supposed to go on trial for tax evasion
and fraud on December 2nd. But the judge has granted Marc’s request to delay
the trial until March 2015. The case involves voluminous records, and this
gives both of the Sorrentino brothers more time. They are free on $250,000 bail.”
If only “The
Situation” had spent as much time developing his brain as he did his body.But then if he had he would have never
appeared on a reality tv piece of excrement.
“Extenders
as extortion. The administration
yesterday complicated the negotiations on the extension of the perpetually-expiring
tax provisions by demanding an extension of the refundable child credit and a
permanent expansion of the fraud-ridden earned income tax credit, the New York
Times reports.
It’s obnoxious to throw a new welfare program into the
extender negotiations at this late date, but a lame-duck administration that
will never again face the voters has nothing to lose. While I still think the
$500,000 Section 179 deduction will be extended retroactively to January 1,
this makes me a lot more nervous.
If anything good comes of this extortion attempt, it’s
that it highlights the unwisdom of passing tax provisions temporarily if you
don’t really want them to be temporary. Every time you need to re-enact them,
you open yourself up to just this sort of shakedown.”
Right on, my
brother!
We must also
recognize and acknowledge the “unwisdom” of refundable tax credits and
distributing welfare benefits through the Tax Code.
* And Joe also
provides some excellent advice in another item in the same Roundup (highlight
is mine) –
“Wait for your W-2 before filing. Don’t try
to file off of your pay stub. And if
your preparer offers to prepare a return without waiting for your W-2, find
another preparer.”
“TIGTA's report is the third in a series of
audits it has conducted since 2010 that have documented alarming growth in
prisoner tax refund fraud. The number of
fraudulent tax returns filed using a prisoner's Social Security Number that
were identified by the Internal Revenue Service (IRS) increased from
approximately 37,000 tax returns in Calendar Year 2007 to more than 137,000 tax
returns in Calendar Year 2012. The refunds claimed on these tax returns
increased from $166 million to $1 billion.
Reports issued by TIGTA in December 2010 and December
2012 identified concerns with the IRS's efforts to identify and prevent
prisoner tax fraud. The objective of TIGTA's latest audit was to evaluate the
effectiveness of corrective actions the IRS has taken to address conditions
identified in a prior TIGTA review of prisoner tax fraud.
‘Since TIGTA first began documenting this fraudulent
activity four years ago, refund fraud committed by prisoners has grown to
become a billion dollar problem,’ said J. Russell George, Treasury Inspector
General for Tax Administration. ‘While the IRS has agreed with four of our six
recommendations, more needs to be done, as is explained in our report. It is
incumbent upon the IRS to act aggressively to prevent tax fraud wherever it
occurs, particularly behind bars.’”
“Continuing cuts to the Internal Revenue
Service budget could have deleterious long-term effects on the agency's
knowledge transfer, according to one IRS official.”
On the short-term
front –
“National Taxpayer Advocate Nina Olson also
is worried about a bumpy 2015 filing season. Her main concern, however, is the
problem that budget cuts have caused in the customer service area.
Both individual filers calling the IRS for help filling
out their forms, as well as tax professionals looking for guidance on their
clients' returns could see even longer wait times, warned Olson.”
* Russ Fox has
already seen firsthand “tax professionals
looking for guidance on their clients' returns could see even longer wait times”,
and shares his recent experience with us in “One Ringy Dingy, Two Ringy Dingies…” at TAXABLE TALK (highlight is mine) –
“I was on hold for two hours today on the IRS Practitioner Priority Service before my
call was picked up.”
THE FINAL WORD-
A man asks his wife
what she wants for Christmas. Her reply = A divorce?
Jason’s post grew
out of one of the “things” in the article “Taking on a New Business Client: 18
Things You Need to Know” from the latest issue of NATP’s quarterly Taxpro
Journal written by NJ tax pro Marc Standig, a fellow frequent contributing
author for the NJ-NATP newsletter.
* Are you looking
for a competent and experienced tax professional to help you with year-end tax
planning, and prepare your 2014 returns?Learn “What to Ask A Preparer” at my FIND A TAX PROFESSIONAL website.
* Michael Cohn
tells us about the results of a survey of CPAs about the economy conducted by
the Franklin and Marshall College Center for Opinion Research University on
behalf of the NJSCPA and the PICPA in “N.J. and Pennsylvania CPAs See Economy Stuck in Neutral” at ACCOUNTING TODAY.
Here are some of
the results for NJ (no surprises – highlights are mine) -
ü. . . New Jersey respondents overwhelmingly (71
percent) feel that the business climate
in their state hinders economic growth. Other areas where New Jersey
respondents expect to see stalled growth include clients’ revenues (44
percent), clients’ workforce (65 percent) and clients’ salaries (51 percent).
üIn terms
of taxes, nearly 87 percent of the survey respondents believe that New Jersey’s tax structure is worse than
most states. They cited high taxes as the number one reason (35 percent) for
hindering future economic growth in New Jersey and the number two reason (33
percent) for the state’s high unemployment rate.
üA whopping 83
percent of New Jersey respondents feel estate and inheritance taxes have
prompted clients to leave the state. Not only that, but 71 percent of CPAs in the Garden State have advised their clients to
relocate to another state due to New Jersey’s estate and inheritance taxes.
A large majority (84 percent) think these taxes affect the state’s middle class
just as much as the affluent.
What did this perennial
publicity seeker, who Robert misidentifies as a “Civil rights leader”, do?
“The New York Times paints a picture of poor
planning, with virtually everything paid for and owned by the entities, not by
Mr. Sharpton. That may even extend to the clothes on his back, with the
entities paying for such personal items as his daughters’ private school
tuition.”
It amazes me that “celebrities”
and other public personalities, who are otherwise intelligent (he may be a
self-important idiot, but he is not necessarily stupid), claim to be totally
clueless when it comes to obvious tax fraud or when it comes to hiring tax and
accounting “professionals”.
* Let me end with a
non-tax item. Fellow great mind Peter J Reilly “tweeted” a
great, and totally true, quote from “Idiot America: How Stupidity Became a
Virtue in the Land of the Free” by Charles P. Pierce:
“. . . there is very little that’s real about a reality
show.”
Judging from the
title of the book it discusses the steaming pile of excrement that is so-called
“reality tv” a lot.
THE FINAL WORD-
Uncle Frank was a
staunch Conservative, and voted straight Republican until the day he died in
Chicago.
Friday, November 21, 2014
Once
again I celebrated my natel day in Atlantic City while attending the National
Association of Tax Professionals’ year-end tax update workshops.I travelled to Bally’s Hotel and Casino in
Atlantic City, where I had been earlier this year for the NATP Forum, for 2
days of the 3-day “Taxpro Symposium”.
We
were supposed to be at the Showboat, but that hotel and casino had closed its
doors.I paid the same rate at Bally’s
that I had booked for the Showboat.Once
again I got a lower rate by booking independently online without using the NATP
code.What good is having a special
conference rate if it is not cheaper than the “off the street” internet rate? I expect this only happens with casino hotels.
One
complaint about Bally’s (in addition to the $12.99 per day charge for wifi,
which I passed on again) is there was not a chair in sight – not even in the
hotel lobby, or on the restaurant/conference room floor (although you could sit
in one of the few chairs a small enclosed smoking “closet”).If you wanted, or needed, to sit down you had
to do so at a slot machine in the casino.I mentioned this in passing to one of the floor guards as I was heading
to the elevator of my tower and he gave me directions to the one out of the way
lounge area.By the time I arrived at
the lounge I really needed to sit down!
Sitting
on the Boardwalk one morning I noticed that the arrogant and annoying Chef
Tantrum (aka Gordon Ramsey) is opening a Pub and Grill in Caesar’s.If I return here in the future that is one
place I will avoid – I certainly do not want to put any money in that idiot’s
pocket.
For
Tuesday, my 61st birthday, the topic was “The Essential 1040”, which
covered the “standard features of the
inflation-indexed updates for preparing individuals’ 1040 tax returns, new tax
laws applicable to 2014 and recent developments”.And, of course, included the obligatory
2-hours of redundant ethics preaching.
To
be honest, for someone like me, who has written frequently over the past year
about the 2014 inflation-indexed numbers, and recently about the 2015 numbers,
the basic update component is boring.And, with one exception, there was really nothing of consequence in new
developments (IRS rulings and regulations, court cases, etc.).Any real value was in the discussion of the new
capital vs repair regulations, a complicated issue that basically comes down to
capitalize just about everything, and, of course, the new Affordable Care Act
(aka Obamacare) individual shared responsibility and premium tax credit rules.
During
the inflation updates the workshop leader did make a good point about the
dreaded Alternative Minimum Tax.It is
not all bad – especially when it comes to year-end tax planning.It can be looked at like a “bell curve”.From the point where your situation causes
you to enter “AMTland” up till the point that your applicable AMT exemption is
fully phased out it is bad.But from
that point on, as long as you are still subject to AMT, it is good – because
additional income is taxed at a maximum of 28% and not 33% to 39.6%.
One
thing that I did learn on Tuesday is that if a taxpayer does not self-assess
the Obamacare penalty where applicable, or if a taxpayer does self-assess the
penalty but does not pay it, the only way the IRS can collect the amount of
penalty due is by “garnishing” a future income tax refund.
One
item I use to judge the workshop location is the continental breakfast provided
by the venue.Bally’s offering was
certainly not the best I have ever seen (there have been two or three hotels
over the years that have laid out a true banquet), but it was also certainly
not the worst.
I
had originally planned an expensive meal at the high-end restaurant in nearby
Caesar’s for my birthday dinner - but thought hey, why not celebrate by having
what I really enjoy (and at a closer venue).So I had a greasy cheeseburger, fries, and a banana shake at “Johnny
Rocket’s”, home of the singing and dancing waiters.
Wednesday’s
“Beyond the 1040” was more beneficial, at least to me, as it covered specific
ongoing tax issues.The topics included divorce,
estimated taxes, foreign investment income (the foreign tax credit on Form 1116
and FATCA/FBAR – not to be confused with FUBAR, which is a technical term for
the entire Tax Code), and the “Cohan rule” and documentation of travel, home
office, and charitable contribution deductions.
Here
are some items of interest from Wednesday’s workshop –
*
This was not discussed during the workshop – but it is important to point out
that there are many potential short-term, long-term, and future tax
consequences of actions, payments, and property distributions set forth in a
divorce degree about which neither spouse, nor many divorce lawyers, have no
clue - so it is important to discuss them with, or have the decree reviewed by,
a competent tax professional.I realize
I am showing my age - but while you would certainly want Arnie Becker as your
divorce attorney, you should have the divorce agreement reviewed and approved
by Stuart Markowitz before signing it.
*
Your filing status for the tax year is determined by your marital status on the
last day of the year.If you are legally
married on December 31st you are married for the entire year.If you are legally divorced on December 31st
you are unmarried for the entire year.But under an “annulment” the marriage is treated as never having occurred.If your marriage is annulled you have never
been married – and you can amend any prior years’ returns that you filed as
married, either joint or separate, to recalculate each individual “spouse’s”
tax as unmarried (Single or Head of Household) and therefore void the marriage
tax penalty.
*
It is interesting that, when it comes to preparing a 1040, in one situation the
divorce degree means absolutely nothing (claiming a dependency deduction) while
in another what is stated in the document is very important
(deducting/reporting alimony).
*
While, for purposes of calculating any penalty for underpayment of estimated
tax, income tax withholding is generally considered to have been paid evenly
throughout the year, regardless of when actually withheld, you can, using Form
2210, elect to treat withholding as being withheld when actually withheld to
avoid or reduce a penalty.
*
At one point during the discussion of withholding the question “why is there a
marriage tax penalty?” was posed.The
answer (my answer) is that the concept of filing status and the differing tax
tables were created at a time in history, many years ago, when the husband
worked and the wife stayed home and “kept house”.
Another
set of well-done year-end sessions from NATP.Next fall if they are offered again in Lancaster PA, as they were this
year, I will not be returning to Atlantic City.
Thursday, November 20, 2014
I couldn't come up with anything else to write about, so here is a "rerun" of a post from a few years back with an overview of the history of our federal income tax, updated for more recent developments.
1643: The colony of New Plymouth, Massachusetts levies the first recorded income tax in America.
1861: Congress passed the first income tax law as an emergency measure to fund the Civil War.
1872: Congress repeals the income tax law.
1894: As a response to complaints that excessive reliance on tariffs as a source of revenue resulted in an increase in the cost of imported goods, Congress again passed an income tax law.
1895: The US Supreme Court ruled that the income tax law was unconstitutional.
1913: In February the 16th Amendment, which states "Congress shall have the power to lay and collect tax on incomes, from whatever sources derived, without apportionment among the several states, and without regard to any census or enumeration", was ratified by the necessary 3/4 of the states. On October 3rd Congress passed the Revenue Act of 1913, which created the first permanent US income tax.
Under this act, the first $3000 of income for single persons and $4000 for married couples was exempt from taxation. A "normal" tax of 1% was applied to income above $3000 or $4000, and a "super" tax of from 1-6% was applied to income in excess of $20,000. Deductions were allowed for business expenses (including depreciation), interest paid on "personal indebtedness", all national, state, county, school and municipal taxes paid, casualty losses, and worthless debt. In the first year only 1 out of every 271 American citizens were taxed and $28 Million in revenue was raised.
1916: The Federal Estate Tax was enacted to help generate additional revenue to fund America's anticipated entry into the first World War.
1917: Congress raised tax rates in response to the increasing cost of the war and approved credit for dependents and deductions for charitable contributions.
1922: For the first time preferential tax treatment was provided for capital gains.
1932: The tax law was amended to provide that US presidents were liable for federal income tax on their salaries. Franklin Roosevelt was the first president since Abraham Lincoln to pay federal income tax on his presidential salary.
1935: The Social Security tax, 1% on the first $3000 of wages, was enacted.
1942-1945: New tax laws, in response to the cost of World War 2, created withholding on wages, more tax brackets for lower income taxpayers, the standard deduction, a personal exemption for dependents, a deduction for medical expenses, and increased tax rates. By the end of the war the maximum tax rate was 94%.
1953: The Bureau of Internal Revenue becomes the Internal Revenue Service. And Robert D Flach, who would eventually become the internet's WANDERING TAX PRO, is born.
1961: Taxpayers were required to provide their Social Security or other taxpayer identification number to banks and other financial institutions so they could report interest and dividend payments to the IRS.
1964: Tax rates were reduced from a range of from 20% to 94% to from 16% to 77%. The Income Averaging method of tax computation was introduced.
1970: Congress created a Minimum Tax so high-income individuals could not completely avoid paying taxes through the use of preferential tax shelters, loopholes and deductions.
1972: Robert D Flach, who would later become the internet's WANDERING TAX PRO, prepares his first Form 1040 as a paid preparer.
1975: Low-income taxpayers were allowed to claim a refundable Earned Income Credit (EIC).
1979: Unemployment compensation was made partially taxable.
1981: Tax legislation reduced tax rates by 25% over 3 years, indexed tax brackets for inflation, and applied the same tax rates to earned and unearned income.
1984: For the first time recipients of Social Security and Railroad Retirement benefits were subject to tax on up to 50% of the benefits received, depending on the recipient's income.
1986: The largest revision of the Tax Code since 1954, the Tax Reform Act of 1986, was enacted. The law reduced the number of tax brackets from 14 to 2, decreased the maximum tax rate from 50% to 28%, repealed the dividend exclusion, Income Averaging, the itemized deduction for sales tax paid and the preferential treatment of long-term capital gains, introduced the passive activity rules, the Kiddie Tax, the deduction from gross income for health insurance premiums paid by self-employed individuals, and the 2% of AGI limitation on most miscellaneous itemized deductions, phased out the itemized deduction for personal (credit card, auto loan, etc.) interest, limited the deduction for business meals and entertainment to 80%, and replaced the additional personal exemption s for age 65 and blind with an increased standard deduction.
1987: For the first time taxpayers were required to list the Social Security number of dependent children, age 5 and over.
1990: The Revenue Reconciliation Act of 1990 added a third tax bracket (31%) and instituted the reduction of itemized deductions and phase-out of personal exemptions for high-income taxpayers.
1993: The Omnibus Budget Reconciliation Act added the 36% and 39.6% tax brackets, increased the maximum tax on Social Security benefits from 50% to 85%, and reduced the deduction for business meals and entertaining from 80% to 50%.
1998: In response to abusive treatment of taxpayers by the Internal Revenue Service, the IRS Reform and Restructuring Act of 1998 was enacted.
2001: Congress passed the Economic Growth and Tax Relief Reconciliation Act of 2001, the largest tax cut in over 20 years, with 85 major provisions. All provisions of this act will expire in 2011. And Robert D Flach begins publishing THE WANDERING TAX PRO blog.
2003: To stimulate the economy, Congress passed the Jobs and Growth Tax Relief Reconciliation Act of 2003, the third major tax bill in as many years, and the third largest tax cut in history.
2010: Congress passed the "Patient Protection and Affordable Care Act of 2010 and Health Care and Education Reconciliation Act of 2010", aka "Obamacare", which made the US Tax Code even more of a mucking fess and created more unnecessary work for tax preparers.
2013: Congress passed the "American Taxpayer Relief Act of 2012", which made permanent most of the expiring "Bush" tax cuts )from the Economic Growth and Tax Relief Reconciliation Act of 2001 and the Jobs and Growth Tax Relief Reconciliation Act of 2003), permanently "fixed" the dreaded Alternative Minimum Tax, and brought back PEP and Pease.
Click HERE for reports, information, and charts on major tax legislation over the years.
Wednesday, November 19, 2014
The
November issue of NATP’s TAXPRO Monthly includes an article on “Engaging the
Nonfiler”.
The
article describes a “nonfiler” as “as
taxpayer who simply failed to file his or her tax return – either accidentally
or intentionally”.
Over
the past 40+ years I have come across quite a few clients who did not file on
time. Often I have prepared two or even
three consecutive years' 1040s at the same time.
I
currently have a client who has not filed her 2011, 2012, or 2013 returns.Based on our relationship I know that she
really has not filed, and not just gone to another preparer.
Why
would something like this happen? I have
encountered three reasons:
1.The taxpayer has lost or misplaced
much or all of the information (and/or client-prepared worksheets) needed to
prepare the first return.Because the
first year’s return has not been filed the second year is delayed.By the time the third year comes around the
taxpayer is already two years behind, and things just begin to snowball.This is the reason for the current non-filing
of the client referenced above.
2.The first and second returns are not
filed on time because of ill health – physical or mental (this could include an
addiction to alcohol or drugs), and, again, when it approaches the due date of
the third year’s return the taxpayer is already two years behind, and things
just begin to snowball.
3.The taxpayer just stops filing tax
returns because of the misconception that he/she no longer has to file tax
returns. For some unknown reason there are those who think that you do not have
to file tax returns any more once you reach age 65 or age 72. This is
bull-pucky (technical IRS term). You are required to file a federal income tax
return from the day you are born until the day you die, whether you go to your
final audit at age 66 or 96, as long as you have sufficient net taxable income!
If
three or four years pass since the due date of a return and it is still not
filed there is an excellent chance that the Internal Revenue Service will
reconstruct the return for you, using the information it has available in its
computer “matching” system. Of course this will only happen if you have income
that is reported to the IRS by a third party – such as W-2 wages, gross pension
and annuity distributions, interest, dividends, and gross proceeds from the
sale of assets.
The
problem with the IRS “reconstructing” a tax return is that they prepare the return under the worst possible scenario.
If
you are married they will calculate the tax as Married Filing Separately –
reconstructing two returns if there is income reported under both spouse’s
Social Security numbers. If you should be filing as Head of Household the IRS
will classify you as Single.
You
will not be given any exemptions for dependents, even if you had claimed
dependents in the past. The only personal exemption that will be included in
the calculation is that for yourself, which may be reduced due to a truly
“gross” AGI. If there are no dependents there will be no corresponding Child
Tax Credit.
The
reconstructed return will report the gross amount of income that is in the IRS
computer system under your Social Security number.
üIf
you were issued a Form 1099-R for $50,000 for a distribution from an employer
pension plan or an IRA that was rolled over within the 60-day “grace” period,
and therefore not taxable, the full $50,000 will be included in AGI, and you
will be assessed the 10% premature withdrawal penalty.
üThere
will be no provision for the cost basis of any asset sales reported on a Form
1099-B – 100% of the gross proceeds will be included in income and taxed as
short-term gains.
üNo
deductions will be taken against business income reported as “non-employee
compensation” on a Form 1099-MISC, and self-employment tax will be calculated
on the full amount.
üThere
will be no “above the line” deductions (i.e. qualified tuition and fees, IRA
contributions, health insurance premiums for self-employed), with the one
exception of 1/2 of any self-employment tax assessment.
üThe
tax liability will be calculated using the Standard Deduction.
It
is not unusual to receive a bill from the IRS for assessed tax, penalties and
interest on a reconstructed federal income tax return for $50,000 - $100,000!
Let’s
look at a real life example of a reconstructed return from my client files.
FYI, in this case returns were file late because of medical reasons –
*
Although the taxpayer had consistently filed his tax returns as Married Filing
Joint in the past, his filing status for the reconstruction was Married Filing
Separately.
*
The calculation showed $260,610 in “total income reported by payers”, which
included $205,000 in gross proceeds from the sale of investments reported on a
Form 1099-B.
*
There were no “adjustments to income”, the one personal exemption allowed was
totally phased out due to the inflated AGI, and the Standard Deduction allowed
for Married Filing Separately was claimed.
*
The total tax assessment was $84,459, which after deducting withholding became
$71,375 in net tax due. Adding interest of $4,823 and penalties for late
filing, late payment and underpayment of estimated tax of $23,392 the total
amount due came to $99,590!
The
spouse only had W-2 income reported under her Social Security number. Her
withholding was enough to cover the tax liability, even filing separately with
no deductions, so she did not get a bill from the IRS. She also did not receive
a refund for the overpayment on the reconstructed return.
Here
are the facts from the Form 1040 that I eventually prepared –
*
The joint total income was $128,751.
*
After subtracting the cost basis of the investments sold there was a net
capital loss, and the $3,000 maximum loss deduction was claimed.
*
There were adjustments to income for the educator expenses and qualified
tuition and fees.
*
Schedule A was filed to report $40,961 in total itemized deductions.
*
The couple had two dependent daughters and personal exemptions of $3000 each
were claimed in full for 4 individuals.
*
The final tax liability was $15,552, which was more than covered by
withholding.
While
we eventually got everything straightened out with the IRS, as a result of the
late filing the taxpayer became subject to “back-up withholding” and for
several years had 28% in federal income tax withheld from his interest and
dividend income, which reduced the potential accrual of earnings on this money.
You
should note that even if there is an overpayment on the properly constructed
return the taxpayer will not receive a refund if that return is not received by
the IRS within 3 years of the original due date for the return. While the
three-year clock for audit purposes begins on the date the return is actually
filed, the clock for refunds begins on the actual due date for the return.
In
another real life case from the practice of my mentor, from several decades
ago, the taxpayer was convinced that he no longer had to file once he turned
age 65. He became our client only after
the IRS attempted to foreclose on his home to pay the tax due on a
reconstructed return!
If
you receive a reconstructed return, which is referred to by the IRS as a
“Substitute For Return” (SFR), you should not file an amended Form 1040-X to
report the correct information. While an SFR is considered to be a valid tax
return it does not constitute an original return filed by the taxpayer. In the
above detailed example I filed an original Form 1040 for the reconstructed
year.
So
if you want to avoid hassles with the IRS, possible excessive penalties and
interest, and a potential lien on your personal residence you should always file your income tax returns on time, even if you
do not have the money to actually pay the tax due. It is much “more better” to submit a balance due return with no payment
than to submit nothing at all.
And
if you have not filed your 1040 for the past year or two or three, get thee to a tax professional!
Tuesday, November 18, 2014
You
have several options available for investing your current, retirement, college,
and health savings.It is important to
understand the tax aspects of each option, and the tax treatment of the various
types of investment accounts – currently taxable, tax-deferred, and tax-exempt
- to maximize your “after-tax” earnings from your investments.
DOMESTIC
STOCKS
Investment
in shares of stock, both domestic and foreign, can generate qualified dividends
while held and, if held for more than a year, long-term capital gains when sold.Qualified dividends and long-term capital
gains are taxed at a special lower rate, from 0% (no federal income tax) to
20%, depending on your level of overall net taxable income.Short-term capital gains (from the sale of
stock held for one year or less) are taxed at ordinary income rates, from 10%
to 39.6%.
Qualified
dividends and long-term capital gains are also taxed at the special lower rate
under the dreaded Alternative Minimum Tax (AMT).However this type of income increases your
Alternative Minimum Taxable Income (AMTI) and may cause you to become a victim
of AMT and/or reduce your AMT exemption.
And,
depending on your level of Adjusted Gross Income (AGI), all dividends and
capital gains may be subject to the 3.8% Net Investment Income Tax.
Distributions
from tax-deferred accounts, retirement accounts like a traditional IRA or
401(k) and the various self-employed retirement accounts, are taxed at ordinary
income rates regardless of the source of the income within the account - so
qualified dividends and long-term capital gains earned within a tax-deferred
retirement account are taxed at ordinary income rates when the money is
withdrawn from the account.
While
taxable distributions from a tax-deferred account will increase AMTI, these
distributions are not subject to the Net Investment Income Tax.
Stock
investments that will generate qualified dividends and long-term capital gains
are taxed less if held in currently taxable accounts.
If
you, or your broker, are more of a day trader, and invest in some stocks for
quick turn-over short-term gains, these stocks could ultimately generate more
net after-tax income if held in tax-deferred accounts.
Regardless
of where held the gains will be taxed at ordinary income rates, but holding
these investments in retirement accounts will defer the taxation of gains to
the future, in future dollars, when distributions are made after retirement
(and when your marginal tax rate, or all tax rates, could be less than they are
now).And holding them in deferred
accounts will allow for greater eventual growth as a result of the tax
deferral.
I
am not telling you not to invest
tax-deferred funds in stocks that generate qualified dividends and long-term
capital gains.You obviously want to
earn as much as possible within a tax-deferred account.Even though you may lose the benefit of the
lower tax rate, you may make up for this by the increased tax-deferred
accumulation of income that will ultimately be taxed in the future in future
dollars.
What
I am saying is that when considering how to invest funds in currently taxable
accounts it is more “tax efficient” to choose investments that will generate
income taxed at the lower capital gain rates.
INTERNATIONAL
STOCK
While
the same considerations I discussed under domestic stocks apply to
international, or foreign, stock (the stock of a company organized and located
outside of the United States), the dividends from international stock will
often have foreign tax withheld.
Foreign
tax withheld from dividends generated by currently taxed investments can be
taken as a credit - often a 100% dollar for dollar credit against current
income tax liability.Unused credits can
be carried forward to be used in future years.
While
foreign tax withheld from dividends generated by investments held in a
tax-deferred retirement account will reduce the income that is eventually
taxed, you do not get the benefit of the tax credit.
You
should hold investments in international stock in currently taxable accounts.
TAXABLE
BONDS
Bonds
pay interest.Interest is always taxable
at ordinary income rates.
Interest
on bonds and other direct obligations of the US Government (such as savings
bonds and Treasury bonds and notes), while fully taxed at ordinary rates on the
1040, are exempt from state income tax.
Taxable
bonds are a good investment for tax-deferred retirement accounts.
TAX-EXEMPT
BONDS
The
interest from municipal bonds (issued by the 50 states and the District of
Columbia, and the bonds of US possessions like Guam, Puerto Rico and the Virgin
Islands) are exempt from the “regular” federal income tax, and the state income
tax of a “resident” state (interest from bonds issued by the state of NJ or a
NJ municipality, and US possessions, are exempt from NJ state income tax).Interest from certain “private activity”
municipal bonds are taxable under the dreaded AMT.
You
should never purchase tax-exempt
bonds in a tax-deferred account.
Distributions
from a tax-deferred retirement account are subject to federal income tax at
ordinary income rates regardless of the source of the income within the account
- so interest on tax-exempt municipal bonds earned within a tax-deferred
retirement account are taxed at ordinary income rates when the money is
withdrawn from the account.
REAL
ESTATE INVESTMENT TRUSTS
INVESTOPEDIA
tells us that a Real Estate Investment Trust, or REIT, is a security that sells
like a stock on the major exchanges and invests in real estate directly, either
through properties or mortgages.
Generally
the dividend payments issued by a REIT are taxed at ordinary income rates.
REITs
should be held in tax deferred accounts.
LIMITED
PARTNERSHIPS
My
personal, albeit selfish, advice is never
invest in limited partnerships in a currently taxable account.
Long-time
readers of TWTP know that I hate K-1s from limited partnership
investments.Properly reporting all the
items from the K-1, including those buried in attached statements, on the
taxpayer’s Form 1040, and keeping track of suspensions, carry forwards and tax
basis, causes considerable pain in various parts of the anatomy of a tax
preparer.And the additional tax
preparation costs that result can be more than, or at least take a large bite
out of, any eventual tax and financial benefits from the investment.I truly believe that a carefully researched
mutual fund will provide the same potential tax and financial benefit as any
limited partnership investment (and welcome the comments of brokers on this
statement).
If
your broker insists that you must purchase units in a limited partnership, and
no mutual fund will provide the same tax and financial benefits, then purchase
the partnership in your IRA, traditional or ROTH, or another tax-deferred or
tax-exempt account, so you tax professional does not have to deal with it on
the 1040.
MUTUAL
FUNDS
Mutual
funds invest in all types of investments – domestic and international stocks,
taxable and tax-exempt bonds, real estate, and limited partnerships.
The
taxability of dividends issued by mutual funds is determined by the rules for
taxing the individual investments in the fund. Choosing what types of funds you
purchase in currently taxed and tax-deferred accounts should be governed by the
types of investments held in the fund.
Mutual
funds can issue qualified dividends, non-qualified dividends, tax-exempt
dividends, return of capital, and capital gain distributions.Non-qualified dividends are taxed at ordinary
income rates.Return of capital
distributions are not currently taxed as income – they reduce your cost basis
in the fund.Capital gain distributions
are taxed at the lower capital gain tax rates.
There
are “tax-efficient” mutual funds.These
funds can keep it's turnover low, especially if the fund invests in stock, and
avoid or limit income-generating assets, such as dividend-paying stocks.These funds should be held long-term in
currently taxable accounts.
It
really does not matter how you invest funds held in accounts whose
distributions will never be taxed.
Qualified
distributions from a ROTH IRA or 401(k) account, a Section 529 qualified
tuition program, a Coverdell Education IRA, a Health Savings Account, or a
Medical Savings Account are totally tax
free.So taxes are not a
consideration in determining where to invest the money.Obviously you want to make sure that all
distributions from these types of accounts are qualified distributions.
Before
you invest you should consult a tax professional.Do not
rely on a broker for tax advice.
Monday, November 17, 2014
I do believe that indexing of tax
items for inflation began with the Economic Recovery Tax Act of 1981, the first
major tax act of the Reagan Administration.ERTA called for the indexing of personal exemptions and rate brackets,
effective in 1985, based on changes in the Consumer Price Index (CPI) for years
ending in September of the calendar year preceding the tax year.The landmark Tax Reform Act of 1986 indexed
the Standard Deduction for inflation beginning in 1989.
As subsequent tax acts continued the
concept of reducing deductions and credits based on AGI that began with TRA 86
most of the phase-out thresholds were indexed for inflation.The American Taxpayer Relief Act of 2012
finally permanently indexed the dreaded Alternative Minimum Tax (AMT) for
inflation
Today many items on the 1040 are
indexed for inflation – but not all.
“Retirement Distributions: Finding
the Sweet Spot” by Michael McGilligan in the Fall 2014 issue of NATP’s Taxpro
Journal discusses the taxation of Social Security and Railroad Retirement
benefits.Michael provides background on
this issue and tells us that when the taxation of up to 50% of these benefits
was first enacted, effective with tax year 1984 –
“. . . it was estimated that only 10% of Social Security recipients would be
affected by the tax.By the time the law
was first amended in 1993 {to make up to 85% of benefits taxable – rdf}, about 18% of beneficiaries were affected.”
This change apparently “did not increase the number of beneficiaries
subject to the tax, but did increase the amount of taxes for over half of the
18%”.
Michael goes on to say –
“The
Congressional Budget Office estimated that by 2005, 39% of beneficiaries had a
portion of their benefits subject to tax.”
What changed?
“The
answer lies in what didn’t change – the thresholds used to calculate the
taxable portion were not indexed for inflation and remain at the levels in
effect in 1984 and 1994, respectively.Therefore, without indexing, we can expect the percentage of
beneficiaries subject to tax to continue increasing.”
“Social Security: Calculation and
History of Taxing Benefits” Noah P. Meyerson, published by the Congressional
Research Service on August 4, 2014, provides the following update -
“According
to the Congressional Budget Office (CBO), 49% of Social Security beneficiaries
(25.5 million people) will be affected by the income taxation of Social
Security benefits this year.”
An article from the March-June 2003
issue of Enrolled Agent Doug Thorburn’s “Wealth Creation Strategies” newsletter
suggested that had the threshold numbers ($25,000 for single taxpayers and
$32,000 for married couples for the 50% level. and $34,000 and $44,000 for the
85% level) been indexed for inflation since day one the adjusted numbers for tax
year 2002 would have been $44,422
and $56,861 and $60,415 and $78,183. .
Another item that has not been
indexed for inflation is the $3,000 maximum current capital gains deduction.If capital losses exceed capital gains on
Schedule D you are only allowed to deduct up to $3,000 against other
income.Net losses in excess of this
$3,000 limit can be carried forward to apply against net gains in future years.
When I first started preparing 1040s
back in 1972 the maximum capital loss deduction was $1,000.In went to $3,000 in 1978.According to Doug Thorburn, the inflation
adjusted amount for tax year 2002
would have been $8.759.
The maximum amount of rental loss that
can be currently deducted on Schedule E under the passive activity rules
created by TRA 86 has been $25,000, and the phase-out range for this deduction $100,000-$150,000,
since 1987. According to Doug indexing
would have brought these numbers to $40,344 and $161,378-242,067 for tax year
2002.
Doug’s index-inflation estimates are
for 2002.Imagine what they would have
been for 2014!
One final example.The maximum deduction for a business gift has
been $25.00 per person for the 40+ years I have been preparing 1040s.The $25.00 limit was actually set by Congress
in 1962!That was 52 years ago.In 1962 the median annual family income was $6,000,
a new house cost $15,000, a gallon of gas was 25 cents, and a 1st
class postage stamp was 4 cents.
The result of the lack of indexing
for many important numbers on the 1040 is annual “back-door” tax increases for
many taxpayers.
If it is appropriate to index some
tax items for inflation why shouldn’t ALL
deductions, credits, thresholds, etc. be indexed for inflation?
AIN'T THAT THE TRUTH!
DONALD T RUMP HAS NOT DONE A SINGLE THING THAT IS "APPROPRIATE" OR "ACCEPTABLE" FOR A CANDIDATE OR A PRESIDENT SINCE THROWING HIS HAT INTO THE RING.EVERY SINGLE DAY TRUMP PROVIDES MORE PROOF THAT HE IS AN IGNORANT, SELF-ABSORBED, UNFIT, MENTALLY UNSTABLE IDIOT, AND A DEPLORABLE AND DESPICABLE HUMAN BEING.TRUMP MUST BE REMOVED FROM OFFICE FOR MENTAL INCOMPETENCE ASAP! PLEASE READ AND SHARE THIS - THE TRUTH ABOUT TRUMP'S MENTAL CONDITION
Donald T Rump has not done a single thing that anyone with intelligence would consider “appropriate” or “acceptable” for a President since deciding to run for office.
Every single day Trump provides more proof that he is an ignorant, self-absorbed, unfit, mentally unstable idiot, and a deplorable and despicable human being, who must be removed from office ASAP.
VERY IMPORTANT -
(1) Before contacting me with questions about how a blog post relates to your specific situation, please be aware that I do not give free tax advice to non-clients by e-mail, comment response, or phone. So don't waste your time and mine.
(2) I am winding down my tax practice, and I will not, under any circumstances, accept any new clients. Period. I am actually trying to "thin the herd".
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Antipneumococcal seroprevalence and pneumococcal carriage during a meningococcal epidemic in Burkina Faso, 2006.
To better understand the high incidence of pneumococcal meningitis in the African meningitis belt, we conducted a pneumococcal seroprevalence study during a meningococcal meningitis epidemic in Western Burkina Faso, March 2006. In 3 villages experiencing epidemics, we included 624 healthy persons (1-39 years) by cluster sampling. We determined pneumococcal serum immunoglobulin G (IgG) antibody concentrations against 12 serotypes contained in 13-valent pneumococcal conjugate vaccine, and evaluated determinants for IgG ≥ 0.35 μg/mL by multivariate logistic regression. The percentage of subjects with serotype-specific IgG concentrations ≥0.35 μg/mL increased with age and was similar for the different serotypes: it was 20%-43% among 1-4-year-olds and 56%-90% among 20-39-year-olds. Prevalence of IgG ≥ 0.35 μg/mL against serotype 1 was up to 71% after age 10 years. During multivariate analyses, determinants of IgG concentrations ≥0.35 μg/mL varied by serotype; for 5 and 6 serotypes, respectively, female sex (around 2-fold increased odds) and cigarette smoking (about 5-fold reduced odds) predicted elevated titers. Despite a substantially higher historical pneumococcal meningitis incidence in Burkina Faso, the general population has an antibody seroprevalence against 12 pneumococcal serotypes similar to that reported from the United Kingdom. The role of putatively protective antibody seroprevalence in preventing pneumococcal meningitis in the meningitis belt requires more thorough evaluation.
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The majority of lymphomas are treatable and many, with modern treatment approaches are potentially curable. There is, however, a need to mitigate the risk of life-threatening treatment toxicity and to overcome the poor prognosis associated with treatment failure. This has led to interest in the development of biomarkers that report the efficacy/toxicity of treatments and inform the development of individualised treatment strategies. Tissue-based biomarkers are the traditional 'gold standards\' used in drug development, but circulating biomarker analyses are more readily applicable to most clinical settings. A biomarker detected in the blood and measured serially could provide a real-time assessment of a patient\'s disease course and response to treatment ([@bib22]). Here, as a first step towards biomarker qualification, a panel of circulating biomarkers of cell death and myelosuppression has been evaluated for its potential to predict and/or report drug efficacy and toxicity in patients with lymphoma receiving standard chemotherapies.
During apoptosis, chromatin is cleaved into nucleosomal strands by caspase-activated endonucleases ([@bib17]). Nucleosomes reside in membrane-bound apoptotic fragments, undergo macrophage phagocytosis and are released into the blood as nucleosomal DNA (nDNA) ([@bib14]). Circulating nDNA levels are elevated in patients with cancer including lymphoma ([@bib7]) compared with healthy controls, and have demonstrated prognostic and pharmacodynamic utility in studies of lung cancer and other tumour types ([@bib12]; [@bib13]). In this study of nDNA in patients with lymphoma, we postulated that baseline nDNA predicts treatment outcome and that changes in nDNA levels during therapy act as pharmacodynamic and surrogate biomarkers of response.
Cytokeratin 18 (CK18), a major component of an epithelial cell cytoskeleton, is not expressed in cells of lymphoid origin. Consequently, elevated circulating CK18 levels reflect epithelial damage as reported in several non-malignant conditions including sepsis, hepatitis and ischaemic heart disease ([@bib1]; [@bib8]; [@bib11]). Here, we postulated that circulating CK18 levels measured soon after initiating treatment for lymphoma would be useful for monitoring and/or predicting subsequent epithelial toxicity.
Myelosuppression and potentially life-threatening sepsis are common complications of chemotherapy ([@bib15]). FLT3 ligand, a cytokine that binds the FLT3 receptor, acts synergistically with other cytokines such as stem cell factor and granulocyte colony-stimulating factor in the maintenance of the stem cell compartment and in progenitor expansion/differentiation during haematopoiesis. FLT3 ligand may be particularly important in expansion of the stem cell compartment in response to bone marrow stress ([@bib4]). In this study, we postulated that changes in soluble FLT3 ligand in patients receiving standard doses of chemotherapy would provide an early warning of severe myelosuppression that could in future be used to guide early intervention with growth factors.
In this study, we have evaluated the utility of nDNA to monitor treatment response, CK18 to monitor and/or predict epithelial toxicity and FLT3 ligand to monitor myelotoxicity in patients with lymphoma receiving standard chemotherapy. This study was also designed with a view to exploring the potential utility of these circulating biomarkers in future clinical trials investigating individualised treatment strategies.
Patients and Methods
====================
Patients and blood sample collection
------------------------------------
The study received ethical approval from the North Manchester Research Ethics Committee in December 2006 and was conducted according to good clinical practice. Patients with a history of auto-immune disease, viral hepatitis or HIV were excluded as these conditions are known to be associated with high levels of the biomarkers under investigation. Fifty patients with histologically confirmed Hodgkin (HL), follicular (FL) or diffuse large B-cell lymphoma (DLBCL) were recruited between 2007 and 2009 before starting chemotherapy. One patient found to be ineligible after consent was withdrawn. The clinical characteristics of the remaining 49 patients are shown in [Table 1](#tbl1){ref-type="table"}. Patients had standard clinical follow-up (median 479 days (range 10--748 days)).
Control samples were obtained from 61 healthy volunteers at a single time-point. Blood samples were collected from lymphoma patients on days 1, 3, 8 and 15 of the first treatment cycle and days 1 and 3 of subsequent cycles of chemotherapy ([Figure 1](#fig1){ref-type="fig"}). On treatment days, samples were taken immediately before the administration of chemotherapy. All samples for serum were processed within 2 h of collection as previously described and all samples analysed within 3 months of collection. These conditions have been shown not to have any significant effect on CK18 levels ([@bib9]).
Treatment
---------
Patients were treated with standard chemotherapy according to histological sub-type ([Figure 1](#fig1){ref-type="fig"}). Patients with HL were treated with ABVD (doxorubicin 25 mg m^--2^, bleomycin 10 000 IU, vinblastine 6 mg m^--2^ and dacarbazine 375 mg m^--2^) every 14 days. Patients with FL were treated with R-CVP (rituximab 375 mg m^--2^, cyclophosphamide 750 mg m^--2^, vincristine 2 mg and prednisone 40 mg m^--2^ (for 5 days)) every 21 days; in the event of insufficient radiological or clinical response doxorubicin 50 mg m^--2^ was added to the regimen (*n*=1). Patients with DLBCL were treated with R-CHOP (rituximab 375 mg m^--2^, cyclophosphamide 750 mg m^--2^, vincristine 2 mg, doxorubicin 50 mg m^--2^ and prednisone 40 mg m^--2^ (for 5 days)) every 21 days.
Circulating biomarkers
----------------------
Serum was analysed for (a) nDNA using the Cell Death Detection ELISA (Roche, Basel, Switzerland), (b) CK18 using the M65 ELISA (Peviva, Sweden) and (c) FLT3 ligand using an ELISA (R&D, Abingdon, UK; \#DFK00). All analyses followed the manufacturer\'s instructions and were conducted according to Good Clinical Laboratory Practice. As nDNA is a quasi-quantative ELISA, data are expressed as optical density readings. The M65 and FLT3 ligand ELISAs are semi-quantative assays and data are expressed in U l^--1^ and pg ml^--1^, respectively. Both full blood count and lactate dehydrogenase (LDH) were determined independently by the Christie Pathology Laboratories (Manchester, UK).
Image analysis
--------------
X-ray computed tomography (CT) examination was performed before and after therapy. Patients were imaged on a LightSpeed Plus CT scanner (GE Medical Systems, Slough, UK) with typical clinical helical acquisition variables (tube voltage 120 kV, tube current 40 mA). Images were acquired following intravenous injection of 200 ml Omnipaque-140 (GE Medical Systems) and reformatted to produce contiguous slices with no overlap. Slice thickness was 5 mm in all but four cases, where initial data constraints necessitated a slice thickness of 7.5 mm. Nodal tumour burden was assessed by measuring enlarged lymph nodes. Two types of measurements were made based on (a) the sum of the bi-dimensional measurements (units cm^2^) as recommended in the Cheson criteria ([@bib6]) and (b) tumour volume, calculated as the product of the number of voxels and voxel volume (units mm^3^). Extra-nodal disease in solid organs and bone was excluded. All measurements were performed by a radiologist blinded to the serological biomarker data.
Toxicity recording
------------------
Epithelial toxicity was assessed when the patient attended for their second cycle of chemotherapy. The maximum experience of vomiting, diarrhoea, stomatitis, inter-current infection and hepatotoxicity was graded according to the Common Terminology for Classification of Adverse Events (CTCAE) version 3.0 ([@bib21]) and individual grades summed to give an overall subjective toxicity score. Multifactorial symptoms of nausea and constipation were not included in the determination of the epithelial toxicity score, as it can be difficult to attribute these symptoms directly to epithelial damage. Nausea following chemotherapy may be initiated by cerebral signalling, and constipation result from changes to autonomic control of intestinal motility.
Statistics
----------
Statistical analysis was performed using GraphPad Prism version 5.02 for Windows, (GraphPad Software, San Diego, CA, USA, [www.graphpad.com](http://www.graphpad.com)) and probability values of *P*⩽0.05 considered significant throughout. As the biomarkers were positively skewed, non-parametric tests were used to satisfy the assumptions of variance between sample groups. Differences between groups were tested using Mann--Whitney (M--W) *U*-test if comparing two groups or Kruskal--Wallis (K--W) (followed by Dunn\'s *post hoc* test if significant) if comparing more than two groups. Relationships were examined using the non-parametric Spearman\'s rho bivariate correlation with a two-tailed test for significance. The Wilcoxin signed test (WST) was used to assess whether changes in biomarkers following chemotherapy were significantly different from 0%. The area under the curve (AUC) was calculated using the trapezoid method. In the absence of pre-existing data, no formal statistical power calculations were performed.
Results
=======
Utility of circulating nDNA as a biomarker in lymphoma
------------------------------------------------------
Baseline levels of nDNA were significantly higher in the 49 patients with lymphoma (geometric mean 1.58) compared with 61 healthy volunteers (geometric mean 0.33; K--W *P*\<0.001) and 193 patients with metastatic carcinomas (small cell lung cancer (*n*=67), non-small cell lung cancer (*n*=35) or colorectal cancer (*n*=80) (geometric mean 0.94; K--W *P*\<0.05 see [Figure 2A](#fig2){ref-type="fig"}). No statistically significant relationship was found between lymphoma histology and baseline nDNA levels (K--W *P*=0.99) although the patients with the four highest values all had DLBCL. No stage-related trend in baseline nDNA was identified, there was no difference between patients with/without 'B\' symptoms (defined as weight loss \>10% of baseline, drenching night sweats or unexplained fevers \>38 °C; see [Table 2](#tbl2){ref-type="table"}) and no correlation with LDH levels was seen (*R*=0.26). Baseline levels of nDNA did not correlate with baseline nodal tumour burden using 2D or volumetric measurements.
Prognostic significance of baseline circulating nDNA
----------------------------------------------------
In the DLBCL cohort, disease progression occurred in nine patients with a median time to progression of 115 days (range 10--277). The median follow-up in non-progressors was 517 days (range 274--851). Baseline nDNA levels were significantly higher in progressors (geometric mean 3.62, range 0.50--11.65) compared with non-progressors (geometric mean 1.0, range 0.06--21.5; *P*\<0.05). No progression was seen in patients with nDNA levels below the median observed in healthy subjects (nDNA\<0.3). In patients with levels above the maximum observed in healthy subjects (nDNA \>2.9; *n*=11), the median PFS was 83 days (log-rank; *P*\<0.001) and five patients died with a median OS of 63 days; *P*\<0.05) ([Figure 2B](#fig2){ref-type="fig"}). There was no significant difference between baseline LDH levels in progressors/non-progressors with DLBCL (geometric mean 598 U ml^--1^ in progressors *vs* 681 U ml^--1^ in non-progressors (*P*=0.37).
In the patients with FL, 5 of 10 patients progressed with a median time to progression of 295 days (range 147--627) and a median PFS in the remainder of 707 days (range 493--8570). There was no difference between the baseline levels of nDNA in progressors (geometric mean 1.37) *vs* non-progressors (geometric mean 2.55).
Only one patient with HL progressed and subsequently died; prognostic outcome analysis was not therefore performed in this cohort.
Changes in circulating nDNA following therapy
---------------------------------------------
In patients with HL, nDNA levels had decreased by 48 h following therapy. By day 15, levels were similar to healthy controls (0.24 *vs* 0.29; M--W NS with mean change of −2.86, range −9.142 to 0.028; WST *P*\<0.001) ([Figure 3](#fig3){ref-type="fig"}).
In patients with DLBCL, nDNA levels did not decrease until day 8 ([Figure 3](#fig3){ref-type="fig"}) (mean change −3.64, range −20.01 to 1.62; WST *P*\<0.01). Nucleosomal DNA levels were lowest at this point, significantly lower than baseline and no longer significantly elevated compared with healthy controls (0.32 *vs* 0.29 M--W NS). Initial decreases in nDNA were observed following therapy in all patients with elevated levels, however, in those who subsequently progressed, significantly higher levels of nDNA (geometric means 1.58 *vs* 0.15; *P*\<0.01) were seen by the end of therapy.
In patients with FL, nDNA levels changed more gradually during the first three weeks of treatment. Nucleosomal DNA levels were declining by day 3 ([Figure 3](#fig3){ref-type="fig"}), a decline sustained throughout the first cycle (mean change at day 22 −1.53 (range −8.73 to 1.49); WST *P*=0.23). However, in contrast to HL and DLBCL, these changes from baseline did not reach statistical significance and levels of nDNA at day 22 remained elevated compared with healthy controls (geometric means baseline 1.87; day 22 0.95; controls 0.29; K--W *P*\<0.005). Patients with FL where the nDNA levels failed to fall during the first week of therapy had a significantly worse outcome with median PFS of 405 days, while only one patient with a falling nDNA at day 8 had progressed with a median follow-up of 658 days (*P*\<0.05; Supplementary Figure 1).
Analyses were performed to assess whether early changes in nDNA levels following treatment could predict subsequent radiological response to therapy. Overall, 26 patients had evaluable CT examinations at baseline and after therapy (Supplementary Table 1). Levels of nDNA during the first week of therapy (assessed by the AUC days 1--8) correlated strongly with initial nodal tumour burden measured in both 2-D and volume (*R*=0.53 and 0.49, respectively; *P*\<0.01), however, there was no statistically significant relationship between early changes in nDNA and changes in tumour volume on completion of therapy.
Taken together, these results suggest that circulating nDNA levels may have utility as a pharmacodynamic biomarker based on the rapid decreases in nDNA levels observed following therapy. Patients with DLBCL/FL and persistent elevation or rising levels in during therapy had a poorer outcome.
Utility of circulating CK18 as an epithelial toxicity biomarker
---------------------------------------------------------------
Baseline levels of circulating CK18 in patients with lymphoma (geometric mean 348 U l^--1^) were no greater than observed in healthy subjects (geometric mean 347 U l^--1^) and were significantly lower than patients with epithelial cancers (geometric mean 712 U l^--1^, *P*\<0.001).
Following therapy, CK18 increases peaked at day 3 (HL, mean change +33%, range −17 to 113% DLBCL mean change +33%, range −44 to 113%) and were recovering by day 15 (HL mean change 14%, range −6 to 64% DLBCL mean change +15%, range −52 to 84%). No significant elevation was seen in CK18 following chemotherapy in patients with FL (day 3 change +9%, range −27 to 39% and day 15 change −4%, range −52 to 26%), in keeping with the lower incidence of epithelial toxicity observed with this chemotherapy regimen.
In patients with more severe toxicities, higher and more durable peaks in CK18 were seen following therapy ([Figure 4](#fig4){ref-type="fig"}). The change in CK18 at day 3 gave the greatest power to discriminate patients experiencing the worst epithelial toxicity. The largest changes in CK18 were seen in patients with a CTCAE score \>3 (mean increase in CK18 at day 3 for a toxicity score 0=+12% score 1=−7% score 2=+3% score 3=+38% score 4=+69% and score 5=+73%). This showed significant differences with a mean change in CK18 of 62% in patients with toxicity score ⩾3 (*n*=9) compared with 12% in patients with toxicity score \<3 (*n*=37; M--W *P*\<0.005) with an estimated AUC for the receiver operating characteristics (ROCs) curve to detect an epithelial toxicity CTCAE score of ⩾3 of 0.85 (*P*\<0.005). These data suggest that CK18 can be used to predict epithelial toxicity in patients with lymphoma.
Utility of FLT3 ligand as a biomarker of myelosuppression
---------------------------------------------------------
FLT3 ligand was evaluable in 43 patients (10 HL, 10 FL and 23 DLBCL). Baseline levels were low for all three lymphoma types, although higher levels were seen in FL than DLBCL. There was no significant difference between patients with documented bone marrow involvement and those without (geometric mean involved 115 pg ml^--1^ (range 26--411 pg ml^--1^) *vs* uninvolved 90 pg ml^--1^ (range 29--229 pg ml^--1^)).
In patients with HL treated with AVBD, an elevation in FLT3 ligand was seen by day 3 (mean change +166%, range +46 to 356%), which resolved by day 15 (mean change −2%, range −40 to 36%). In patients with DLBCL, increases in FLT3 ligand did not occur until day 8 (mean change +261%, range 16 to 796%), and while in most was resolving by day 22, some patients had persistent elevations (mean change +92%, range −11 to 1280%).
In patients with FL, smaller elevations were seen compared with other lymphoma subtypes, in keeping with the lower myelosuppressive potential of R-CVP. As in DLBCL maximum elevation was seen at day 8 (mean change +57%, range 24--111%) with resolution by day 22 (mean change +3%, range −54 to 51%).
The correlation of increases in FLT3 ligand at day 8 with myelosuppression was assessed in all 43 patients, and a significant correlation with neutropenia (assessed at day 15) was observed (*R*=−0.34; *P*\<0.05). Nine patients (21%) developed neutropenic sepsis (NPS) at some point during treatment. These patients had significantly higher increases in FLT3 ligand at day 8 of their first cycle of chemotherapy (mean 349%, range 111 to 796%) than patients who did not develop NPS (mean 131%, range −13 to 482%), ([Figure 5A](#fig5){ref-type="fig"}). There was no difference between the nadir neutrophil count in the two groups (mean 5.9, range 0.3--24.4) in patients with NPS *vs* 4.3, range 0.2--13.9 in patients without NPS. Therefore, measurement of circulating FLT3 ligand at day 8 was a better predictor of a future episode of NPS then nadir neutrophil count routinely checked at day 15 (AUC for the ROC curves 0.83 (95% CI 0.70--0.97) for FLT3 ligand *vs* 0.53 (95% CI 0.34--0.72) for the neutrophil count, [Figure 5B](#fig5){ref-type="fig"}).
Discussion
==========
Improving the efficacy and reducing the toxicity of cancer treatment are amongst the biggest challenges facing oncologists. Developing readily measurable biomarkers to accurately predict and report these endpoints is therefore desirable. The study presents for the first time, a potential clinical role for a panel of circulating biomarkers in patients with lymphoma and suggests the need to further evaluate their utility.
In this study, pretreatment nDNA levels in DLBCL correlated strongly with PFS and overall survival. Prognosis in this disease can be estimated by determination of the revised-international prognostic index, which incorporates both clinical parameters and LDH ([@bib20]). This study was not designed or powered to assess the additional prognostic information given by determination of nDNA, however, the strong association with poor outcome in the absence of any observed association with traditional prognostic parameters (e.g. tumour stage and B symptoms), suggests a putative role for nDNA as a new independent prognostic biomarker. This should now be assessed in larger cohorts.
The use of nDNA as a PD biomarker following therapy may give the most additional clinical utility. In all lymphoma sub-types rapid falls in nDNA were seen following the initiation of therapy. Notably, in FL, persistent elevations in nDNA during the first week of treatment were associated with a significantly shorter time to progression. These findings suggest that nDNA could be used both to guide early changes in therapy if an insufficient biomarker response is observed, and also allow comparison between different regimens or dosing schedules in early clinical trials. Although other cell death products have been assessed as PD biomarkers in lymphoma such as caspase 3 and cytochrome *c* ([@bib2]; [@bib7]), nDNA has the advantage that extensive validation and qualification of this assay has already been performed in other tumour types ([@bib12]).
As nDNA is a quasi-quantitative assay ([@bib5]), it is difficult to directly compare results between laboratories. Results in healthy volunteers are consistently and reproducibly low ([@bib13]). The average values derived in a large cohort of healthy controls offers a surrogate measure so that other laboratories can indirectly compare their results with those presented here.
Prediction of toxicity may allow pre-emptive action such as early clinical review and the potential use of supportive care medication. We did not examine a detailed temporal relationship between a rise in CK18 levels and the appearance of epithelial toxicity. Instead, a maximum peak in CK18 was recorded at day 3 and an obvious difference in CK18 profiles between patients with and without subsequent toxicity was observed before the clinical manifestation of toxicity appeared (which typically occurred at days 7--14). Strategies that increase the dose intensity of chemotherapy would be facilitated by a measure of epithelial toxicity and the potential of this approach is currently being tested in patients with HL treated in a phase I dose-escalation study.
Other biomarkers of small bowel toxicity have been evaluated including carbon^13^-sucrose breath tests, plasma citrulline and faecal calprotectin and lactoferrin ([@bib16]; [@bib10]). However, the clinical utility of these biomarkers has yet to be fully determined. In addition, CK18 gives a global measure of epithelial damage, as compared with the specificity to small bowel damage of the assays described above.
The mechanism of increasing FLT3 ligand in response to bone marrow stress remains unclear but local release in the bone marrow, potentially by the stroma, is the leading hypothesis ([@bib19]). In mice, circulating FLT3 ligand levels at day 3 following radiotherapy predicted the length and depth of subsequent pancytopenia ([@bib18]). In humans, FLT3 ligand levels rose within 24 h of radiotherapy and before the effect on peripheral blood counts ([@bib3]). Similarly, in this study the rise in FLT3 Ligand was observed before the neutrophil nadir.
An observed increase in FLT3 ligand at day 8 in cycle 1 would potentially allow the use of growth factors or antibiotics to prevent NPS, even during this cycle of therapy. However, it may be particularly useful to allow interventions in patients who first experience NPS in later cycles of therapy (four patients in this study) or have repeated episodes of NPS (1 patient). As CK18 and FLT3 ligand appear to measure host toxicity objectively, potentially they could be used to adjust doses in an individual patient in order to maximise the therapeutic index, or in early clinical trials to assess the potential for future problematic toxicity. The different chemotherapy regimens examined in this study demonstrated that these biomarkers are useful in predicting the patients who will experience side effects both from therapies where relatively high rates of toxicity can be observed (e.g., ABVD and R-CHOP), but also from better tolerated regimens (e.g., R-CVP). The utility of these biomarkers in patients receiving mechanism-based agents is now being examined.
Our results show the potential utility of a panel of validated biomarker assays in patients with three sub-types of lymphoma. The data suggest first that baseline nDNA in DLBCL gives additional prognostic information over LDH and early changes during therapy may predict subsequent outcome in FL, second, that rises in CK18 on chemotherapy give early warning of epithelial toxicity and third, that substantial elevation in FLT3 ligand identifies patients at risk of NPS. These results argue for inclusion of these biomarkers in larger studies testing individualised treatment strategies.
Supported by a Clinical Pharmacology Fellowship from Cancer Research UK and AstraZeneca Ltd (to AG) by Cancer Research UK (Grant C147/A6058) and by Christie Hospital Foundation Trust Charitable Funds.
Supplementary Information accompanies the paper on British Journal of Cancer website (http://www.nature.com/bjc)
Supplementary Material {#sup1}
======================
######
Click here for additional data file.
{#fig1}
{#fig2}
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{#fig4}
{#fig5}
###### Clinical characteristics of patients with Hodgkin lymphoma, follicular lymphoma and diffuse large B-cell lymphoma
**HL** **FL** **DLBCL**
------------------------------ ---------------- ------------------------------------ -------------------------------------
Number 13 10 26
Age (median (range)) 38 (18--61) 60 (47--85) 69 (22--90)
Gender male: female (% male) 4:9 (31%) 5:5 (50%) 14:12 (54%)
*PS*
0 5 (38%) 4 (40%) 4 (15%)
1 7 (54%) 6 (60%) 16 (62%)
2 1(8%) 0 2 (8%)
3 0 0 4 (15%)
*Stage*
I 1 (8%) 0 4 (15%)
II 6 (46%) 2 (20%) 7 (27%)
III 2 (15%) 2 (20%) 3 (11%)
IV 4 (31%) 6 (60%) 12 (46%)
'B\' symptoms 7 (54%) 0 13 (50%)
Therapy ABVD ( × 3) 1 R-CVP 10 (two converted to R-CHOP) R-CHOP 24 (two converted to R-GCVP)
ABVD ( × 6) 12 R-GCVP 2
Abbreviations: ABVD=therapy with doxorubicin, bleomycin, vinblastine and dacarbazine; 'B\' symptoms=weight loss \>10%, drenching night sweats or unexplained fevers \>38 °C; DLBCL=diffuse large B-cell lymphoma; FL=follicular lymphoma; HL=Hodgkin lymphoma; PS=performance status; stage=as defined by Ann-Arbor Staging System; R-CVP=therapy with rituximab, cyclophosphamide, vincristine and prednisone; R-CHOP=therapy with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone; R-GCVP=therapy with rituximab, gemcitabine, cyclophosphamide, vincristine and prednisone.
###### Baseline levels of circulating biomarkers in patients with lymphoma by histology and stage
**LDH (U ml^−1^)** **nDNA (OD)** **CK18 (U l^−1^)** **FLT3 ligand (pg ml^−1^)**
-------------------------------- -------------------- -------------------- -------------------- -----------------------------
*Histology*
Hodgkin lymphoma 403 (236--621) 1.59 (0.06--9.62) 267 (126--470) 96 (40--229)
Follicular lymphoma 404 (301--699) 1.87 (0.57--10.90) 331 (220--580) 153 (90--411)
Diffuse large B-cell lymphoma 626 (310--4030) 1.60 (0.06--21.5) 405 (192--1982) 85 (26--181)
*Stage*
I 448 (310--653) 2.05 (0.21--8.91) 301 (194--472) 94 (55--134)
II 406 (236--915) 1.56 (0.22--19.05) 338 (193--665) 86 (29--162)
III 650 (360--1331) 2.35 (0.62--21.5) 299 (126--571) 132 (74--229)
IV 555 (270--4030) 1.42 (0.05--11.65) 390 (192--1982) 105 (26--411)
A 454 (236--1311) 1.66 (0.06--21.5) 325 (192--580) 112 (47--411)
B 605 (288--4030) 1.64 (0.06--19.05) 386 (126--1982) 88 (26--229)
Abbreviations: CK18=cytokeratin 18; LDH=lactate dehydrogenase; nDNA=nucleosomal DNA.
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This invention relates to systems and methods for treatment of cancer, and particularly to the use of hyperthermia in cancer treatment.
Present modalities for treatment of malignant tumors include surgery, radiation therapy, chemotherapy, and immunotherapy which apply a physical or chemical force to alter the biological function of a tumor in order to affect its viability. Despite the medical advances that these modalities represent, most solid cancerous tumors carry with them a very poor prognosis for survival. Quality of life during and after treatment for survivors is often poor. The dismal prognosis for malignant solid tumors has led to continuing research for more effective treatment modalities with a lesser degree of disability and fewer side effects. In vitro and in vivo evidence indicates hyperthermia produces a significant anti-cancer activity through alteration of the physical environment of the tumor caused by increasing the temperature. Hyperthermia is more cytotoxic to tumor cells than normal cells because cancer cells are oxygen deprived, nutritionally deficient, and low in pH making them incapable of tolerating the stress imposed by elevated temperature. Tumor vasculature is immature, lacking the smooth muscle and vasoactivity which allows mature vessels to dilate, increasing blood flow to carry away heat, therefore intratumor temperatures exceed those in normal tissue. The mechanisms of selective cancer cell eradication by hyperthermia is not completely understood. However, four cellular effects of hyperthermia on cancerous tissue have been proposed: 1) changes in cell or nuclear membrane permeability or fluidity, 2) cytoplasmic lysomal disintegration, causing release of digestive enzymes, 3) protein thermal damage affecting cell respiration and the synthesis of DNA and RNA and 4) potential excitation of immunologic systems.
The major forms of energy for generating hyperthermia to date include microwaves, radio frequency induction, radio frequency localized current, and ultrasound. Most of the techniques used to dispense these are non-invasive, i.e,. the heat generating source is external to the body and does not invade the body. Consequently, the energy must pass through the skin surface and substantial power absorption by normal peripheral body tissue is unavoidable. Currently available external heating sources result in nonuniform temperature profiles throughout the tumor and increased temperatures in normal tissue. It is desirable to selectively heat tissues deep in a patient's body, i.e., to heat the tumor mass without heating cutaneous and normal tissue.
Others have attempted the use of interstitial techniques to obtain local hyperthermia, with limited success. Interstitial heating of brain tumors through an implantable microwave antenna has been investigated. However, microwave probes are ineffective in producing precisely controlled heating of tumors. Temperatures may deviate as much as 10 degrees Celcius from the desired target temperature. Besides, microwave activity adversely affects cellular structures and their integration, regardless of other thermal effects. The result is nonuniform temperatures throughout the tumor. Studies indicate that tumor mass reduction by hyperthermia is related to the thermal dose. Thermal dose is the minimum effective temperature applied throughout the tumor mass for a defined period of time. Hot spots and cold spots which occur with microwave hyperthermia may cause increased cell death at the hot spot, but ineffective treatment at cold spots resulting in future tumor growth. Such variations are a result of the microwave antenna's inability to evenly deposit energy throughout the tissue.
Since efferent blood flow is the major mechanism of heat loss for tumors being heated and blood flow varies throughout the tumor, more even heating of tumor tissue is needed to ensure more effective treatment.
To be effective, the application and deposition of thermal energy to the tumor must be precisely controlled to compensate for the variations in blood flow. In addition, the therapy itself will perturb the tumor's vascular system during treatment causing variations in local perfusion around the probe. Thus, heat loss from a tumor will be time dependent and affected by the hyperthermia treatment. This demonstrates the need to both monitor and control the temperature of the tumor throughout treatment.
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by Déborah Mesquita
Big Picture Machine Learning: Classifying Text with Neural Networks and TensorFlow
Developers often say that if you want to get started with machine learning, you should first learn how the algorithms work. But my experience shows otherwise.
I say you should first be able to see the big picture: how the applications work. Once you understand this, it becomes much easier to dive in deep and explore the inner workings of the algorithms.
So how do you develop an intuition and achieve this big-picture understanding of machine learning? A good way to do this is by creating machine learning models.
Assuming you still don’t know how to create all these algorithms from scratch, you’ll want to use a library that has all these algorithms already implemented for you. And that library is TensorFlow.
In this article, we’ll create a machine learning model to classify texts into categories. We’ll cover the following topics:
How TensorFlow works What is a machine learning model What is a Neural Network How the Neural Network learns How to manipulate data and pass it to the Neural Network inputs How to run the model and get the prediction results
You will probably learn a lot of new things, so let’s start! ?
TensorFlow
TensorFlow is an open-source library for machine learning, first created by Google. The name of the library help us understand how we work with it: tensors are multidimensional arrays that flow through the nodes of a graph.
tf.Graph
Every computation in TensorFlow is represented as a dataflow graph. This graph has two elements:
a set of tf.Operation , that represents units of computation
, that represents units of computation a set of tf.Tensor , that represents units of data
To see how all this works you will create this dataflow graph:
A graph that computes x+y
You’ll define x = [1,3,6] and y = [1,1,1] . As the graph works with tf.Tensor to represent units of data, you will create constant tensors:
import tensorflow as tf
x = tf.constant([1,3,6]) y = tf.constant([1,1,1])
Now you’ll define the operation unit:
import tensorflow as tf
x = tf.constant([1,3,6]) y = tf.constant([1,1,1])
op = tf.add(x,y)
You have all the graph elements. Now you need to build the graph:
import tensorflow as tf
my_graph = tf.Graph()
with my_graph.as_default(): x = tf.constant([1,3,6]) y = tf.constant([1,1,1])
op = tf.add(x,y)
This is how the TensorFlow workflow works: you first create a graph, and only then can you make the computations (really ‘running’ the graph nodes with operations). To run the graph you’ll need to create a tf.Session .
tf.Session
A tf.Session object encapsulates the environment in which Operation objects are executed, and Tensor objects are evaluated (from the docs). To do that, we need to define which graph will be used in the Session:
import tensorflow as tf
my_graph = tf.Graph()
with tf.Session(graph=my_graph) as sess: x = tf.constant([1,3,6]) y = tf.constant([1,1,1])
op = tf.add(x,y)
To execute the operations, you’ll use the method tf.Session.run() . This method executes one ‘step’ of the TensorFlow computation, by running the necessary graph fragment to execute every Operation objects and evaluate every Tensor passed in the argument fetches . In your case you will run a step of the sum operations:
import tensorflow as tf
my_graph = tf.Graph()
with tf.Session(graph=my_graph) as sess: x = tf.constant([1,3,6]) y = tf.constant([1,1,1])
op = tf.add(x,y) result = sess.run(fetches=op) print(result)
>>>; [2 4 7]
A Predictive Model
Now that you know how TensorFlow works, you have to learn how to create a predictive model. In short,
Machine learning algorithm + data = predictive model
The process to construct a model is like this:
The process to create a predictive model
As you can see, the model consists of a machine learning algorithm ‘trained’ with data. When you have the model you will get results like this:
Prediction workflow
The goal of the model you will create is to classify texts in categories, we define that:
input: text, result: category
We have a training dataset with all the texts labeled (every text has a label indicating to which category it belongs). In machine learning this type of task is denominated Supervised learning.
“We know the correct answers. The algorithm iteratively makes predictions on the training data and is corrected by the teacher.” — Jason Brownlee
You’ll classify data into categories, so it’s also a Classification task.
To create the model, we’re going to use Neural Networks.
Neural Networks
A neural network is a computational model (a way to describe a system using mathematical language and mathematical concepts). These systems are self-learning and trained, rather than explicitly programmed.
Neural networks are inspired by our central nervous system. They have connected nodes that are similar to our neurons.
A neural network
The Perceptron was the first neural network algorithm. This article explains really well the inner working of a perceptron (the “Inside an artificial neuron” animation is fantastic).
To understand how a neural network works we will actually build a neural network architecture with TensorFlow. This architecture was used by Aymeric Damien in this example.
Neural Network architecture
The neural network will have 2 hidden layers (you have to choose how many hidden layers the network will have, is part of the architecture design). The job of each hidden layer is to transform the inputs into something that the output layer can use.
Hidden layer 1
Input layer and 1st hidden layer
You also need to define how many nodes the 1st hidden layer will have. These nodes are also called features or neurons, and in the image above they are represented by each circle.
In the input layer every node corresponds to a word of the dataset (we will see how this works later).
As explained here, each node (neuron) is multiplied by a weight. Every node has a weight value, and during the training phase the neural network adjusts these values in order to produce a correct output (wait, we will learn more about this in a minute).
In addition to multiplying each input node by a weight, the network also adds a bias (role of bias in neural networks).
In your architecture after multiplying the inputs by the weights and sum the values to the bias, the data also pass by an activation function. This activation function defines the final output of each node. An analogy: imagine that each node is a lamp, the activation function tells if the lamp will light or not.
There are many types of activation functions. You will use the rectified linear unit (ReLu). This function is defined this way:
f(x) = max(0,x) [the output is x or 0 (zero), whichever is larger]
Examples: ifx = -1, then f(x) = 0(zero); if x = 0.7, then f(x) = 0.7.
Hidden layer 2
The 2nd hidden layer does exactly what the 1st hidden layer does, but now the input of the 2nd hidden layer is the output of the 1st one.
1st and 2nd hidden layers
Output layer
And we finally got to the last layer, the output layer. You will use the one-hot encoding to get the results of this layer. In this encoding only one bit has the value 1 and all the other ones got a zero value. For example, if we want to encode three categories (sports, space and computer graphics):
+-------------------+-----------+| category | value |+-------------------|-----------+| sports | 001 || space | 010 || computer graphics | 100 ||-------------------|-----------|
So the number of output nodes is the number of classes of the input dataset.
The output layer values are also multiplied by the weights and we also add the bias, but now the activation function is different.
You want to label each text with a category, and these categories are mutually exclusive (a text doesn’t belong to two categories at the same time). To consider this, instead of using the ReLu activation function you will use the Softmax function. This function transforms the output of each unity to a value between 0 and 1 and also makes sure that the sum of all units equals 1. This way the output will tell us the probability of each text for each category.
| 1.2 0.46|| 0.9 -> [softmax] -> 0.34|| 0.4 0.20|
And now you have the data flow graph of your neural network. Translating everything we saw so far into code, the result is:
# Network Parametersn_hidden_1 = 10 # 1st layer number of featuresn_hidden_2 = 5 # 2nd layer number of featuresn_input = total_words # Words in vocabn_classes = 3 # Categories: graphics, space and baseball
def multilayer_perceptron(input_tensor, weights, biases): layer_1_multiplication = tf.matmul(input_tensor, weights['h1']) layer_1_addition = tf.add(layer_1_multiplication, biases['b1']) layer_1_activation = tf.nn.relu(layer_1_addition)
# Hidden layer with RELU activation layer_2_multiplication = tf.matmul(layer_1_activation, weights['h2']) layer_2_addition = tf.add(layer_2_multiplication, biases['b2']) layer_2_activation = tf.nn.relu(layer_2_addition)
# Output layer with linear activation out_layer_multiplication = tf.matmul(layer_2_activation, weights['out']) out_layer_addition = out_layer_multiplication + biases['out']
return out_layer_addition
(We’ll talk about the code for the output layer activation function later.)
How the neural network learns
As we saw earlier the weight values are updated while the network is trained. Now we will see how this happens in the TensorFlow environment.
tf.Variable
The weights and biases are stored in variables ( tf.Variable ). These variables maintain state in the graph across calls to run() . In machine learning we usually start the weight and bias values through a normal distribution.
weights = { 'h1': tf.Variable(tf.random_normal([n_input, n_hidden_1])), 'h2': tf.Variable(tf.random_normal([n_hidden_1, n_hidden_2])), 'out': tf.Variable(tf.random_normal([n_hidden_2, n_classes]))}biases = { 'b1': tf.Variable(tf.random_normal([n_hidden_1])), 'b2': tf.Variable(tf.random_normal([n_hidden_2])), 'out': tf.Variable(tf.random_normal([n_classes]))}
When we run the network for the first time (that is, the weight values are the ones defined by the normal distribution):
input values: xweights: wbias: boutput values: z
expected values: expected
To know if the network is learning or not, you need to compare the output values (z) with the expected values (expected). And how do we compute this difference (loss)? There are many methods to do that. Because we are working with a classification task, the best measure for the loss is the cross-entropy error.
James D. McCaffrey wrote a brilliant explanation about why this is the best method for this kind of task.
With TensorFlow you will compute the cross-entropy error using the tf.nn.softmax_cross_entropy_with_logits() method (here is the softmax activation function) and calculate the mean error ( tf.reduce_mean() ).
# Construct modelprediction = multilayer_perceptron(input_tensor, weights, biases)
# Define lossentropy_loss = tf.nn.softmax_cross_entropy_with_logits(logits=prediction, labels=output_tensor)loss = tf.reduce_mean(entropy_loss)
You want to find the best values for the weights and biases in order to minimize the output error (the difference between the value we got and the correct value). To do that you will use the gradient descent method. To be more specific, you will use the stochastic gradient descent.
There are also many algorithms to compute the gradient descent, you will use the Adaptive Moment Estimation (Adam). To use this algorithm in TensorFlow you need to pass the learning_rate value, that determines the incremental steps of the values to find the best weight values.
The method tf.train.AdamOptimizer(learning_rate).minimize(loss) is a syntactic sugar that does two things:
compute_gradients(loss, <list of variables>) apply_gradients(<list of variables>)
The method updates all the tf.Variables with the new values, so we don’t need to pass the list of variables. And now you have the code to train the network:
learning_rate = 0.001
# Construct modelprediction = multilayer_perceptron(input_tensor, weights, biases)
# Define lossentropy_loss = tf.nn.softmax_cross_entropy_with_logits(logits=prediction, labels=output_tensor)loss = tf.reduce_mean(entropy_loss)
optimizer = tf.train.AdamOptimizer(learning_rate=learning_rate).minimize(loss)
Data manipulation
The dataset you will use has many texts in English and we need to manipulate this data to pass it to the neural network. To do that you will do two things:
Create an index for each word Create a matrix for each text, where the values are 1 if a word is in the text and 0 if not
Let’s see the code to understand this process:
import numpy as np #numpy is a package for scientific computingfrom collections import Counter
vocab = Counter()
text = "Hi from Brazil"
#Get all wordsfor word in text.split(' '): vocab[word]+=1 #Convert words to indexesdef get_word_2_index(vocab): word2index = {} for i,word in enumerate(vocab): word2index[word] = i return word2index
#Now we have an indexword2index = get_word_2_index(vocab)
total_words = len(vocab)
#This is how we create a numpy array (our matrix)matrix = np.zeros((total_words),dtype=float)
#Now we fill the valuesfor word in text.split(): matrix[word2index[word]] += 1
print(matrix)
>>> [ 1. 1. 1.]
In the example above the text was ‘Hi from Brazil’ and the matrix was [ 1. 1. 1.]. What if the text was only ‘Hi’?
matrix = np.zeros((total_words),dtype=float)
text = "Hi"
for word in text.split(): matrix[word2index[word.lower()]] += 1
print(matrix)
>>> [ 1. 0. 0.]
You will to the same with the labels (categories of the texts), but now you will use the one-hot encoding:
y = np.zeros((3),dtype=float)
if category == 0: y[0] = 1. # [ 1. 0. 0.]elif category == 1: y[1] = 1. # [ 0. 1. 0.]else: y[2] = 1. # [ 0. 0. 1.]
Running the graph and getting the results
Now comes the best part: getting the results from the model. First let’s take a closer look at the input dataset.
The dataset
You will use the 20 Newsgroups, a dataset with 18.000 posts about 20 topics. To load this dataset you will use the scikit-learn library. We will use only 3 categories: comp.graphics, sci.space and rec.sport.baseball. The scikit-learn has two subsets: one for training and one for testing. The recommendation is that you should never look at the test data, because this can interfere in your choices while creating the model. You don’t want to create a model to predict this specific test data, you want to create a model with a good generalization.
This is how you will load the datasets:
from sklearn.datasets import fetch_20newsgroups
categories = ["comp.graphics","sci.space","rec.sport.baseball"]
newsgroups_train = fetch_20newsgroups(subset='train', categories=categories)newsgroups_test = fetch_20newsgroups(subset='test', categories=categories)
Training the model
In the neural network terminology, one epoch = one forward pass (getting the output values) and one backward pass (updating the weights) of all the training examples.
Remember the tf.Session.run() method? Let’s take a closer look at it:
tf.Session.run(fetches, feed_dict=None, options=None, run_metadata=None)
In the dataflow graph of the beginning of this article you used the sum operation, but we can also pass a list of things to run. In this neural network run you will pass two things: the loss calculation and the optimization step.
The feed_dict parameter is where we pass the data for each run step. To pass this data we need to define tf.placeholders (to feed the feed_dict ).
As the TensorFlow documentation says:
“A placeholder exists solely to serve as the target of feeds. It is not initialized and contains no data.” — Source
So you will define your placeholders like this:
n_input = total_words # Words in vocabn_classes = 3 # Categories: graphics, sci.space and baseball
input_tensor = tf.placeholder(tf.float32,[None, n_input],name="input")output_tensor = tf.placeholder(tf.float32,[None, n_classes],name="output")
You will separate the training data in batches:
“If you use placeholders for feeding input, you can specify a variable batch dimension by creating the placeholder with tf.placeholder(…, shape=[None, …]). The None element of the shape corresponds to a variable-sized dimension.” — Source
We will feed the dict with a larger batch while testing the model, that’s why you need to the define a variable batch dimension.
The get_batches() function gives us the number of texts with the size of the batch. And now we can run the model:
training_epochs = 10
# Launch the graphwith tf.Session() as sess: sess.run(init) #inits the variables (normal distribution, remember?)
# Training cycle for epoch in range(training_epochs): avg_cost = 0. total_batch = int(len(newsgroups_train.data)/batch_size) # Loop over all batches for i in range(total_batch): batch_x,batch_y = get_batch(newsgroups_train,i,batch_size) # Run optimization op (backprop) and cost op (to get loss value) c,_ = sess.run([loss,optimizer], feed_dict={input_tensor: batch_x, output_tensor:batch_y})
Now you have the model, trained. To test it, you’ll also need to create graph elements. We’ll measure the accuracy of the model, so you need to get the index of the predicted value and the index of the correct value (because we are using the one-hot encoding), check is they are equal and calculate the mean to all the test dataset:
# Test model index_prediction = tf.argmax(prediction, 1) index_correct = tf.argmax(output_tensor, 1) correct_prediction = tf.equal(index_prediction, index_correct)
# Calculate accuracy accuracy = tf.reduce_mean(tf.cast(correct_prediction, "float")) total_test_data = len(newsgroups_test.target) batch_x_test,batch_y_test = get_batch(newsgroups_test,0,total_test_data) print("Accuracy:", accuracy.eval({input_tensor: batch_x_test, output_tensor: batch_y_test}))
>>> Epoch: 0001 loss= 1133.908114347 Epoch: 0002 loss= 329.093700409 Epoch: 0003 loss= 111.876660109 Epoch: 0004 loss= 72.552971845 Epoch: 0005 loss= 16.673050320 Epoch: 0006 loss= 16.481995190 Epoch: 0007 loss= 4.848220565 Epoch: 0008 loss= 0.759822878 Epoch: 0009 loss= 0.000000000 Epoch: 0010 loss= 0.079848485 Optimization Finished!
Accuracy: 0.75
And that’s it! You created a model using a neural network to classify texts into categories. Congratulations! ?
You can see the notebook with the final code here.
Tip: modify the values we defined to see how the changes affect the training time and the model accuracy.
Any questions or suggestions? Leave them in the comments. Oh, and thank’s for reading! ? ✌?
Did you found this article helpful? I try my best to write a deep dive article each month, you can receive an email when I publish a new one.
It would mean a lot if you click the ? and share with friends. Follow me for more articles about Data Science and Machine Learning.
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Q:
c# class written in simple terms for wpf mvvm
I've come across an example tutorial online and saw a property for a class written like this for a wpf project. I was wondering what this would translate to in simpler terms since i've never seen a class written like this before.
Is the translation i attempted correct? Meaning is it doing the same thing the tutorial class is doing?
Tutorial Class
public ObservableCollection<string> Directories
{
get { return Get<ObservableCollection<string>>(); }
set { Set(value); }
}
My attempted guess at what it would be written as
private ObservableCollection<string> _directories;
public ObservableCollection<string> Directories
{
get
{
return this._directories;
}
set
{
this._directories= value;
}
}
A:
We'd have to see the Get/Set implementations to answer this question properly but my guess is it's been done to improve code readability. There are two things you often see in MVVM properties: auto-initialization in the getter and property change notification in the setter...
private ObservableCollection<string> _directories;
public ObservableCollection<string> Directories
{
get
{
// auto-initialize
if (this._directories == null)
this._directories = new ObservableCollection<string>();
return this._directories;
}
set
{
this._directories = value;
RaisePropertyChanged("Directories"); // INPC
}
}
This leads to a lot of code bloat if you're doing it for a large number of properties. My guess is the Set and Get methods in your tutorial class are probably being used to implement these features automatically, or at least something similar. The following code would provide that functionality for properties such as the one you posted:
private Dictionary<string, object> Properties = new Dictionary<string, object>();
public T Get<T>([CallerMemberName] string propertyName = null)
{
if (!Properties.ContainsKey(propertyName))
Properties[propertyName] = default(T);
return (T)Properties[propertyName];
}
public void Set<T>(T value, [CallerMemberName] string propertyName = null)
{
Properties[propertyName] = value;
RaisePropertyChanged(propertyName);
}
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Pain outcomes in patients with advanced breast cancer and bone metastases: results from a randomized, double-blind study of denosumab and zoledronic acid.
In this study, the authors evaluated the effect of denosumab versus zoledronic acid (ZA) on pain in patients with advanced breast cancer and bone metastases. The prevention of pain, reduction in pain interference with daily life activities, and the proportion of patients requiring strong opioid analgesics were assessed in a randomized, double-blind, double-dummy phase 3 study comparing denosumab with ZA for preventing skeletal-related events in 2046 patients who had breast cancer and bone metastases. Patients completed the Brief Pain Inventory-Short Form at baseline and monthly thereafter. Fewer patients who received denosumab reported a clinically meaningful worsening of pain severity (≥2-point increase) from baseline compared with patients who received ZA, and a trend was observed toward delayed time to pain worsening with denosumab versus ZA (denosumab, 8.5 months; ZA, 7.4 months; P = .08). In patients who had no/mild pain at baseline, a 4-month delay in progression to moderate/severe pain was observed with denosumab compared with ZA (9.7 months vs 5.8 months; P = .002). Denosumab delayed the time to increased pain interference by approximately 1 month compared with ZA (denosumab, 16.0 months; ZA, 14.9 months; P = .09). The time to pain improvement (P = .72) and the time to decreased pain interference (P = .92) were similar between the groups. Fewer denosumab-treated patients reported increased analgesic use from no/low use at baseline to strong opioid use. Denosumab demonstrated improved pain prevention and comparable pain palliation compared with ZA. In addition, fewer denosumab-treated patients shifted to strong opioid analgesic use.
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Starbreeze announces asymmetrical horror game Dead by Daylight
Brothers: A Tale of Two Sons developer and Payday 2 publisher Starbreeze is partnering with developer Behaviour Interactive (Naughty Bear, Wet) to make an asymmetrical competitive horror game called Dead By Daylight.
The premise pits four survivors against one killer in a game of cat and mouse. On paper it sounds very similar to the recently Kickstarted Friday the 13th game in which a player assumed the role of Jason Voorhees attempting to slay seven camp counselors. The difference with Dead by Daylight is that the killer plays from first-person, while the survivors have a third-person camera granting them more environmental awareness.
The killer will be more powerful, however, with special abilities like swifter movement. They won't all be teen horror slashers either, as some monsters will be of a more paranormal nature.
Stages will be procedurally-generated too, so you can't rely on the regular camping spots as you must scour the scenery for items to craft and traps to set.
It's certainly a promising premise and while Behaviour's extensive track record is hit-or-miss, Starbreeze is fairly exacting in what projects it chooses to pick up. Definitely one to keep an eye on anyway.
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Peebles to Build Hotel at 5th and I Streets
WI Web Staff |
5/11/2014, 11:31 p.m.
R. Donahue “Don” Peebles, a multi-millionaire developer of luxury hotels, high residential and commercial projects in Miami Beach, Florida, Las Vegas, New York City and Philadelphia, who got his first deal in the District, has been selected by The Walker Group to redevelop a property at 5th and Eye streets in Northwest into an upscale hotel and residences.
Peebles, who’s at the helm of the largest black-owned real estate development company in the country, will also work with WDG Architecture of Northwest Washington, the California-based MacFarlane Partners which is led by Victor MacFarlane -- a wealthy African American commercial real estate developer -- and Standard Group of Alexandria, Virginia, to build a 13-story hotel and 59 market-rate residences and 100 affordable housing units. The project is expected to hundreds of jobs and more than $8 million in new revenue for the District.
Peebles, a District native who’s been listed as one of the 10 wealthiest black Americans, is happy that the project is taking shape.
"We are humbled by all of the community support and look forward to bringing to District residents culturally and economically transformative project, one that will bring tourism and revenue to the city," said Peebles, 54, owner of the Courtyard by Marriott-Convention Center in Northwest.
D.C. Mayor Vincent Gray (D) praised the Peebles project.
"The Peebles Corporation and Walker Group will transform this valuable site into a thriving and productive asset that will serve the District of Columbia and beyond," Gray said.
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Adding Content to your web content
A web page made victimization HTML includes a basic and fundamental structure. The Software Education HTML Page continuously begins with the beginning tag of the HTML component and continuously terminates with the tip tag of the HTML component as follows:Thecomponent primarily tells your laptop that this is often an HTML document. All different component tags are 'nested' among the beginning and finish HTML tags. the net page is then more divided into 2 main sections that are the 'head' and therefore the 'body'.The head section start with the
HTML document
...
begin tag and before the
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Q:
JSON.parse in Wordpress Backend returns error
i have a dropdown list of WordPress pages with the class of 'page_id' (they repeat and can be added.. hence the livequery). on the change event i want to send an ajax request to pull out the title, excerpt and thumbnail for the page selected. at the moment I am just trying to get ANY kind of valid json response. i am using json_encode in my callback to return a json object back to my .ajax function. and then in the .ajax success function I am trying to parse this json object into something usable, but get an error every time instead.
the error seems to be a problem with JSON.parse:
JSON.parse
success(response="{"title":"bacon title","message":"bacon text"}0")
handleError(a=Object { url="http://localhost/single/wp-admin/admin-ajax.php", global=true, more...}, b=XMLHttpRequest { onreadystatechange=[xpconnect wrapped nsIDOMEventListener], readyState=4, more...}, d="success", e="{"title":"bacon title","message":"bacon text"}0") onreadystatechange(m=readystatechange )
[Break On This Error] var json = JSON.parse(response);
firebug console says the response looks like:
{"title":"bacon title","message":"bacon text"}0
i don't know if that extra 0 is the trouble-maker, but i also don't know how it is getting there. chrome reports the error as:
Uncaught SyntaxError: Unexpected number
$.livequery.$.change.$.ajax.success/single/wp-admin/post.php?post=2&action=edit&message=1:917
here is my jquery function:
<script type="text/javascript">
//<![CDATA[
jQuery(function($) {
$('.page_id').livequery(function(){
var loading = $(this).next('img.ajax-loader');
$(this).change( function() {
var value = $(this).val();
if (value.length) {
$(loading).fadeIn();
var data = {
action: 'featured_meta_action',
data: value,
security: '<?php echo wp_create_nonce('featured-ajax-nonce'); ?>',
};
$.ajax({
type: "POST",
data: data,
url: ajaxurl,
complete: function(){
$(loading).fadeOut();
},
success: function(response){
var str = '{ "title": "bar" }';
var json = JSON.parse(response);
alert(json['title']);
},
error: function(){
alert('fail');
}
});
}//end if
}); //end change
}); //end livequery
}); //end ready
/* ]]> */
</script>
my php callback looks like:
function featured_meta_action_callback(){
$responseVar = array(
'title'=>'bacon title',
'message'=>'bacon text',
);
echo json_encode($responseVar);
}
add_action('wp_ajax_featured_meta_action', 'featured_meta_action_callback');
this has turned out to be way harder than it seems like it ought to be. i just need to get the data back from the PHP function so I can use it in the jquery. where am i going wrong?
A:
I think it could be this:
function featured_meta_action_callback(){
$responseVar = array(
'title'=>'bacon title',
'message'=>'bacon text' // <<< Here you do not need another comma
);
echo json_encode($responseVar);
}
See the comment within the array.
EDIT: Although unnecessary, in a test it doesn't output a 0 at the end of the JSON object if I leave it in there. So it's probable that it is coming from somewhere else (in other words, the WordPress code).
EDIT v2: Run these through Firebug's console. The second throws an error, but if you don't include the 0, it works just fine.
console.log(JSON.parse('{"title":"bacon title","message":"bacon text"}'));
console.log(JSON.parse('{"title":"bacon title","message":"bacon text"}0'));
EDIT v3: Ok, you apparently need to short circuit the rest of the WP process by calling exit/die at the end of the function, like so:
function featured_meta_action_callback(){
$responseVar = array(
'title'=>'bacon title',
'message'=>'bacon text' // <<< Here you do not need another comma
);
echo json_encode($responseVar);
exit;
}
Suggested here:
http://codex.wordpress.org/AJAX_in_Plugins#Ajax_on_the_Administration_Side
And here:
http://amiworks.co.in/talk/simplified-ajax-for-wordpress-plugin-developers-using-jquery/
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mit
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If the 2016 presidential election were held today, Hillary Clinton Hillary Diane Rodham ClintonJoe Biden looks to expand election battleground into Trump country Biden leads Trump by 12 points among Catholic voters: poll The Hill's Campaign Report: Biden goes on offense MORE would still lose the election.
Despite her continued effort to cast blame, Clinton’s loss should not have come as such a surprise. Her lack of a clear, focused agenda, distasteful attacks on Republicans, and apathy toward stagnating wages among middle class Americans all contributed to her defeat.
ADVERTISEMENT
The Washington Post
that "when you lose to somebody who has 40 percent popularity, you don't blame other things — Comey, Russia — you blame yourself."
Clinton should recognize that her time has passed. She will soon be 73 years old and her home state of New York has two others strong prospective 2020 candidates in Senator Kirsten Gillibrand Kirsten GillibrandSunday shows preview: Justice Ginsburg dies, sparking partisan battle over vacancy before election Suburban moms are going to decide the 2020 election Jon Stewart urges Congress to help veterans exposed to burn pits MORE and Governor Andrew Cuomo.
For these reasons among others, Hillary Clinton should not run again.
The Democratic Party must move on from the 2016 loss and find both a new strategy and a new leader.
To be sure, the party’s new narrative must move beyond resistance to President Trump Donald John TrumpBubba Wallace to be driver of Michael Jordan, Denny Hamlin NASCAR team Graham: GOP will confirm Trump's Supreme Court nominee before the election Southwest Airlines, unions call for six-month extension of government aid MORE and suspicion that the election was somehow stolen from Clinton.
In a poll conducted by The Washington Post earlier this month, only 37 percent of Americans said that the party “currently stands for something,” while 52 percent said it “just stands against Trump.”
Resistance has been an ineffective strategy for Democrats in special elections, and there is no indication that it will help win back seats during the 2018 midterm elections.
Instead of dwelling on the 2016 election, the Democrats need to push forward.
Rather than turning to Hillary Clinton for answers, Democrats can trust Special Counsel Robert Mueller, who is both qualified and has bipartisan support, to conduct a thorough investigation into the Trump campaign and its alleged collusion with Russian officials.
Through Mueller’s investigation, the American people will get the answers they deserve.
In the meantime, the constant speculation into these matters detracts from focusing on critical issues that hardworking Americans face daily.
The Democratic Party is finally seeing this, prompting the launch of “A Better Deal,” the party’s new policy agenda.
“A Better Deal’s” proposed mission is “to help build an America in which working people know that somebody has their back.”
House Minority Leader Nancy Pelosi, Schumer, and other top Democrats have rolled out this plan in an effort to present a strong and clear message to voters.
Schumer has described “A Better Deal” as “not about moving the party left or right,” nor “about appealing to one coalition or another.” Pelosi has followed by clarifying that the new focus “is not a course correction, but it’s a presentation correction.”
The Democrats are doing exactly what Hillary Clinton failed to do throughout her campaign. A change in message is imperative to the Democrats turning their failing strategy around.
In certain areas, such as the economy and job creation, election polls showed Clinton beating Trump with large margins. However, when she shifted her focus to unsubstantiated calls for “unity and opportunity,” she lost crucial support.
“Unity” did not bring the same sense of security as her economic plan. The shift in focus from a concrete plan to an abstract concept was lethal for the Clinton campaign.
With this “Better Deal,” the Democrats are being careful not to make the same mistake.
This agenda signifies a focus on policy and strategy.
As Representative Hakeem Jeffries of New York put it, “Republicans talk in headlines,” but under this new plan, “Democrats speak in fine print.”
“A Better Deal” has the potential to be exactly what the Democrats are looking for in their effort to convince the American people that they are the party of the working class. It will also remind the country that they are a party of substance, and not simply a resistance movement.
Democratic Senator Al Franken Alan (Al) Stuart FrankenGOP Senate candidate says Trump, Republicans will surprise in Minnesota Peterson faces fight of his career in deep-red Minnesota district Getting tight — the psychology of cancel culture MORE says, “we have to move on by proving we are the party that cares about a lot of the people who voted for Donald Trump.”
By having some of Congress’ most established Democrats finally admit to the party’s shortcomings in 2016, they are signaling that they are ready to move past some of last year’s deadweight in Democratic politics and pursue an agenda that benefits hardworking Americans.
Douglas E. Schoen (@DouglasESchoen) served as a pollster for President Bill Clinton. A longtime political consultant and pollster, he is also a Fox News contributor and the author of 11 books. His latest book is Putin’s Master Plan: To Destroy Europe, Divide NATO, and Restore Russian Power and Global Influence (Encounter, 2016).
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Synergistic effect of angiotensin II type-1 receptor 1166A/C with angiotensin-converting enzyme polymorphism on risk of acute myocardial infarction in north Indians.
This first study from north India investigated the synergistic effect of AT1R 1166A/C with the ACE I/D polymorphism on risk of acute myocardial infarction (AMI). Traditional coronary risk factors, ACE I/D and AT1R 1166A/C polymorphism were analyzed in 350 patients with AMI and 350 matched controls. In univariate analysis, hypertension (52.9% vs. 11.1%; OR=8.9; 95%CI 6.0-13.3), diabetes mellitus (16.0% vs. 0.6%; OR=33.1; 95%CI 8.0-137), smoking (43.7% vs. 20.9%; OR=2.9; 95%CI 2.1-4.1), family history of coronary artery disease (22.3% vs. 14.0%; OR=1.8; 95%CI 1.2-2.6), high body mass index (64.3% vs. 51.4%; OR=1.7; 95%CI 1.3-2.3), high waist-hip ratio (46.2% vs. 2.3%; OR=37; 95%CI 16-85.8) and AT1R 1166AC genotype (20.6% vs. 12%; OR=1.9; 95%CI 1.3-2.9) were associated with AMI. In multivariate analysis, all these factors were found to be independent risk predictors for AMI. Subjects carrying the AT1R 1166AC+CC and ACE ID+DD combined genotype showed a twofold increased association (OR=2.1; 95%CI 1.2-3.5) compared with the AT1R 1166AA-ACE II combined genotype. Patients who smoked and who carried the ACE ID+DD genotype had 2.4-fold (OR=2.4; 95%CI 1.5-3.8), and with the AT1R 1166AC+CC genotype had 15-fold (OR=14.9; 95%CI 5.2-42.8) increased risk of AMI compared with non-smoking non-carriers. The AT1R 1166A/C polymorphism has association with AMI among north Indian patients, particularly if integrated with ACE I/D polymorphism and smoking.
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Introduction {#s1}
============
Fear serves as an alert mechanism which is vital for survival, because adaptive fear reactions enable an individual to cope with survival threats by escaping or avoiding them. However, fear reactions can become maladaptive when they are no longer appropriate to the actual situation. The ability to readjust behavior is especially important in a rapidly changing environment. In anxiety disorders, this ability usually is impaired (e.g., Rauch et al., [@B52]; Schiller et al., [@B54]). The high prevalence of anxiety disorders (\~14% within 12 months; BGS98 2000) has led to extensive research in the field of fear and the neural systems involved in fear learning and its extinction.
Fear learning in animal and human research is mainly examined on the basis of Pavlovian fear conditioning (Pavlov, [@B42]). In this paradigm, an initially neutral conditioned stimulus (CS), such as a tone or a light, is paired with an aversive unconditioned stimulus (US), such as a shock. After several pairings, the CS is associated with the US and evokes a conditioned fear response (CR) on its own. In differential fear conditioning, there are two initially neutral stimuli. One of them, the CS+, is paired with the aversive US, while the other one, the CS−, is not. In this case, only the CS+ elicits a CR after several pairings with the US. Based on timing aspects of the CS--US pairing we have to distinguish two forms of classical fear conditioning: In delay conditioning, the US follows the CS with no temporal gap (e.g, either the US directly follows the CS or the CS and the US coterminate meaning that there is a overlap in time), while in trace conditioning there is a temporal gap, called the trace interval, between CS offset and US onset. In the latter case, a "memory trace" is necessary to learn the association between the CS and the US (Pavlov, [@B42]).
This small difference in timing was found to be associated with differences in the neural structures underlying the acquisition of fear. The most prominent structure reported to be crucial for fear learning is the amygdala. Information about the CS and the US is transmitted from sensory cortices via the thalamus to the amygdala, which controls the expression of the fear reaction via projections to the brainstem (e.g., LeDoux, [@B27]). Other areas found to be involved in fear acquisition in delay conditioning are the anterior cingulate cortex (ACC) and the insular cortex. Activation in the ACC has been linked to the anticipation of pain. For example, Büchel et al. ([@B9]) assumed that the ACC, together with activation of the anterior insula, might integrate nociceptive input with memory and therefore allows for appropriate responses to subsequent stimuli, as has been shown in pain studies (e.g., Coghill et al., [@B12]). The insula has been reported to be involved in conveying a cortical representation of fear to the amygdala (Phelps et al., [@B44]). Knight et al. ([@B24]) and Milad et al. ([@B32]) proposed that the ACC is involved in the expression of the fear response. It has often been reported that the hippocampus is involved in fear conditioning. The current opinion is, that contextual information, which can be of spatial or temporal nature, is represented in the hippocampus (O\'Keefe and Dostrovsky, [@B69]). Hippocampal activation has been shown to be crucial for trace conditioning since the trace interval requires the formation of a temporal context. This has been found both in fear conditioning (Büchel et al., [@B8]; Knight et al., [@B24]) as well as in eyeblink conditioning (Cheng et al., [@B10]). Also, Clark et al. ([@B11]) reported that trace eyeblink conditioning additionally depends on the hippocampus and the neocortex, whereas for delay eyeblink conditioning activations of the cerebellum and the brainstem are sufficient. Human delay fear conditioning has been reported to occur without explicit hippocampal activity, too (see e.g., LaBar et al., [@B25]; Phelps et al., [@B43]; Schiller et al., [@B53]). Taken together, we conclude that the main neural structures involved in delay fear conditioning are the amygdala, the insula, and the ACC (see review by Sehlmeyer et al., [@B56]). However, for both fear and eyeblink trace conditioning, declarative memory, which is associated with hippocampus activity, is formed additionally.
To understand the mechanisms of fear and the development and maintenance of anxiety disorders, it is not sufficient to study the acquisition of fear only. Inappropriate fear can also result from a deficit in extinction of the fear (Baas et al., [@B4]). Extinction occurs if the CS is presented several times without the US. Importantly, there is convincing evidence that extinction does not lead to forgetting or unlearning, but rather to the formation of a new memory inhibiting the acquired fear memory (Bouton, [@B6], [@B7]; Milad and Quirk, [@B31]; Myers and Davis, [@B40]; Quirk, [@B48]). To date, the neural structures involved in the extinction of fear are less understood than those involved in fear acquisition. The amygdala has been shown to play an important role in both acquisition and extinction. In addition, it is assumed that, as extinction, i.e., new learning, takes place, the prefrontal cortex inhibits the expression of conditioned fear (Quirk et al., [@B49]). Rodent models of extinction suggest that during extinction, an inhibitory memory trace between ventromedial prefrontal cortex (vmPFC) and amygdala is established (Sotres-Bayon et al., [@B58], [@B59]), by means of which the expression of fear is inhibited. The vmPFC activates GABAergic intercalated cells in the amygdala which in turn inhibit the central nucleus of the amygdala (Quirk et al., [@B49]; Sotres-Bayon et al., [@B59]). Evidence for this model has been provided by lesion studies (Morgan and LeDoux, [@B33]; Quirk et al., [@B50]; Morgan et al., [@B34]; Lebron et al., [@B26]). For example Morgan and LeDoux ([@B33]) showed that rats with lesions of the medial PFC were resistant to extinction learning in a delay fear conditioning paradigm. Human studies on the extinction of fear memory acquired through classical delay conditioning have confirmed the role of the amygdala and the vmPFC (Phelps et al., [@B43]; Milad et al., [@B32]). The insula and the ACC have also been shown to be involved in extinction learning in humans (Gottfried and Dolan, [@B18]; Phelps et al., [@B43]). Additionally, extinction learning was found to be highly context dependent. Kalisch et al. ([@B21]) for example showed that a network containing the vmPFC and the hippocampus is activated during context dependent recall of extinction memory.
The striatum seems to be involved in affective learning, too, more precisely in the processing of prediction errors which occur when the expected result does not match the actual result (e.g., Delgado et al., [@B15]). Besides clear evidence that the striatum is involved in appetitive conditioning (e.g., O\'Doherty et al., [@B41]), there is also growing evidence for its involvement in the processing of prediction errors in aversive conditioning such as classical fear conditioning (Jensen et al., [@B20]; Delgado et al., [@B15]). Importantly, the absence of the US during extinction resembles a positive prediction error, meaning that a negative outcome, which is expected, does not occur. Thus, the striatum has to be considered as an important region in the extinction of conditioned fear.
Imaging studies examining extinction so far focused on delay conditioning, and little is known about extinction following trace conditioning in humans. Since differences in involved brain regions have been found during acquisition of delay and trace fear conditioning (see discussion above) it is likely that different brain areas are involved during extinction, too. Many situations in real life, in which fear is acquired, are closer to the trace than the delay conditioning paradigm, since there is often a temporal gap between a CS and the predicted US. Given the relevance of fear extinction for the maintenance of anxiety disorders, it seems very important to detect possible differences in the neural networks involved in these different types of fear conditioning.
Based on prior results in humans, we expected that extinction of delay and trace fear conditioning is associated with activations of the amygdala and the vmPFC as well as involvement of the insular and the anterior cingulate cortices and the striatum. In contrast, we assumed differences between delay and trace conditioning regarding activations in the prefrontal cortex. One model of the functional organization of the lateral PFC postulates that the ventrolateral part of the PFC is mainly involved in the maintenance of information, such as retaining a sequence of letters in working memory, whereas the dorsal part is more important when it comes to manipulation of information, such as reordering the sequence into alphabetical order (D\'Esposito et al., [@B67]). There are also findings that nonhuman primates and humans with lesions of the dorsolateral PFC (dlPFC) are less able to adjust behavior appropriately in delayed response tasks (e.g., D\'Esposito et al., [@B14]). In these tasks it is necessary to hold information in working memory over a short temporal gap before making choices and decisions. This demand is similar to forming and changing a memory trace bridging the trace interval in trace fear conditioning. Therefore, in contrast to vmPFC contributions to the extinction of delay memory, the dlPFC might---exclusively or additionally---be involved in the extinction of trace memory. Evidence for the involvement of the dlPFC in trace eyeblink conditioning also comes from animal models. Weiss and Disterhoft ([@B64]) propose a neural network in which the dlPFC, together with prelimbic areas, orchestrates neural activity that bridges the trace interval. Finally, since the hippocampus is assumed to be involved in the formation of declarative memory in a conditioning process, we expected it to play a greater role in the extinction of trace memory compared to delay memory.
The present study used virtual reality (VR) to implement both the delay and the trace fear conditioning paradigm. VR is a powerful tool for studying fear reactions in ecologically valid environments (Mühlberger et al., [@B35],[@B38], [@B39],[@B36]). It has successfully been applied in treatment of specific phobias (Mühlberger et al., [@B37]) as well as in conditioning studies (Baas et al., [@B3], [@B4]; Alvarez et al., [@B1]; Glotzbach et al., [@B17]; Tröger et al., [@B63]). The use of VR allows for the full control of a fear conditioning situation that is closer to the complexity of learning situations in real life than most laboratory designs. The used virtual environment consisted of a corridor and an office, through which subjects were passively guided while lying in the scanner. In both the delay (DCG) and the trace conditioning group (TCG), a blue and a yellow light in the office served as CS+ and CS−, respectively, and a mildly painful electric stimulus as US. Besides ratings of valence, arousal, fear, and contingency assessed after acquisition and extinction phases, the BOLD response differences between the CS+ and CS− served as indices of brain responses related to learning. Specifically, differences between delay and trace fear conditioning during extinction were analyzed. Bold responses during acquisition were not analyzed because of an overlap of brain responses to the CS and US in the learning phase due to their temporal closeness.
Methods and materials {#s2}
=====================
Participants
------------
The final sample consisted of 26 participants, 13 in the DCG (5 male, 8 female, mean age = 23.1 years, *SD* = 3.0 years) and 13 in the TCG (4 male, 9 female, mean age = 23.5 years, *SD* = 2.5 years). All participants gave their written informed consent and received 12 €/h for participation. The study protocol was approved by the Ethics Committee of the Medical Faculty of the University of Würzburg.
To reach this sample, a total of 43 right-handed volunteers (14 male, 29 female; age 19--29) had to be recruited. They were randomly assigned to the DCG or the TCG. Excluding criteria were past or present psychiatric disorders, use of antipsychotic drugs, regular alcohol or drug consumption (one subject was excluded), and allochromasia (for blue and yellow) assessed by self-report. Twelve subjects had to be excluded due to technical problems, and one subject because of extensive head movements during scanning. Three participants did not explicitly learn the contingency between the CSs and the US (only DCG, see below). They were also excluded because the small group size did not allow for a separate analysis to investigate distinct neuronal patterns in aware vs. unaware participants.
Stimuli and apparatus
---------------------
### VR environment
For creating the virtual environment of the experiment we used the Source Engine SDK (Valve Corporation, Bellevue, Washington, USA). Some office models were used from the free Source Engine Modification "Weekday Warrior" (<http://www.moddb.com/mods/weekday-warrior>). The virtual environment consisted of an office and an associated corridor evenly illuminated in a neutral white light. In the office, a lamp situated in the middle of the room could be switched on and off. The color of the light illuminated by the lamp was either blue or yellow and served as CS+ or CS−, respectively (see Figure [1](#F1){ref-type="fig"}). If turned on, the lamp illuminated the whole room. One light (CS+) was followed by a mildly painful electric stimulus (US) with 100% contingency; the other light (CS−) was never followed by an US. Colors of CS+ and CS− were counterbalanced across participants and groups. In the delay conditioning paradigm, the lights were always switched on for 8 s, and the US was presented simultaneously with the offset of the CS+. In the trace paradigm, the lights were presented for 4 s, and the US was presented 8 s after CS+ onset; thus, the trace interval between CS and US lasted 4 s. To avoid movement in the scanner and enhance control over the course of events during the experiment, participants were guided through the VR environment on a prerecorded path. We used the in-house written VR simulation software CyberSession to manipulate the VR environment during the experiment (e.g., switching on the light, delivering the electrical pulse). VR rendering was done by an image generator running the in-house written Source SDK modification VRSessionMod 0.3. The virtual environment was displayed via MRI-compatible goggles (VisuaStim; Magnetic Resonance Technologies, Northridge, CA, USA).
{#F1}
### Electric stimuli
The US was a mildly painful electric stimulus generated by a current stimulator (Digitimer DS7A, Digitimer Ltd, Hertfordshire, England). It was delivered at the left index finger through surface bar electrodes consisting of two durable gold-pasted stainless steel disk electrodes with 9mm diameter, 30mm spacing, and with an impedance of max. 5 Ω. Electric stimuli associated with the CS+ were triggered automatically by CyberSession during conditioning for 200 ms with a frequency of 50 Hz. Current intensity was determined individually for each participant in the beginning of the experiment (for a detailed description of the adjustment of individual current intensity see Andreatta et al., [@B2]). It was adjusted at the individual pain threshold and increased by 30% to prevent habituation to the US. Both conditioning groups did not differ in current intensity (delay group: *M* = 2.25 mA, *SD* = 0.99; trace group: *M* = 2.18, *SD* = 0.90), *t*~(23)~ = 0.19, *p* = 0.853, and pain ratings (delay group: *M* = 5.00, *SD* = 0.84; trace group: *M* = 5.04, *SD* = 1.57), *t*~(23)~ = −0.08, *p* = 0.934, of the US.
Psychometric measures
---------------------
### Ratings
At several times during the experiment, ratings of valence (from "very negative" to "very positive"), arousal (from "not arousing at all" to "very arousing"), fear (from "no fear" to "extreme fear"), and CS--US contingency (from "not likely at all" to "very likely") were collected, each on scales from 0 to 100.
### Awareness
Explicit knowledge of contingencies between CS and US was assessed on the basis of the question "During which light presentation did you receive electric shocks?" Participants who were able to state the correct color of light after the second acquisition run were labeled "aware," the others were labeled "unaware." While 26 participants met these criteria of awareness; three participants in the DCG failed and were labeled "unaware." There were no unaware participants in the TCG.
Procedure
---------
After reading information about the scanning procedure, participants completed the questionnaires on personal information and excluding criteria. Then they received written instructions related to the experiment and gave their written informed consent. The electrode for electric stimulation was attached when participants were already positioned in the scanner room. After that, the individual pain threshold was determined and adjusted as explained earlier. This preparation phase was followed by a pre-acquisition block, in which participants were guided through the virtual office once to get used to the environment and the two different lights (each light was presented once). After that, participants were told that they would be able to predict the electric stimuli if they paid close attention to the experiment.
The following acquisition phase consisted of two blocks. Each block included two passages through the office. During one passage, participants were guided through the office once and were exposed to four CS+ and four CS−. The CS+ was always followed by the US. One room visit lasted 172 s; accordingly one block lasted approximately 6 min. During the complete acquisition participants were exposed to 16 CS+ and 16 CS− and received 16 US.
The extinction phase consisted of one block including two visits with the same duration and CS frequencies as the acquisition trials (i.e., 8 CS+ and 8 CS−). No US was applied during extinction.
Classification of lights as CS+ and CS− as well as order of stimuli was pseudo-randomized across participants. In total, there were four different courses of events, two of them with the blue light and two with the yellow light serving as CS+. The length of the interstimulus interval (ISI) was also pseudo-randomized and varied between 11 and 13 s in steps of 250ms.
Ratings
-------
After each of the two acquisition blocks, awareness was measured by posing free recall questions as described above. Participants rated screenshots of the room with either the CS+ or the CS− light switched on regarding valence, arousal and fear after pre-acquisition, each acquisition block and extinction. Additionally, after both acquisition phases and extinction, contingency of CS+ and CS− with the US was measured. For all ratings, questions and screenshots were presented via the goggles. Participants were told to relate their answers to the way they felt during the last phase of the experiment. Answers were given orally via the speaker system of the scanner room and recorded by the investigator.
Magnetic resonance imaging
--------------------------
A 1.5-T whole-body magnetic resonance tomograph (MagnetomAvanto, SiemensHealthcare, Erlangen, Germany) with standard 12-channel head coil and integrated head holder was used for acquisition of structural and functional brain images. Structural imaging consisted of 160 T1-weighted sagittal magnetization-prepared rapid gradient-echo imaging (MP-RAGE) 3D MRI sequence (MPRAGE, 1mm slice thickness, TR = 2250 ms, TE = 3.93 ms, flip angle: 8°, FOV: 256mm, matrix: 256 × 256, voxel size: 1 × 1 × 1mm^3^). The acquisition of structural images was situated at the end of the experiment.
Functional imaging was conducted in four phases (pre-acquisition, first and second acquisition phase and extinction). During subjective ratings after each of the experimental phases, imaging was intermitted. A total of 161 volumes was registered using a T^\*^2-weighted gradient echo-planar imaging sequence (EPI) with 25 axial slices \[slice thickness 5-mm with 1-mm gap, interleaved (descending) order\] covering the whole brain (TR: 2500 ms; TE: 40 ms; flip angle: 90°; FOV: 240 × 240 mm; matrix size: 64 × 64; voxel size: 3.1 × 3.1 × 3mm^3^) for functional imaging. Orientation of axial slices was parallel to the AC-PC line. The first 8 images of each phase were excluded from analysis to allow for T1 equilibration.
Image preprocessing and statistical analysis
--------------------------------------------
### Imaging
Analysis of fMRI data was performed with Statistical Parametric Mapping (SPM8, Wellcome Department of Cognitive Neurology, London) integraded in MatLab 7.0 (Mathworks Inc., Sherborn, MA). After slice time correction, functional images were realigned. T1-scans were coregistered to each participant\'s mean image of the realigned. The mean functional images were normalized to the Montreal Neurological Institute (MNI) single-subject template (Evans et al., [@B16]). Normalization parameters obtained from the previous segmentation procedure of coregistered T1 images were applied and images were resampled (voxel size 2 × 2 × 2mm^3^). Subsequently, EPI images were spatially smoothed with an 8-mm full-width-half-maximum (FWHM) Gaussian kernel and filtered with a 128 ms high pass filter.
The different experimental conditions were modeled using a boxcar reference vector convolved with a canonical hemodynamic response function (general linear model, Kiebel and Holmes, [@B22]). The six movement parameters of the rigid body transformation, applied by the realignment procedure, were included to regard variance caused by residual movement. Low-frequency signal drift was filtered using a first-order autoregressive model. Parameter estimates were subsequently calculated for each voxel using weighted least squares to provide maximum likelihood estimates based on the non-sphericity assumption in order to get identical and independently distributed error terms. Since we were especially interested in the activation related to the extinction of fear reactions, the extinction phase served as main test phase and the BOLD signal was calculated at the onset of the colored lights. In a conditioning paradigm with a CS--US contingency of 100% during acquisition we expected rapid decrease of fear reactions. To account for this, the extinction phase was divided into two parts of equal duration and the first and second half were analyzed separately (early and late extinction). In a second step we compared activation during early extinction (first to fourth CS+) with activation during late extinction (fifth to eighth CS+).
First level individual contrast images (CS+ \> CS−) were used in a second-level analysis (one sample *t*-test). Conditioning groups (delay and trace) were analyzed separately for the contrast CS+ \> CS−. We also analyzed the contrast early extinction (CS+ \> CS−) \> late extinction (CS+ \> CS−). ROI analyses were carried out for the amygdala, the hippocampus, the insula, the ACC (Brodmann areas 24, 32, and 33) the striatum (caudate and putamen) and the ventromedial (medial orbital frontal gyrus) and dorsolateral prefrontal cortices (middle frontal gyrus) at an uncorrected threshold of *p* = 0.005 with a minimum cluster size of ten voxel. Additionally, we conducted an explorative whole brain analysis which also included the cerebellum (*p* = 0.001, uncorrected, minimum cluster size of five voxel). ROIs were based on masks of the WFU Pick Atlas (Maldjian et al., [@B28]) and Brodmann Areas (BA).
### Ratings
For the ratings of valence, arousal, and fear, ANOVAs were conducted for pre-acquisition and extinction with the between factors stimulus (CS+, CS−) and group (delay, trace). Ratings during acquisition were analyzed with repeated measures ANOVAs with the between factors stimulus (CS+, CS−) and group (delay, trace) and the additional within factor phase (Acquisition 1, Acquisition 2). Contingency ratings were not collected after pre-acquisition and thus were only analyzed for acquisition and extinction.
All rating data were analyzed using SPSS for Windows (Release 17.0). Alpha was set at.05 for all statistical tests, effect sizes are reported as η^2^~*p*~ scores.
Results {#s3}
=======
Ratings
-------
### Pre-acquisition
As expected, after the pre-acquisition phase, CS+ and CS− did not differ in valence, arousal, or fear in any group (all *p*s \> 0.23).
### Acquisition
***Valence ratings***. For valence ratings, we found a significant main effect of stimulus, *F*~(1,\ 24)~ = 13.49, *p* = 0.001, η^2^~*p*~ = 0.360, as well as a marginally significant interaction of Phase × Stimulus, *F*~(1,\ 24)~ = 3.92, *p* = 0.059, η^2^~*p*~ = 0.140. The CS+ was rated overall more negative than the CS− (CS+: *M* = 36.83, *SD* = 20.49; CS−: *M* = 65.38, *SD* = 27.88). Significant effects involving the factor group were not detected.
***Arousal rating***. The analysis revealed a significant main effect of stimulus, *F*~(1,\ 24)~ = 33.05, *p* \< 0.001, η^2^~*p*~ = 0.579, and a significant three way interaction of Phase × Stimulus × Group, *F*~(1,\ 24)~ = 4.30, *p* = 0.049, η^2^~*p*~ = 0.152. After the first acquisition phase, the CS+ elicited more arousal than the CS− in both the delay group, *t*~(12)~ = 2.578, *p* = 0.024 (CS+: *M* = 40.38, *SD* = 25.70; CS−: *M* = 14.62, *SD* = 19.84), and the trace group, *t*~(12)~ = 5.41, *p* \< 0.001 (CS+: *M* = 51.15, *SD* = 28.88; CS−: *M* = 8.46, *SD* = 9.87). After the second phase, we found a similar pattern as after the first phase in the trace group, *t*~(12)~ = 4.38, *p* = 0.001 (CS+: *M* = 40.77, *SD* = 26.91; CS−: *M* = 6.92, *SD* = 9.47), while in the delay group the effect was more pronounced than after the first phase, *t*~(12)~ = 3.534, *p* = 0.004, (CS+: *M* = 43.46, *SD* = 27.03; CS−: *M* = 9.62, *SD* = 18.76).
***Fear ratings***. For fear ratings, we again found a significant main effect of stimulus, *F*~(1,\ 24)~ = 22.32, *p* \< 0.000, η^2^~*p*~ = 0.482, indicating that the CS+ elicited overall more fear than the CS− in both groups (CS+: *M* = 36.25, *SD* = 30.40; CS−: *M* = 5.58, *SD* = 11.57).
***Contingency ratings***. The CS+ was clearly perceived as more likely to be followed by an electric stimulus during acquisition, main effect of stimulus, *F*~(1,\ 24)~ = 201.45, *p* \< 0.001, η^2^~*p*~ = 0.894 (CS+: *M* = 88.65, *SD* = 18.72; CS−: *M* = 9.62, *SD* = 14.69). Additionally, we found a significant interaction of Phase × Stimulus, *F*~(1,\ 24)~ = 5.66, *p* = 0.026, η^2^~*p*~ = 0.191. Already after the first phase, the CS+ was rated as more likely to be followed by the US than the CS−, *t*~(25)~ = 6.681, *p* \< 0.001 (CS+: *M* = 81.73, *SD* = 29.29; CS−: *M* = 15.00, *SD* = 27.75), but after the second phase this difference between CS+ and CS− further increased, *t*~(25)~ = 26.239, *p* \< 0.001 (CS+: *M* = 95.58, *SD* = 14.45; CS−: *M* = 4.23, *SD* = 11.38). The contingency between the CS+ and the US was rated higher after the second than after the first phase, *t*~(25)~ = −2.612, *p* = 0.015.
### Extinction
***Valence ratings***. The CS+ and the CS− did no differ significantly in their valence after extinction, *F*~(1,\ 24)~ = 3.48, *p* = 0.075, η^2^~*p*~ = 0.127, although a marginal difference was still present.
***Arousal ratings***. The analysis revealed a significant main effect of stimulus, *F*~(1,\ 24)~ = 20.30, *p* \< 0.001, η^2^~*p*~ = 0.458, as well as a significant interaction of Stimulus × Group, *F*~(1,\ 24)~ = 5.26, *p* = 0.050, η^2^~*p*~ = 0.151. In the delay group, arousal ratings of CS+ and the CS− did not differ significantly. However, in the trace group, the CS+ was still rated as more arousing than the CS−, *t*~(12)~ = 4.368, *p* = 0.001 (CS+: *M* = 21.76, *SD* = 6.03; CS−: *M* = 10.05, *SD* = 2.91).
***Fear ratings***. The main effect of stimulus was still significant after the extinction phase, *F*~(1,\ 24)~ = 11.61, *p* = 0.002, η^2^~*p*~ = 0.326. We also found a marginal interaction of Stimulus x Group, *F*~(1,\ 24)~ = 3.76, *p* = 0.064, η^2^~*p*~ = 0.136, suggesting similar results as for arousal ratings: In the trace group, the CS+ was after extinction still associated with more fear than the CS−, *t*~(12)~ = 3.726, *p* = 0.003 (CS+: *M* = 25.77, *SD* = 21.39; CS−: *M* = 6.15, *SD* = 10.44), while there was no such difference in the delay group, *t*~(12)~ = 1.054, *p* = 0.313.
***Contingency ratings***. As for fear ratings, the main effect of stimulus persisted during the extinction phase, *F*~(1,\ 24)~ = 18.39, *p* \< 0.001, η^2^~*p*~ = 0.434. After extinction, the CS+ was still more associated with the US than the CS− (CS+: *M* = 47.31, *SD* = 35.98; CS−: *M* = 12.31, *SD* = 20.06).
In sum, arousal and fear ratings suggest that extinction proceeded more slowly in the trace group compared to the delay group. After extinction, the trace group rated the CS+ still as more arousing and more frightening than the CS−, whereas in the delay group these differences were no longer present after extinction.
Imaging data
------------
### Early extinction
***ROI analysis***. Regarding the contrast of CS+ minus CS−, both groups showed insular and striatal activation during early extinction. Interestingly, the two groups differed in their prefrontal activation (see Figure [2](#F2){ref-type="fig"}). In the DCG, we observed significant activation of the vmPFC (medial orbital frontal gyrus R), while in the TCG the dlPFC was significantly activated (middle frontal gyrus R). Additionally, the trace group showed activation of the dorsal part of the ACC (BA 33).
{#F2}
***Whole brain analysis***. In addition to the areas defined as ROIs, we also found significant activation in several other regions. In the DCG, the cuneus (L), the left motor cortex (precentral gyrus L), and the middle occipital gyrus (R) were activated. In the TCG, we found activations in the somatosensory cortex (postcentral gyrus L), the calcarine (R), the rolandic operculum (R), and the ventral ACC (middle cingulate cortex L, BA 24).
For exact coordinates see Table [1](#T1){ref-type="table"}.
######
**Significant activations revealed by whole brain (WB) and regions of interest (ROI) analysis for contrast CS+ \> CS− during early extinction**.
**Group** **Brain structure** ***x*** ***y*** ***z*** ***Z*** **Cluster size** ***P***
----------- -------------------------------------- --------- --------- --------- --------- ------------------ ---------
Delay Cuneus R (WB) 12 −76 24 3.7 29 \<0.001
Precentral gyrus L (WB) −22 −14 62 3.67 26 \<0.001
Caudate body L (WB) −18 20 8 3.53 7 \<0.001
Medial orbital frontal gyrus R (WB) 12 58 −12 3.43 5 \<0.001
Middle occipital gyrus L (WB) −34 −66 18 3.32 7 \<0.001
Insula R (ROI) 44 2 0 3.01 10 0.001
Caudate L (ROI) −18 20 8 3.53 17 \<0.001
Putamen R (ROI) 36 −12 −8 3.07 14 \<0.001
Medial orbital frontal gyrus R (ROI) 12 58 −12 3.43 11 \<0.001
Trace Postcentral gyrus L (WB) −42 −32 54 4.7 6 \<0.001
Rolandic operculum R (WB) 42 −22 26 4.37 86 \<0.001
Putamen L (WB) −30 −14 2 4.11 8 \<0.001
Calcarine R (WB) 12 −92 12 4.09 19 \<0.001
Middle frontal gyrus R (WB) 40 6 40 3.55 7 \<0.001
Ventral ACC L (WB) −12 10 30 3.55 8 \<0.001
Insula R (ROI) 36 −18 22 3.78 15 \<0.001
Dorsal ACC R (ROI) 4 22 34 2.93 11 0.002
Putamen L (ROI) −30 −14 2 4.11 14 \<0.001
Middle frontal gyrus R (ROI) 40 6 40 3.55 12 \<0.001
α \< 0.001 uncorrected for whole brain analysis (WB) and α \< 0.005, uncorrected ROI analyses, with a minimum cluster size of k = 5 (WB) or k = 10 (ROI); L, left; R, right hemisphere. The cluster with the largest number of significant voxels within each region is reported. Coordinates x, y, and z of the peak voxels are given in Montreal Neurological Institute space.
### Late extinction
***ROI analysis***. For the second phase of extinction, we observed significant activation in the ventral part of the ACC in the DCG only.
***Whole brain analysis***. In the DCG, the ventral ACC (R), the inferior frontal gyrus (R), and the supramarginal gyrus (R) were activated during late extinction. In the TCG however, we found significant activation of the precuneus (L and R).
For exact coordinates see Table [2](#T2){ref-type="table"}.
######
**Significant activations revealed by whole brain (WB) and regions of interest (ROI) analysis for contrast CS+ \> CS− during late extinction**.
**Group** **Brain structure** ***x*** ***y*** ***z*** ***Z*** **Cluster size** ***P***
----------- -------------------------------------------------- --------- --------- --------- --------- ------------------ ---------
Delay ventral ACC R (WB) 6 10 30 4.84 13 \<0.001
Triangular part of inferior frontal gyrus R (WB) 50 18 14 3.67 35 \<0.001
Supramarginal gyrus R (WB) 60 −34 28 3.66 25 \<0.001
Ventral ACC R (ROI) 6 10 30 4.84 17 \<0.001
Trace Precuneus R (WB) 14 −58 24 3.74 62 \<0.001
Precuneus L (WB) −10 −62 30 3.37 10 \<0.001
ROI analysis: no significant voxel
α \< 0.001 uncorrected for whole brain analysis (WB) and α \< 0.005, uncorrected ROI analyses, with a minimum cluster size of k = 5 (WB) or k = 10 (ROI); L, left; R, right hemisphere. The cluster with the largest number of significant voxels within each region is reported. Coordinates x, y, and z of the peak voxels are given in Montreal Neurological Institute space.
### Early extinction \> late extinction
***ROI analysis***. We also tested for areas, which showed stronger activation in the early extinction than in the late extinction. In the delay group, this was the case for the insula (L), whereas in the trace group the hippocampus (R) and the striatum (putamen L) showed greater activation in the first part of the extinction compared to the second part.
***Whole brain analysis***. Whole brain analysis revealed additional activation of the ACC (ventral anterior cingulate area), the precentral gyrus (L), and the transverse temporal gyrus (Heschl L) in the DCG. In the TCG, the ventral ACC (L) was activated as in the delay group, and additionally we found activation in the parahippocampal area.
For exact coordinates see Table [3](#T3){ref-type="table"}.
######
**Early extinction (CS+ \> CS−) \> late extinction (CS+ \> CS−): significant activations revealed by whole brain (WB) and regions of interest (ROI) analysis**.
**Group** **Brain structure** ***x*** ***y*** ***z*** ***Z*** **Cluster size** ***P***
----------- ------------------------- --------- --------- --------- --------- ------------------ ---------
Delay Precentral gyrus L (WB) −22 −14 62 4.10 59 \<0.001
Ventral ACC L (WB) −16 0 44 3.65 14 \<0.001
Heschl L (WB) −32 −28 16 3.29 5 0.001
Insula L (ROI) −38 −20 14 3.05 25 0.001
Trace Putamen L (WB) −30 −14 2 3.74 13 \<0.001
Ventral ACC L (WB) −10 14 30 3.61 10 \<0.001
Parahippocampus R (WB) 32 −34 −12 3.51 11 \<0.001
Hippocampus R (ROI) 30 −32 −8 3.87 11 \<0.001
Putamen L (ROI) −30 −14 2 3.74 23 \<0.001
α \< 0.001 uncorrected for whole brain analysis (WB) and α \< 0.005, uncorrected ROI analyses, with a minimum cluster size of k = 5 (WB) or k = 10 (ROI); L, left; R, right hemisphere. The cluster with the largest number of significant voxels within each region is reported. Coordinates x, y, and z of the peak voxels are given in Montreal Neurological Institute space.
Discussion {#s4}
==========
To our knowledge, this is the first study investigating neural substrates of extinction of both delay and trace fear memory in humans. In both conditioning groups we found activation in the insular cortex and the striatum during early extinction. Interestingly, extinction of delay and trace memory differed in prefrontal activation. The vmPFC was activated during extinction in the DCG, while the dlPFC was activated during extinction in the TCG. These results point to different PFC activity involved in early extinction of delay vs. trace fear conditioning. In the late part of the extinction process, the delay group only showed significant activation of the ventral ACC. No other activation could be found in our predefined regions of interest during the second half of extinction. However, when comparing the early with the late part of extinction, we found greater activation in the insula (delay group), the hippocampus, and the striatum (trace group) during early extinction.
Prefrontal cortex
-----------------
The most prominent result of our study is the dissociation of prefrontal activation in delay vs. trace conditioning during early extinction. As has been shown in previous human fear conditioning studies, the vmPFC plays an important role in the extinction of fear memory (e.g., Phelps et al., [@B43]). In accordance with findings from the animal model it is assumed that, during extinction, an inhibitory memory trace is formed between the vmPFC and the amygdala (Sotres-Bayon et al., [@B58], [@B59]), which allows for the modulation of the fear response. However, existing evidence from human studies for this model comes exclusively from delay fear conditioning. Significant activation of the vmPFC in the DCG of our study provides further evidence for this model. Milad et al. ([@B30]) indicated that the vmPFC is not only involved in extinction learning, but also in the retention of extinction memory. They reported a significant correlation between the thickness of the medial orbitofrontal cortex and skin conductance response (SCR) in extinction recall assessed one day after extinction training. More precisely, a thicker medial orbitofrontal cortex was associated with a lower SCR in extinction recall, that is, with greater extinction memory.
However, in the TCG, we did not find significant activation of the vmPFC, but instead of the dlPFC. This finding points to different processes during extinction in delay and trace conditioning. VmPFC activation in delay conditioning reflects the inhibition of the fear response already during early extinction. According to a model of functional organization of the lateral PFC, the vmPFC is mainly involved in the mere maintenance of information, whereas the dorsal part is assumed to be involved in the manipulation of information, requiring more working memory capacities (D\'Esposito et al., [@B67]; Postle et al., [@B47]). As mentioned in the introduction, lesion studies in nonhuman and human primates indicate that the dlPFC is important for adjusting behavior appropriately in delayed response tasks (e.g., D\'Esposito et al., [@B14]), in which information has to be kept in working memory for a short period of time before making choices and decisions on the basis of this information. This interpretation is in line with activation of the dlPFC during the extinction of trace conditioning. In contrast to delay conditioning, trace conditioning and its extinction afford higher working memory contribution to bridge the trace interval and hold information in short term memory. Results of ratings indicate that extinction proceeded more slowly in the trace group compared to the delay group. In the DCG, we did no longer find differences in arousal and fear ratings of the CS+ compared to the CS− after extinction. However, the CS+ was still rated more arousing and more frightening than the CS− in the TCG. A slower extinction process in the trace group can be seen as an indication for a higher working memory contribution in the extinction of trace conditioning and therefore may account for different processes of extinction in delay and trace conditioning. There is also an interesting connection between our findings and evidence from trace eyeblink conditioning in rabbits (Weiss and Disterhoft, [@B64]). They assume an important role of the dlPFC in the acquisition of trace conditioning: Activation of dlPFC and hippocampus potentiates the effect of the CS at pontine nuclei on the way to the cerebellum and thus bridge the trace interval during acquisition. After consolidation of the CS+/US association, structures mediating the conditioned response reorganize. While the hippocampus becomes less important, the dlPFC becomes more important. Further research with regard to both acquisition and extinction is necessary for investigating to what extend these findings from the animal model can be transferred to classical conditioning in humans.
Insula and ACC
--------------
In both conditioning groups we found activation of the insular cortex during early extinction. Additionally, the insula showed greater activation in the early compared to the late extinction in the delay group. Evidence for the involvement of the insula in classical fear conditioning comes, among others, from Phelps et al. ([@B44]): In contrast to the instructed fear paradigm, in which the insula was activated already in early trials, the activation in the conditioning paradigm occurred not until the later trials of acquisition, when participants were consciously aware of the association between the CS+ and the US. These findings are consistent with evidence coming from pain research showing that the insula plays an important role in the anticipation of pain (e.g., Ploghaus et al., [@B46]; Wiech et al., [@B65]). Phelps et al. ([@B43]) suggest that the anticipation of pain leads to a cortical representation of fear, which is transmitted to the amygdala via the insular cortex. During early extinction of fear memory, the CS+ is no longer followed by a painful stimulus. However, it is still associated with the US and thus leads to the anticipation of pain, resulting in the observed activation of the insular cortex. Interestingly, during extinction of both trace and delay fear conditioning, insular activation was limited to the right hemisphere. The left insula has been associated with semantic processing which is necessary in instructed fear conditioning. In contrast, the right insula has been associated with the response to a sensory aversive US like for example the mildly painful electric stimulus we applied (for further information see for example Craig, [@B13]).
In addition to the insula, the dorsal ACC was activated during early extinction in the TCG. Moreover, we found stronger activation of the ACC during early extinction compared to late extinction in both groups. The combined activation of the ACC and the insula has been discussed to represent a pathway for the integration of nociceptive input in memory processes (Coghill et al., [@B12]). According to this model, both structures are involved in the adjustment of behavior in response to a stimulus predicting pain (see also Büchel et al., [@B9]). The ACC has also been associated with sustained attention toward a stimulus that might be followed by pain (Yaguez et al., [@B66]). There is broad evidence that sustained attention is necessary for trace fear conditioning, but not for delay conditioning. In a trace conditioning paradigm, fear memory is only established when subjects are consciously aware of the CS--US contingency. However, in delay paradigm, conditioning can also occur when participants have not formed declarative memory and thus are unaware of the association between the CS and the US (e.g., Manns et al., [@B29]; Clark et al., [@B11]; Weike et al., [@B70]). Han et al. ([@B19]) have shown that attention-distracting stimuli interfere with trace but not delay or contextual fear conditioning in mice. Moreover, they found a higher density of c-fos-positive cells in the ACC of mice that had undergone trace fear conditioning compared to delay conditioning. In the same study, lesions of the ACC selectively impaired trace conditioning. These results give additional evidence for the association of ACC activation and sustained attention during trace fear conditioning, and also offer an explanation why we found combined activation of the insula and the ACC only during the early extinction of trace but not delay fear memory.
Striatum
--------
During acquisition of fear memory, the expectation is formed that an initially neutral stimulus is followed by a negative event such as an electric stimulus. During extinction, this expectation is violated: The negative event does no longer occur. This discrepancy between the expected and the actual outcome is referred to as prediction error (e.g., Schultz et al., [@B55]). The striatum has been shown to be involved in the coding of prediction errors in both appetitive and also aversive classical and instrumental conditioning. This applies to primary reinforcers such as pain (Phelps et al., [@B43]; Seymour et al., [@B57]), but also to secondary ones such as monetary gains (e.g., Delgado, [@B68]). In our study, we found striatal activation in both the DCG and the TCG. These results provide further evidence for the important role of the striatum not only in the acquistion (Jensen et al., [@B20]; Delgado et al., [@B15]; Klucken et al., [@B23]; Tabbert et al., [@B61]), but also in the extinction of fear memory and therefore in the coding of prediction errors characterized by the omission of a negative outcome. Raczka et al. ([@B51]) recently showed that a functional polymorphism of the dopamine transporter gene, which is mainly expressed in the striatum, influences extinction learning. The 9-repeat allele is associated with enhanced phasic dopamine release and with higher learning rates in the extinction of conditioned fear. In 9R carriers they also found stronger activation of the ventral striatum in response to prediction errors during extinction. In relation to these findings they assumed that extinction is an appetitive-like learning process mediated by the mesostriatal dopamine system, rather than a learning process driven by an aversive prediction error.
Hippocampus
-----------
The hippocampus has been found to be involved in the representation of the temporal context in a conditioning process, which plays an important role in a trace paradigm including a temporal gap between CS+ and US (e.g., Phillips and LeDoux, [@B45]). In the TCG, we found greater hippocampal activation in the comparison of early vs. late extinction. Clark et al. ([@B11]) stated that the hippocampus is crucial for explicit or declarative memory processes. According to them, trace conditioning requires declarative knowledge and therefore hippocampus activity because the temporal gap between the CS and the US makes it difficult to process the CS--US relationship in an automatic, reflexive way. Knight et al. ([@B24]) found hippocampal activation during extinction as well. They also reported a rapid decrease of its activation during the early trials of extinction.
Amydala
-------
We did not find significant activation of the amygdala in either one of the two groups. One possible reason for this is the rapid habituation of amygdala activity during extinction learning, especially in a conditioning paradigm with 100% contingency of CS+ and US during acquisition (e.g., LaBar et al., [@B25]). Secondly, EPI is highly vulnerable to susceptibility artifacts, which occur near the interfaces of substance of different magnetic susceptibility and thus are likely in structures of the medial temporal lobe, like the amygdala (Bellgowan et al., [@B5]; Stocker et al., [@B60]).
Conclusion
----------
In sum, our results add further evidence for the involvement of the PFC, insula, ACC, striatum, and hippocampus in the extinction of conditioned fear memory. We could also confirm that the ACC and the hippocampus are mainly involved in trace conditioning processes. The ACC has been associated with sustained attention, which is necessary for trace but not delay conditioning. The hippocampus is assumed to be necessary for the processing of the temporal context necessary to bridge the trace interval. Most important, our results indicate that different parts of the PFC are activated during extinction of delay vs. trace fear conditioning, the vmPFC vs. the dlPFC, respectively. These results point to different underlying processes during extinction of these two types of conditioning. Due to limited power of our study and a relatively liberal level of significance, results have to be interpreted with care. More evidence is needed to elucidate the role of the PFC in the extinction of trace conditioning in more detail and to translate results from the animal model to human trace fear conditioning
Conflict of interest statement
------------------------------
Prof. Paul Pauli and Prof. Andreas Mühlberger are Shareholders of a commercial company that develops virtual environment research systems for empirical studies in the field of psychology, psychiatry, and psychotherapy. Mathias Müller is shareholder and executive of the same company. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG): Collaborative Research Center "Fear, Anxiety, Anxiety Disorders," SFB-TRR 58 project B1 to Paul Pauli and Andreas Mühlberger. We thank Christoph Horzella and Jonas Wirbelauer for assistance in data collection. This work is part of the dissertation of Heike Ewald. Prof. Andreas Mühlberger and Prof. Paul Pauli are shareholders of a company that develops virtual environment research systems for empirical studies in the field of psychology, psychiatry, and psychotherapy. Matthias Müller is shareholder and executive officer of this company.
[^1]: Edited by: Hauke R. Heekeren, Freie Universität Berlin, Germany
[^2]: Reviewed by: Bram Vervliet, KU Leuven, Belgium; Dagmar Timmann, University Clinic Essen, Germany
[^3]: This article was submitted to the journal Frontiers in Human Neuroscience.
[^4]: ^†^These authors have contributed equally to this work.
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1. Overview of Some Molecular Events Occurring during Meiosis in Yeast {#sec1-ijms-21-04510}
======================================================================
The budding yeast *Saccharomyces cerevisiae* has a strong dependence on the presence of nutrients in its surroundings. As long as there is enough nutrient availability, cells proceed to mitosis, allowing them to proliferate by budding \[[@B1-ijms-21-04510]\]. Provided that diploid cells are subjected to some nutrient starvation, they undergo a differentiation process called sporulation, where meiosis plays an essential role \[[@B2-ijms-21-04510]\]. This specialized cell division process generates four haploid daughter cells by means of two consecutive cell divisions \[[@B3-ijms-21-04510]\]. Meiosis is piloted by a cascade of transcriptional events as soon as mating-type and nutrition signals are brought together \[[@B4-ijms-21-04510]\].
1.1. Transcriptional Events Leading to Meiosis {#sec1dot1-ijms-21-04510}
----------------------------------------------
The ability to sporulate, which is unique to diploid cells, requires the expression of both MATa and MATα alleles. The products of this locus give rise to an a1/α2 heterodimer, without which sporulation is unable to take place \[[@B1-ijms-21-04510]\]. Nonetheless, the decision to undergo this process depends mainly on several extracellular factors, including the presence of nitrogen and glucose, among others.
Most of these elements are able to control the transcription of the master regulator Ime1. The regulation of the expression of this factor is mediated by the Rme1 repressor \[[@B5-ijms-21-04510]\], which is inhibited in the presence of the a1/α2 heterodimer that is expressed in haploid cells \[[@B6-ijms-21-04510]\]. Consequently, in these cells, the a1/α2 heterodimer is able to bind to an Rme1 Repressor Element (RRE) situated within the promoter region of *IME1*. This promoter is much larger than that of the majority of yeast genes and contains regulatory elements for all the different factors that could potentially affect *IME1* expression. These regulatory elements include binding sites for transcription factors responding to mating type, nitrogen deprivation, and others \[[@B7-ijms-21-04510]\]. In haploid cells, Rme1 is able to activate the expression of a long non-coding RNA (lncRNA), called *IRT1*, which nearly encompasses the totality of the *IME1* promoter. This lncRNA acts as a *cis*-element, inhibiting the binding of transcription factors to this region \[[@B8-ijms-21-04510]\].
Furthermore, not only does mating affect the Rme1-dependent pathway, but it modulates the expression of the RNA-methyltransferase Ime4, needed for correctly inducing *IME1* \[[@B9-ijms-21-04510]\]. What makes this factor especially interesting is the fact that its transcripts are different in concordance to the cell type; haploid cells express a non-coding anti-sense *IME4* RNA (known as *IME4-AS* or *RME2*), whereas in diploids, its anti-sense transcription is impeded by the aforementioned a1/α2 heterodimer. As a result, the Ime4 protein is produced, activating *IME1* by the N6-adenosine methylation of its RNA \[[@B10-ijms-21-04510],[@B11-ijms-21-04510],[@B12-ijms-21-04510]\].
Once Ime1 is activated, a first transcriptional wave is triggered, consisting of the so-called "early" genes, which exhibit in their promoters a common regulatory element: the *URS1* site \[[@B13-ijms-21-04510],[@B14-ijms-21-04510]\]. This *cis*-element is the target of the Ume6 protein, which after binding to the *URS1* site displays the capacity of repressing the transcription of these genes during growth, by the recruitment of the Rpd3/HDAC complex and the chromatin remodeler Isw2 \[[@B13-ijms-21-04510],[@B15-ijms-21-04510],[@B16-ijms-21-04510],[@B17-ijms-21-04510]\].
The Ume6 protein has the ability to act as a repressor, but also as an activator. The conversion of Ume6 from repressor to activator was initially thought to be due to impeded interaction with the Rpd3/HDAC complex by the presence of Ime1 \[[@B18-ijms-21-04510]\]. However, more recent studies have shown that this conversion is mediated in a different way. In order to bring about the expression of early genes, Ume6 must undergo a two-step degradation process, mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase \[[@B19-ijms-21-04510],[@B20-ijms-21-04510]\]. The function of Ume6 is regulated by its acetylation by the acetyltransferase Gcn5, belonging to the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex \[[@B21-ijms-21-04510]\], as well as by deacetylation by Rpd3 \[[@B22-ijms-21-04510]\]. Ume6 acetylation reduces its DNA-binding activity, which provokes its disassociation from promoters and triggers its targeted destruction. As a consequence, transcription of early genes is activated \[[@B23-ijms-21-04510]\]. Later on, when Ime1 is present, it binds to Ume6, which is further acetylated by Gcn5 in a second lysine cluster, which promotes its second destruction step, enabling the full activation of early genes \[[@B22-ijms-21-04510],[@B23-ijms-21-04510]\]. As previously mentioned, these genes present a common *URS1* site to which Ume6 is bound, and whose expression is indispensable for the entrance into the premeiotic S phase \[[@B24-ijms-21-04510]\]. Among these early genes, we can find *IME2*, a key regulator in meiosis by mediating Ime1 stability; *INO1*, which is involved in lipid biosynthesis; or *SPO13*, which is linked to sister chromatid cohesion \[[@B13-ijms-21-04510],[@B16-ijms-21-04510],[@B23-ijms-21-04510],[@B25-ijms-21-04510]\].
1.2. Meiotic Recombination: DSB Formation, Repair, and Distribution {#sec1dot2-ijms-21-04510}
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The segregation of homologous chromosomes occurs during the first meiotic division, while the second division is comprised of the separation of sister chromatids. The physical location where homologous chromosomes have exchanged their information is termed the chiasma. It is in this place where the spindle microtubules attach to kinetochores, generating tension. This tension is required for proper segregation at meiosis I \[[@B26-ijms-21-04510]\]. It is in the first step where the process of interhomologous recombination becomes crucial, in order to guarantee genetically diverse gametes \[[@B26-ijms-21-04510],[@B27-ijms-21-04510]\]. Problems during this recombination may give rise to germline mutations or even aberrant chromosome arrangements in gametes \[[@B28-ijms-21-04510]\].
The recombination characterising meiosis consists of four consecutive steps ([Figure 1](#ijms-21-04510-f001){ref-type="fig"}A); (i) the initiation, where programmed DNA double-strand breaks (DSBs) are formed---a maximum of 200 per yeast nucleus \[[@B29-ijms-21-04510],[@B30-ijms-21-04510]\]; (ii) the processing of these DSBs, giving rise to single-strand regions; (iii) a homologous repair of the DSBs, mainly by means of the homologous recombination pathway, as well as meiosis-specific modulators; and (iv) these double-Holliday junctions needing to be resolved and dissolved, thus mainly forming reciprocal crossovers (COs). In the context of meiosis, however, the majority of non-crossover events (NCO) arise from other pathways \[[@B31-ijms-21-04510]\].
As stated, programmed DSBs are formed during the first meiotic division, a reaction catalyzed by the meiosis-specific, topoisomerase-like DNA transesterase Spo11 \[[@B32-ijms-21-04510],[@B33-ijms-21-04510]\] ([Figure 1](#ijms-21-04510-f001){ref-type="fig"}A). Apart from this protein, nine DSB proteins have been described so far in *Saccharomyces cerevisiae* \[[@B34-ijms-21-04510],[@B35-ijms-21-04510],[@B36-ijms-21-04510]\]. The majority of proteins associated to Spo11 form sub-complexes, including the Mer2--Mei4--Rec114 (RMM) subcomplex, which attaches to DSB sites in chromatin loops, permitting them to be cleaved by Spo11 \[[@B34-ijms-21-04510]\]. Among these regulating factors, we can also encounter the Mre11--Rad50--Xrs2 (MRX) complex, Rec102, Rec104, and Ski8 \[[@B37-ijms-21-04510]\].
After these steps, crossover recombination and homologous pairing are linked by the synaptonemal complex (SC), which implicates the multimeric assembly of coiled-coil proteins between aligned homologous chromosomes \[[@B38-ijms-21-04510]\]. In spite of the mechanisms not being completely understood, the SC plays an important role for crossover recombination, and several links between the SC and recombination have been discussed \[[@B39-ijms-21-04510]\]. Among its described subunits in yeast, we can find, on the one hand, Rec8, Red1, and Hop1, which conform the two parallel lateral elements (LEs) \[[@B40-ijms-21-04510],[@B41-ijms-21-04510]\]. On the other hand, the central element (CE) proteins Ecm11 and Gmc2, and the traverse filament component Zip1, localize to centromeres in early prophase \[[@B42-ijms-21-04510]\], mediating the coupling of homologous centromeres, and afterwards, the correct pairing of whole homologous chromosomes \[[@B43-ijms-21-04510]\]. In order to properly build the SC, the assembly of the synapsis initiation complex (SIC) is urged, comprising the Zip2, Zip3, Zip4, and Spo16 proteins \[[@B44-ijms-21-04510],[@B45-ijms-21-04510]\] ([Figure 1](#ijms-21-04510-f001){ref-type="fig"}B).
Once the recombination process has ended, the expression of Ndt80 implies entering into the middle phase \[[@B2-ijms-21-04510]\]. The next main cytological event consists in dividing into four haploid cells. For this purpose, the spindle pole body (SPB), the sole microtubule-organizing centre in both budding and fission yeast, plays a key role. Its architecture is similar to the centrosome present in animal cells, and coordinates microtubule attachment and chromosome segregation \[[@B46-ijms-21-04510]\]. SPBs duplicate twice: at the beginning of both meiosis I and II. The attachment of the spindle to the kinetochore is facilitated by the Dam1 (also named DASH) complex and carried out during metaphase, while chromosomes are pulled apart in anaphase \[[@B47-ijms-21-04510],[@B48-ijms-21-04510]\]. In addition, the phosphorylation of some components of the SPB has been anticipated to stimulate the interaction with DSB to promote an efficient DNA repair \[[@B49-ijms-21-04510]\].
In conclusion, DSB formation and repair are precisely regulated to ensure that this step is performed in a coordinated manner during meiotic recombination.
Nonetheless, the distribution of DSBs in *S. cerevisiae* does not occur at random; it is rather localized into specific regions, called recombination hotspots, where DSBs preferentially form. These short regions are, at the same time, contained within larger regions known as DSB-hot domains---these are opposed to DSB-cold domains, which mainly devoid of DSBs \[[@B30-ijms-21-04510]\]. Among the mechanisms to control the distribution along the genome of these DSBs, chromatin accessibility becomes essential, allowing protein--DNA contacts that serve as docking sites for the recombination machinery \[[@B50-ijms-21-04510]\]. To correctly accomplish it, several DNA-binding proteins, such as Atf-Pcr1, Bas1, or even Prdm9, in mammals, can participate in their regulation \[[@B51-ijms-21-04510]\]. Of interest for this review, we would like to remark that these factors are able to incite some posttranslational modifications (PTMs) of histones, which in turn tempers chromatin disposition (see later section) \[[@B52-ijms-21-04510]\].
2. Different Histone Modifications That Are Linked to Meiotic Events {#sec2-ijms-21-04510}
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As explained above, DSBs occur in DNA. In eukaryote cells, DNA is found wrapped around specialized proteins called histones, making up nucleosomes, the fundamental unit of chromatin \[[@B53-ijms-21-04510]\]. The core particle of nucleosomes contains two copies of the four histones: H2A, H2B, H3, and H4, whose tails are susceptible to be modified by PTMs to regulate the chromatin state \[[@B52-ijms-21-04510]\]. The involvement of these PTMs in different molecular events during meiosis appears to be undisputable, for it is one of the most precise ways to regulate the whole process \[[@B54-ijms-21-04510]\]. Now turning our attention to yeast, we will highlight several PTMs that have been linked to meiosis, though there are many more in other organisms that can be found in relatively recent review articles and elsewhere \[[@B55-ijms-21-04510]\].
Among others, H3K9ac has been reported in DSB hotspots, and H4K16ac is an important checkpoint for meiotic recombination, which is also facilitated by H4K44ac \[[@B56-ijms-21-04510],[@B57-ijms-21-04510],[@B58-ijms-21-04510]\]. H3K4me3 is linked to recombination initiation and programmed DSB formation, not only in yeast, but also in mouse spermatocytes and oocytes \[[@B59-ijms-21-04510],[@B60-ijms-21-04510],[@B61-ijms-21-04510]\]. H2BK123ub---as well as its homologue in higher eukaryotes, H2BK120ub---participates in meiotic recombination \[[@B62-ijms-21-04510]\]. Moreover, the protection of centromeric cohesion is regulated by H2AS121ph \[[@B63-ijms-21-04510]\].
Focusing on DSB hotspots, they are characterized by a nucleosome-depleted region (NDR), featuring a noticeable histone mark arrangement directing their activity where H3K9ac is flanking them. After that, H3K4me3 centres on the +1, +2, and +3 nucleosomes. In contrast, H3K4me1/2, H3K36me3, H3K79me2, and H3R2me are found next to the 3' ends of genes \[[@B36-ijms-21-04510],[@B64-ijms-21-04510]\] ([Figure 2](#ijms-21-04510-f002){ref-type="fig"}).
Due to our research interests, we will discuss in the next subsections (i) the trimethylation of histone H3 on the lysine 4 (H3K4me3), which has been the one most correlated with meiotic DSBs in *S. cerevisiae*, despite being a PTM normally linked to several features of the process of transcription \[[@B65-ijms-21-04510],[@B66-ijms-21-04510]\]; and (ii) the monoubiquitination of histone H2BK123, which has been well studied in transcription regulation and has been implicated in recruiting several DSB factors during meiosis.
2.1. H3K4 Trimethylation by the COMPASS Complex Plays an Important Role in Recombination Initiation during Meiosis and DSB Generation {#sec2dot1-ijms-21-04510}
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In humans and higher eukaryotes, H3K4me3 is deposited by a family of histone methyltransferases, known as SET1/MLL. It is made up of six different members: SETD1A, SETD1B, and MLL1, 2, 3 and 4 \[[@B67-ijms-21-04510]\]. These complexes present the ability to mark H3K4 with different levels of methylation (that is, H3K4me1, H3K4me2, and H3K4me3), using S-adenosine--methionine (SAM) as donor molecule \[[@B68-ijms-21-04510]\].
In budding yeast, all H3K4 methylation is exclusively mediated by the lysine methyltransferase Set1, by being part of a complex called the Complex of Proteins Associated with Set1 (COMPASS) \[[@B68-ijms-21-04510],[@B69-ijms-21-04510]\] or Set1C \[[@B70-ijms-21-04510]\]. In contrast, the reverse process---namely, de-methylation---is uniquely accomplished by the Jhd2 protein, a subtype of demethylase containing a Jumonji domain. This factor cooperates with COMPASS in order to finely regulate chromatin dynamics and ensure a correct H3K4me3 distribution \[[@B71-ijms-21-04510],[@B72-ijms-21-04510]\].
The COMPASS complex consists in yeast of eight subunits, being the catalytic subunit Set1 (containing an active C-terminal SET domain--Su(var)3--9, Enhancer-of-zeste, and Trithorax), as well as seven additional components: Swd1 (Cps50) and Swd3 (Cps30) (necessary for H3K4 mono-, di-, and trimethylation), Swd2 (Cps35), Sdc1 (Cps25), Bre2 (Cps60) (required for di- and trimethylation), Spp1 (Cps40) (required solely for trimethylation), and a little subunit, Shg1, of which little information is available \[[@B67-ijms-21-04510],[@B70-ijms-21-04510]\].
Two different studies published in 2018 shed light into its structure and mechanisms \[[@B73-ijms-21-04510],[@B74-ijms-21-04510]\]. According to these works, the catalytic module (CM) of COMPASS is organized by Swd1, which has a long C-terminal tail able to surround the other three subunits: Swd3, Bre2, and Set1. Swd3---responsible for the regulation of the trimethylating activity---is situated just beside Swd1, forming something like a set of "arms" ([Figure 3](#ijms-21-04510-f003){ref-type="fig"}).
In the opposite part of the complex, Bre2 and Sdc1 form a trimeric subcomplex, with one copy of the first and two of the latter. This dimerization of Sdc1 is necessary for the correct stabilization of Bre2. Spp1, Swd2, and Shg1, on the other hand, can be found interacting with the N-terminal SET domain and the RNA-recognition motif (RRM) of Set1 \[[@B75-ijms-21-04510],[@B76-ijms-21-04510]\]. Since the cryo-EM structure resolved by Qu et al. \[[@B74-ijms-21-04510]\] contains a truncated version of Set1 (762-1080 aa), neither its N-terminal tail, nor Swd2 or Shg1 appear in it. Strikingly, Swd2 is also associated with the Pta1 (APT) complex, as part of the cleavage polyadenylation factor \[[@B77-ijms-21-04510],[@B78-ijms-21-04510],[@B79-ijms-21-04510]\].
The COMPASS plays an important role in recombination initiation during meiosis and DSB generation. Though originally described as a complex involved in transcription, the identification of Spp1 in these events opened up this new function for COMPASS. Spp1 is a subunit of COMPASS that contains a plant homeodomain (PHD) finger and is able to physically interact with H3K4me3/2, as well as with the Mer2 protein (from the RMM subcomplex) \[[@B60-ijms-21-04510]\]. The interaction between Spp1 and Mer2 enables their anchoring to DSB hotspots, which conducts DSB formation with dependence on Spo11 \[[@B60-ijms-21-04510],[@B80-ijms-21-04510]\]. Spp1 functions are, therefore, not limited to regulating the catalytic activity of COMPASS, since this protein is also able to recognize the methylation state of H3K4 and act in consequence in other processes, such as meiotic recombination. For this reason, Spp1 can be found both around actively transcribed genes and in chromosome axial sites, with independence from Set1 \[[@B81-ijms-21-04510]\]. Furthermore, *set1∆* strains display a general reduction in most DSBs, which has been seen to correlate with H3K4me3 levels \[[@B82-ijms-21-04510],[@B83-ijms-21-04510]\]. This particular strain has also been reported to present a synthetic defect with Rec114 of the RRM subcomplex \[[@B84-ijms-21-04510]\]. Moreover, when Spo11 is targeted to either hot or cold recombination sites, it is unable to induce DSBs on its own \[[@B85-ijms-21-04510]\]. These results suggest that the role played by H3K4 methylation in DSB formation is not limited to simply recruiting the endonuclease.
A conserved proof that there is a relationship between H3K4me3 and meiotic-driven DSBs can be found in mammals, where the histone methylase Prdm9 leads DSBs to occur in DNA motifs recognized by its zinc finger domain \[[@B86-ijms-21-04510]\].
2.2. H2B Ubiquitination Is Important for DSB Formation {#sec2dot2-ijms-21-04510}
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Monoubiquitination of histone H2B on its lysine 123 (K120 in higher eukaryotes; from this point on, H2Bub) is controlled by a coordinated action of the E2-conjugating enzyme Rad6, the E3 ligase Bre1, and the regulatory cofactor Lge1 \[[@B87-ijms-21-04510],[@B88-ijms-21-04510]\]. Meanwhile, in yeast H2B deubiquitination is performed by two proteases: Ubp10 and Ubp8. Interestingly, Ubp8 belongs to the SAGA complex, and in particular to its deubiquitination module (DUBm) \[[@B89-ijms-21-04510],[@B90-ijms-21-04510]\]. H2B ubiquitination also requires the participation of other factors and complexes like, for instance, PAF1c \[[@B91-ijms-21-04510],[@B92-ijms-21-04510]\] and FACT (facilitates chromatin transcription) \[[@B93-ijms-21-04510]\], which will be addressed later.
In addition to its role in gene expression (see later), H2B ubiquitination has also been related to the formation of DSBs \[[@B94-ijms-21-04510]\]. The ubiquitination of H2B leads to chromatin relaxation, which, in the context of meiosis, enables the recruitment of several DSB repair factors to their proper positions at hotspots \[[@B94-ijms-21-04510],[@B95-ijms-21-04510],[@B96-ijms-21-04510]\]. As a matter of fact, the involvement of H2Bub in chromatin relaxation is evolutionarily conserved from yeast to mammals \[[@B95-ijms-21-04510]\]. Notably, the Rad6 homolog in mammals, *HR6B*, has been related to defects in male fertility when it is depleted in mice, due to errors during spermatogenesis. The function of Rad6 is thus thought to be involved with both the synaptonemal complex and recombination in meiosis, highly implying a role in chromatin remodelling \[[@B97-ijms-21-04510]\]. Likewise, Bre1 and Lge1 proteins have also been related to meiotic processes; *lge1∆* and *bre1∆* cells initiate meiotic DNA replication in the S-phase, later than the wild-type, and take much longer to complete it, presenting a reduced DSB formation as well \[[@B62-ijms-21-04510],[@B98-ijms-21-04510]\].
Interestingly, not only does the PAF1c play a crucial role during transcription, but also its component Rtf1 has been described as participating in the meiotic process \[[@B99-ijms-21-04510]\]. Rtf1 is important for the formation of DSBs, and this role is independent of the presence of Set1. In these lines, modifications of Dam1, the main subunit of kinetochore DASH complex, depend on PAF1c \[[@B100-ijms-21-04510]\]. Dam1 is methylated by Set1 for proper chromosome segregation, and deletions in Paf1, Bre1, Rad6, or Ubp8 (factors implicated in H2Bub) hinder Dam1 methylation.
Furthermore, Ubp8 from SAGA DUBm is involved in H2B and Cse4 deubiquitination \[[@B101-ijms-21-04510]\]. Cse4 is a centromeric, H3-like histone protein (vertebrates' centromere protein A or CENP-A orthologue). The ubiquitination of Cse4 regulates its localization to centromeres, where the spindle pole body is attached to the chromosome \[[@B102-ijms-21-04510]\]. However, the functional connections between the SAGA DUBm and meiosis are yet to be described. In addition, cells lacking Sgf73, another component of the SAGA DUBm \[[@B103-ijms-21-04510]\], show problems in DNA replication prior to meiosis, and present a reduced expression of *IME1* \[[@B98-ijms-21-04510]\].
3. H3K4me and H2Bub Are Crucial Histone Marks during Transcription: Structural Insights into the Molecular Mechanism behind H3K4me/H2Bub Coordination {#sec3-ijms-21-04510}
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3.1. H3K4me during Transcription {#sec3dot1-ijms-21-04510}
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As noted earlier, H3K4 methylation has been well studied in the context of transcription. H3K4me3 is strongly correlated to transcription activation and active genes, with a prominent peak around the transcription start sites (TSSs) being a feature of its distribution. In addition, this peak has been linked in magnitude to the amount of mRNA for a given gene \[[@B104-ijms-21-04510],[@B105-ijms-21-04510]\]. It is in these TSSs where H3K4me3 serves as a binding site for complexes that initiate transcription in promoters, such as the transcription factor II D (TFIID) or the SAGA complex \[[@B106-ijms-21-04510]\]. However, H3K4me is not only related to transcription activation. This mark is also associated with repression by means of antisense transcription \[[@B107-ijms-21-04510]\]. H3K4me2 and me3 peak around the 3' ends of COMPASS-repressed genes (such as *PHO84*), leading to the expression of antisense transcription at the 3' ends of coding regions. In this study, the authors found that this antisense transcription is promoted by H3K4me3, but is not fully dependent on the mark.
The actual functions for H3K4me in the regulation of transcription, as well as chromatin organization, are not thoroughly known, though it is known that Set1 is mobilized to TSSs by interacting with the RNA polymerase II (RNAPII) when it is phosphorylated at serine 5 of its Rpb1 C-terminal domain (CTD-Ser5P), or via elongation factors \[[@B108-ijms-21-04510],[@B109-ijms-21-04510],[@B110-ijms-21-04510]\]. This modified version of RNAPII is predominant in the first stages of transcription elongation, and its binding to COMPASS allows for high levels of trimethylation at promoter-proximal regions \[[@B105-ijms-21-04510]\]. The association with RNAPII is mediated by the N-terminal domain of Set1, which directly interacts with the Rpb1 CTD, which is connection-dependent on the WD40 domain of the Swd2 subunit \[[@B111-ijms-21-04510]\].
The little impact of this modification on gene expression at the genomic level highlights that the role of methylation on transcription is more complicated than anticipated \[[@B71-ijms-21-04510],[@B112-ijms-21-04510]\]. Although initial investigations have suggested that H3K4me associates with actively transcribed genes \[[@B113-ijms-21-04510]\], RNA-seq experimentation found no change in mRNA levels in steady-state or dynamic conditions when H3K4me3 was eliminated and could serve as a repressive mark \[[@B107-ijms-21-04510]\]. Furthermore, it might seem that H3K4me could be a consequence of transcription, rather than a regulator of it. Either way, extensive work must be carried out in order to ascertain this extent.
The role of COMPASS in transcription can be further understood in light of several recent studies, which show that this complex is able to bind to mRNAs both in vitro and in vivo \[[@B114-ijms-21-04510],[@B115-ijms-21-04510]\]. Not only was the RRM of Set1 found to be important for this process, but also a myriad of surfaces of the other subunits. This RNA-binding capacity was described as fundamental for a correct topology of COMPASS along transcription.
3.2. H2B Monoubiquitination during Transcription {#sec3dot2-ijms-21-04510}
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H2Bub has been described to have several roles in gene transcription regulation, since it has been seen to correlate to more RNAPII processivity, being enriched in promoter regions. It has been proposed that, upon H2B monoubiquitination, inter-nucleosomal interactions are disrupted \[[@B96-ijms-21-04510]\]. This is in part due to the large size of the ubiquitin moiety (76 aa), as well as to its position on the nucleosomal face. As a result, chromatin compaction relaxation facilitates the recruitment of other components to DNA \[[@B116-ijms-21-04510]\]. In fact, H2Bub improves intra-nucleosomal interactions, aided by other components, such as the histone chaperone FACT \[[@B117-ijms-21-04510]\]. Other studies have also made clear the implications of H2Bub in RNAPII stalling, in the event where there are DNA lesions, by participating in a transcription-coupled repair pathway \[[@B118-ijms-21-04510]\]. It has been demonstrated that H2Bub stimulates FACT activity to allow the displacement of the H2A/H2B dimer, which permits RNAPII to continue transcription \[[@B93-ijms-21-04510]\].
H2B ubiquitination also requires the presence of another complex in yeast: PAF1c. This complex is comprised of five different subunits: Rtf1 (the most important subunit, containing the termed histone modification domain, HMD), Cdc73, Paf1, Ctr9, and Leo1 \[[@B119-ijms-21-04510]\]. PAF1c is recruited to chromatin by FACT (as shown in mammalians), which is able to recruit enzymes that ubiquitinate H2B (Rad6 and Bre1) \[[@B93-ijms-21-04510]\]. PAF1c is important for the regulation of RNAPII-transcription elongation by interacting with COMPASS and factors involved in termination \[[@B120-ijms-21-04510]\]. PAF1c has also been related to the phosphorylation state of the C-terminal domain of Rpb1, a promotion of H3K36 trimethylation, as well as histone acetylation on active genes \[[@B121-ijms-21-04510]\].
3.3. Structural Overview of COMPASS Activation upon H2B Ubiquitination {#sec3dot3-ijms-21-04510}
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Notably, H3K4me needs H2Bub in order to occur, since *rad6∆* cells are devoid of methylation \[[@B122-ijms-21-04510]\] and both modifications are highly associated to transcription \[[@B104-ijms-21-04510],[@B123-ijms-21-04510]\]. This kind of interdependency is commonly referred to as "cross-talk", where a histone modification serves as template for the pattern of a second one. Unfortunately, the mechanisms whereby this crosstalk is mediated have been largely unknown, yet several recent structural studies seem to help us understand this mystery. The yeast version of COMPASS presents a catalytic module (CM) able to embrace an H3 tail and made up of five subunits: Set1 (SET domain), Swd1, Swd3, Sdc1, and Bre2 \[[@B124-ijms-21-04510]\]. This module seems to display a component impairing the methyltransferase activity of the complex whenever a H2B-modified nucleosome is absent \[[@B73-ijms-21-04510]\]. However, the presence of the ubiquitin moiety does not modify the affinity of COMPASS for the nucleosome, but is likely to activate the enzyme by inhibiting the CM impairment \[[@B125-ijms-21-04510]\]. Once ubiquitin has been conjugated to H2B, it locates to a cleft between Swd1, Set1, and Bre2 ([Figure 4](#ijms-21-04510-f004){ref-type="fig"}A). The moiety presents several surfaces interacting with COMPASS, including a definite interface comprised of a hydrophobic patch (I44-V70-L8-H68, namely the I44 patch) that contacts a hydrophobic patch consisting of both the N-terminal and C-terminal tails of Swd1, which change their conformation \[[@B125-ijms-21-04510],[@B126-ijms-21-04510],[@B127-ijms-21-04510]\]. This contact is later stabilized by a salt bridge between Lys48 and Arg42 of the ubiquitin and Glu15 and Glu405 of Swd1. Such is the importance of this subunit in ubiquitin recognition that a *swd1* deletion completely abolishes any kind of H3K4 methylation by COMPASS \[[@B127-ijms-21-04510]\].
The main inhibitory region of the COMPASS CM is concentrated on an arginine-rich motif (ARM) that immediately precedes the SET domain of Set1 \[[@B128-ijms-21-04510]\] ([Figure 4](#ijms-21-04510-f004){ref-type="fig"}B). The ARM is found disordered, without a ubiquitinated nucleosome \[[@B74-ijms-21-04510]\]; upon binding to H2Bub, it folds and is constrained into the nucleosomal acidic patch provided by H2A, and adopts an α-helix conformation \[[@B125-ijms-21-04510],[@B128-ijms-21-04510]\]. The anchoring of this ARM into the acidic patch enables the connection between the catalytic SET domain and the ubiquitinated H2B, as well as its closeness to Swd3, Swd1, and Bre2 subunits. This binding also permits a sound restructuration of a Set1 α-helix (residues 926 to 933) into an extended strand, parallel to the ARM; alleviating a steric hindrance in Set1 \[[@B127-ijms-21-04510]\] ([Figure 4](#ijms-21-04510-f004){ref-type="fig"}B). Notably, even though many conformational changes are described in Set1, none of them directly affect the SET catalytic domain. Consequently, it is highly likely that the increased activity is due to the contact to the nucleosome provided by the ARM \[[@B125-ijms-21-04510],[@B127-ijms-21-04510]\].
In summary, these new results help us better understand the molecular mechanism behind the coordination between H2Bub and H3K4me. Further investigations to assess whether this mechanism occurs both in transcription and meiotic recombination and at different genomic positions are required to fully understand the H2Bub/H3K4me connection.
4. Human Diseases Associated to Defects in H3K4me and H2Bub Machineries {#sec4-ijms-21-04510}
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Even if it has been stated several times through this work, a well-defined trait characterizing all aforementioned mechanisms is the fact that they are extensively conserved across evolution. Actually, most of the mentioned factors present well-described orthologues in higher eukaryotes that have been involved in numerous disorders, mainly due to the crucial nature of the processes they regulate. A very short summary of some of the diseases associated to mutations in these factors are given below, and are recapitulated in [Table 1](#ijms-21-04510-t001){ref-type="table"} (more extensive reviews can be found in the literature \[[@B129-ijms-21-04510],[@B130-ijms-21-04510],[@B131-ijms-21-04510],[@B132-ijms-21-04510],[@B133-ijms-21-04510],[@B134-ijms-21-04510]\]).
Set1/MLL has been reported to participate in cancer and ageing \[[@B67-ijms-21-04510]\], being a hallmark for both acute lymphoblastic and acute myeloid leukemia \[[@B130-ijms-21-04510]\], as well as in hematopoiesis \[[@B136-ijms-21-04510]\]. Defects on MLL2/Set1 (also referred to as KMT2D in mammals) trigger the rare autosomal dominant Kabuki syndrome 1 \[[@B135-ijms-21-04510]\]. MLL3 and MLL4 have been connected to cancer development, owing to interactions with p53 \[[@B143-ijms-21-04510]\], which has led to use of them as potential targets in treatments against leukemia \[[@B144-ijms-21-04510],[@B145-ijms-21-04510],[@B146-ijms-21-04510]\]. Other approaches are related to MLL1/MLL2 translocation problems \[[@B147-ijms-21-04510]\].
As stated before, Set1/MLL is implicated in lifespan and ageing as well. High levels of H3K4 methylation are undesirable for an extended longevity, where reactive oxygen species (ROS) play a vital role \[[@B67-ijms-21-04510],[@B148-ijms-21-04510]\]. An increase in ROS is related with a H3K4me3 reduction regulated by Set1, as observed in *C. elegans* and in yeast \[[@B149-ijms-21-04510]\].
Furthermore, Set1 is required for proliferation of ESCs, iPSCs, and neuronal stem cells. Besides participating in leukemia, MLL1/MLL2 are indispensable in embryogenesis \[[@B130-ijms-21-04510]\]. A Set1/COMPASS subunit required for H3K4me3 possesses the CXXC zinc finger protein 1 (CXXC1 or Cfp1, a Spp1 yeast ortholog), which binds to unmethylated CpG islands \[[@B150-ijms-21-04510]\]. CXXC1 has been described as having a pivotal function in oocyte development \[[@B151-ijms-21-04510]\].
As described here, recombination hotspots are triggered by PRDM9-mediated H3K4me3. Deficiencies in this gene are associated with defective synapses and male infertility \[[@B140-ijms-21-04510],[@B141-ijms-21-04510]\]. CXXC1 also interacts with PRDM9 in spermatocytes, but it is not essential to form DSBs \[[@B152-ijms-21-04510]\].
Among others, in the case of H2B ubiquitination, Rad6 (HR6B in mammals) defects result in male infertility \[[@B97-ijms-21-04510],[@B134-ijms-21-04510]\], and Rad6 is overexpressed in ovarian cancer \[[@B142-ijms-21-04510]\]. Apart from PAF1c's involvement in meiosis and H2Bub, it is also correlated with cancer development \[[@B153-ijms-21-04510],[@B154-ijms-21-04510],[@B155-ijms-21-04510]\]. The Paf1 subunit interacts with CXXC1 and PD2 (pancreatic differentiation 2), and is upregulated in ovarian and pancreatic stem cells \[[@B156-ijms-21-04510]\]. PAF1c is also implicated in parathyroid carcinoma (PC) \[[@B137-ijms-21-04510]\], ESC pluripotency maintenance \[[@B138-ijms-21-04510]\], and mitochondrial autophagy \[[@B139-ijms-21-04510]\].
5. Future Directions and Concluding Remarks {#sec5-ijms-21-04510}
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All chromatin-dependent functions are regulated by epigenetic modifications. Among them, histone modifications constitute one of the best-studied regulatory elements that orchestrate specific patterns that will modulate from gene expression to meiosis. The in-depth study of these modifications has provided an extremely interesting view of how different histone modification patterns could lead to the renovation of chromatin structures that allow the signalling required to trigger a specific process. Notably, most of these modifications are executed by multi-subunit complexes that can accomplish different functions depending on their partners and the environment. Our knowledge of how specific histone modifications---for instance, the focus of this review: H3K4me and H2Bub---occur upon a precise signal is far from being complete.
Many questions remain unanswered---for instance, (i) how can the histone writers and erasers differentiate between genomic loci? (ii) How can the histone readers discriminate between the distinct chromatin-based processes? (iii) Which is the interconnection between specific histone modifications that regulates different biological processes? (iv) Are the molecular mechanisms behind each modification shared between molecular events? (v) Is there a set of specific factors that regulate the writers and the erasers only during meiotic recombination? An interesting possibility is that the capacity of different subunits to bind to different partners enables them for specific roles. As a matter of fact, the presence of Spp1 in both processes might indicate its implications in both of them and how they are linked, given that it is localized to COMPASS, allows the deposition of the H3K4me3 mark, and when forming a subcomplex with Mer2, is able to read the mark and conduct DSB formation \[[@B59-ijms-21-04510],[@B81-ijms-21-04510],[@B82-ijms-21-04510]\]. Its presence as differentiated pools might highlight its role as an important interplayer in meiosis and transcription.
Much effort has been focussed on addressing the role of the histone H3K4me and H2Bub writers (COMPASS and Rad6/Bre1/Lge1, respectively); meanwhile less is known about their erasers (Jhd2 and Ubp8/Ubp10, respectively). Jhd2 has been better studied, indicating that it is a general regulator, being essential for delaying transcriptional quiescence during sporulation. In fact, *jhd2∆* cells show a precocious gametogenesis and stress-sensitive spores \[[@B157-ijms-21-04510],[@B158-ijms-21-04510]\]. Additionally, Jhd2 is known to act with COMPASS to ensure a symmetrical H3K4 methylation, and its demethylase activity seems to be hindered by Spp1 \[[@B72-ijms-21-04510],[@B81-ijms-21-04510]\]. In contrast, less is known about the role of Ubp8 or Ubp10 in H2B deubiquitination during meiosis. Interestingly, the enzymatic activity of Ubp8 is carried out as part of the SAGA complex \[[@B159-ijms-21-04510],[@B160-ijms-21-04510],[@B161-ijms-21-04510]\]. SAGA also has the ability to acetylate histones through the activity of Gcn5 \[[@B21-ijms-21-04510]\], which is of extraordinary relevance for transcription activation and the expression of early genes indispensable for entrance into the premeiotic S phase (see above) \[[@B98-ijms-21-04510]\]. Notably, in *S. pombe* the control of a master regulator of cell fate decision, Ste11, depends on different activities of the SAGA complex for the switch from mitotic growth to sexual differentiation \[[@B162-ijms-21-04510]\]. Is Ubp8 activity also essential for early gene activation? Does SAGA participate in meiotic recombination through the coordinated activity of Gcn5 and Ubp8? Future investigations will shed light into these possible roles for SAGA during meiosis.
Everything that has been described here brings our attention to the profound relationship between H3K4me and H2Bub, and the need for understanding more deeply how the machineries that write, read, and erase them are able to discern between the different molecular events occurring in chromatin ([Figure 5](#ijms-21-04510-f005){ref-type="fig"}).
We are grateful to members from the Rodríguez--Navarro lab for their feedback.
Writing-original draft preparation, J.S.-Q. with the help of S.R.-S.; writing-review and editing, S.R.-N.; supervision and funding acquisition, S.R.-N. All authors have read and agreed to the published version of the manuscript.
This research was funded by MICIIN, Spain, grant number PGC2018-099872-B-I00. The APC was funded by MICIIN (PGC2018-099872-B-I00). J.S-Q. was funded by the FPU (Formación de Profesorado Universitario) programme (MICIIN, FPU15/03862).
The authors declare no conflict of interest.
DSB
Double-strand break
PTM
Posttranslational modification
COMPASS
Complex of Proteins Associated with Set1
PAF1c
RNA polymerase II associated factor 1 complex
cryo-EM
Cryo-electron microscopy
RRE
Rme1 repressor element
lncRNA
Long non-coding RNA
APC/C
Anaphase-promoting complex/cyclosome
SAGA
Spt-Ada-Gcn5 acetyltransferase
CO
Crossover
NCO
Non-crossover
RMM
Mer2-Mei4-Rec114
MRX
Mre11-Rad50-Xrs2
SC
Synaptonemal complex
CE
Central element
LE
Lateral elements
SIC
Synapsis initiation complex
SPB
Spindle pole body
NDR
Nucleosome-depleted region
SAM
S-adenosine-methionine
CM
Catalytic module
RRM
RNA-recognition motif
APT
Associated with Pta1
PHD
Plant homeodomain
DUBm
Deubiquitinating module
FACT
Facilitates chromatin transcription
CENP-A
Centromere protein A
TSS
Transcription start site
TFIID
Transcription factor II D
RNAPII
RNA polymerase II
CTD
C-terminal domain
HMD
Histone modification domain
ARM
Arginine-rich motif
ESC
Embryonic stem cell
iPSC
Induced pluripotent stem cell
PC
Parathyroid carcinoma
ROS
Reactive oxygen species
PD2
Pancreatic differentiation 2
{#ijms-21-04510-f001}
{#ijms-21-04510-f002}
![The Complex of Proteins Associated with Set1 (COMPASS). The subunit arrangement model shown here is inspired from the cryo-electron microscopy (EM) structure published by Qu et al. in 2018 \[[@B74-ijms-21-04510]\], which featured a truncated version of Set1, without the N-terminal. This region, as well as Shg1 and Swd2 subunits, have been added to the model (dotted lines). In red (Bre2, Sdc1, and Swd) are the subunits necessary for H3K4me2/3; in blue (Set1, Swd1, and Swd3) are the subunits necessary for H3K4me1/2/3; and in yellow is Spp1, which is necessary for H3K4me3. Little is known about Shg1. The structure is accompanied by several processes in which COMPASS subunits participate.](ijms-21-04510-g003){#ijms-21-04510-f003}
![Structural overview of COMPASS upon H2B ubiquitination (**A**) Change of conformation of the COMPASS complex upon ubiquitylation of H2B. Swd1, Swd3, and Spp1 subunits rotate away from the rest of the catalytic module of COMPASS; Swd1 establishes a series of contacts with the ubiquitin that strengthen the interaction and stabilize the conformational change; (**B**) Schematic representation of the arginine-rich motif (ARM) stabilization. The ARM is stabilized by the contact with the ubiquitin, and is able to interact with several residues of the acidic patch of H2A, after having adopted an α-helix conformation, according to Worden et al. \[[@B127-ijms-21-04510]\]. Upon the conformational change adopted by the ARM, the residues participating in the interaction with the acidic patch vary, as indicated.](ijms-21-04510-g004){#ijms-21-04510-f004}
######
Role of H2Bub and H3K4me3 in transcription and DSB formation. (**A**) General overview of the cross-talk. PAF1c recognizes RNA polymerase II (RNAPII) phosphorylated on the S2 and S5 of its CTD (1), and promotes H2B ubiquitination by Bre1--Lge1--Rad6 (2,3). This modification is recognized by the COMPASS complex (4), which, in turn, trimethylates H3 on its lysine 4 (5). Conversely, H2Bub is removed by the ubiquitin-proteases Ubp10 and Ubp8, the latter belonging to the DUBm of SAGA (6). Trimethylation is eliminated by Jhd2 demethylase (7); (**B**) Spp1 is situated on the chromosome axis, interacting with the RMM subcomplex. In the chromatin loop, the first nucleosome after a nucleosome-depleted region (NDR), situated around the transcription start site (TSS) of a gene, is ubiquitinated by Bre1/Rad6, in coordination with PAF1c (I). H2Bub is read by COMPASS (also containing Spp1), which is now able to methylate H3K4. This H3K4me3 mark on the nucleosome begins to be attracted and protected by COMPASS-free Spp1 situated on the chromosome axis (II). The NDR becomes closer to the chromatin axis, thanks to the interaction with Spp1, and Spo11 is recruited, giving rise to DSBs (III).
ijms-21-04510-t001_Table 1
######
List of some human diseases associated to machineries involved in H3K4me3 and H2Bub.
Complex. Pathologies Associated Reference
------------------------------------------------------------------------------------------------------ ------------------------------------------ -------------------------------------------------
COMPASS/Set1 Lymphoblastic and acute myeloid leukemia \[[@B130-ijms-21-04510]\]
Kabuki syndrome 1 \[[@B135-ijms-21-04510]\]
Hematopoiesis \[[@B136-ijms-21-04510]\]
Embryonic stem cell (ESC), induced pluripotent stem cell (iPSC) and neuronal stem cell proliferation \[[@B130-ijms-21-04510]\]
Embryogenesis \[[@B130-ijms-21-04510]\]
PAF1c Parathyroid carcinoma (PC) \[[@B137-ijms-21-04510]\]
ESC pluripotency maintenance \[[@B138-ijms-21-04510]\]
Mitochondrial autophagy \[[@B139-ijms-21-04510]\]
PRDM9 Defective synapsis and male infertility \[[@B140-ijms-21-04510],[@B141-ijms-21-04510]\]
Rad6 Male infertility \[[@B97-ijms-21-04510],[@B134-ijms-21-04510]\]
Ovarian cancer \[[@B142-ijms-21-04510]\]
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1951 Jordanian general election
General elections were held in Jordan on 29 August 1951. As political parties were banned at the time, all candidates ran as independents, although some affiliated with the Jordanian Communist Party, the Ba'ath Party the Arab Constitutional Party and the Umma Party all won seats.
References
Category:1951 elections in Asia
Category:1951 in Jordan
1951
Category:Elections in Jordan
Category:Non-partisan elections
Category:August 1951 events
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Related branches
Related bugs
Sprints
Whiteboard
MER: Is this implemented?
CM: Yes, a working version is here lp:~corrado-maurini/dolfin/tao. I filed a merge request. But I need some feedback on the interface. It requires the installation on TAO. By the moment I implemented an interface only for a solver of linear problems with bound constraints, which is an extension of PetscKrylovSolver. I added a FindTao.cmake, which works correctly on macosx, but may have some problem on some linux platforms (i had to force the test to true in ubuntu). I am not an expert of cmake and I need help on it.
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If Vladimir Putin fulfills the goals he's set for his new six-year term as president, Russia in 2024 will be far advanced in new technologies and artificial intelligence, many of its notoriously poor roads will be improved, and its people will be living significantly longer.
There's wide doubt about how much of that he'll achieve, if any of it. Analysts assessing the prospects of his term that begins with Monday's inauguration often use the expression "neo-stagnation."
Putin won the new term, which will extend his rule in Russia to a quarter-century if he completes it, with an official tally of 77 percent of the vote in March. Yet, when state pollster VTsIOM asked Russians a month later which politician they trusted to solve the country's problems, only 47 percent chose Putin.
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National Framework of Qualifications
The National Framework of Qualifications (NFQ) is a system used to describe levels of educational qualifications in Ireland. Responsibility for maintaining and developing the framework lies with Quality and Qualifications Ireland (QQI).
Launched in 2003, the NFQ was developed by the National Qualifications Authority of Ireland as a means of comparing training and qualifications between institutions of education at all levels. It encompasses learning at primary and second level, as well as acting as a benchmark for required standards for graduates of courses offered by QQI, and universities. The framework consists of 10 "Levels", ranging from Certificates at Level 1 which signify initial learning to degrees at doctoral level. A 'fan diagram' is used to illustrate the progression of the levels.
Framework
References
External links
National Framework of Qualifications website
Relationship between the Common European Framework of Reference for Languages and the National Framework of Qualifications
Category:Academic transfer
Category:Education in the Republic of Ireland
Category:Educational qualifications in Ireland
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Fall 2016 NewsletterDecember 2016[Download]The Coalition's fall newsletter, Update & Review, can be read at the link above. Featured is reporting on the meeting with North Korea's vice foreign minister - in Pyongyang. Keep an eye out for these other news and issues:* The Coalition Negotiating with North Korea - U.S. remains recovery/air loss sites* The Bring Our Heroes Home Act/ Declassification legislation - Donna Knox* USRJC 20th Plenum/meeting - The search for MIAs taken to the former Soviet Union* Research (John Zimmerlee) and the Cold War (Paul Fees)* The White House, Congress, Defense Department (DPAA Acting Director message)* The Coalition’s Year-In-Review!Summer 2016 NewsletterJuly 2016[Download]The Coalition's summer newsletter, Update & Review, can be read at the link above. As you read through the newsletter, keep an eye out for news on these particular issues:* The Bring Our Heroes Home Act – 2016 (Senate declassification legislation)* Trickle Identifications (New Next Generation DNA Sequencing and the Punch Bowl Unknowns)* House Resolution 799 and Congress’ North Korea Sanctions Enforcement Act/Amendment(Calling on renewed recovery operations in North Korea and protecting them from U.S. sanctions)* DPAA Director Mike Linnington’s resignation and replacement* Research –John Zimmerlee* The Coalition’s new website
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The level from the May/June 2014 OLDC. Run and jump through things in 2D mode and eventually you can climb the big clock tower to reach your goal of... something! The level itself is currently unchanged from the OLDC release, but there are some scripting fixes that might make the level slightly more playable for some.
Changelog:
V2
Updated the version of Axis2D used so the level doesn't crash on load in newer game versions. Also allows use of turn left/right keys for movement.
Improved performance correlating to bouncy chains. (For a measure, I can run the level at full speed on my laptop's low power setting now.)
Slightly reduced Robo-Hoods' firing range further.
Future plans are to give the level a proper update with redone enemy placement, possible new enemies, and general layout tweaks. Possibly with an extra alternate path added? For now, enjoy a version that works on 2.1.11 :v
(The developer time mentioned in the emblems screen is 1'53"45, and you can view a replay that slightly desyncs, sorry by entering "PLAYDEMO DEVTIME" in the console.)
I was just going to go ahead and release this, but then I decided it'd be worth giving a more in-depth review.
I'm sure you don't need me to tell you that you've really made something great here, as any good designer knows it without being told. Nevertheless, I'd like to say it anyways:
This is fantastic. You took several completely original concepts to SRB2 as we knew (2D Axis platforming, real trampolines) and several old ones, and used them all in fantastic and progressive ways, getting more and more interesting and difficult as the level went on. I never thought I'd ENJOY seeing a robohood in SRB2, but your use of them as passive stage hazards a la kirby 64 cannons was a choice that seemed tasteful and fair. You hid secrets all over the place, requiring the players to really think about their path of movement to get to seemingly impossible things. I love that stuff.
My favorite part was the large round tower which you ascended the outside of, each revolution being a little above the path below.
There is one major flaw with your level though, and that is the visual design. That's a topic that is hard to quantify though, so I'll try my best.
This is exactly the example I need. What we have here, is a complete mishmash of textures, all different styles, sizes, and colors. You have the geometrical Sapphire Falls walls textures and grass, the semi realistic clean wooden slabs (all different sizes of it), an uber realism aged brick texture, and a twinkling starry sky that looks like it would be better served to fit some sort of fairytale theme. The sky is the wrong dimensions too, and it stretches horizontally, making your stars oval shaped. Also I know it's vanilla but seriously those flowers are so bad looking in every way
Do I even need to mention the cut in half clocks? I'm honestly not sure how you did that and didn't notice it. :p
Though having a cohesive style of texture patterns would definitely help, I don't think it'd entirely solve it. Some of your level architecture is fundementally boring on the eyes. I mean, your clock towers look like cardboard boxes on posts. Considering the otherwise fantastic nature of your level, I feel it's deserving of majestic towers more on the tier of Big Ben and all.
Spoiler: big ben
Not all parts suffer from this though. A few notable examples of fantastic looking areas with excellent cinematics:
Gotta love those gears, which give a great feeling of motion and progress to the level.
This part is just amazing. The spiral of pillars really draws the player's eyes up and towards the huge clock, which is the backdrop. It feels like there's enough to look at, without it being too much.
Some of the rooms cannot boast this visual impressiveness though.
This one is just plain dull. Same texture plastered all over, and no real visual variation. I think to really fix up the visuals of rooms like these (there's quite a few) you'd have to, first, get a proper floor texture. That alone would jazz it up a lot, and putting some extra woodwork like the stuff in Sphere's Clockwork Towers, would really hit the target dead on.
Visuals aside, there are two small things in particular that bugged me, gameplay wise. The first, is these platforms:
The issue is that they're only used here in this one instance, and it's above a death pit. I didn't expect them to disappear at all, because every other platform in the level stays put, and I died to them instantly. The next time I played, I forgot they existed, and made the jump again, only to have them disappear out from under me. If you're gonna introduce a gimmick like this, it's best to do it in non death situations first, so I can know what I'm up against.
Lastly, this monitor is rather unpredictable, and is instant death if you get it wrong.
I didn't expect the collisions of the spikes to rise above the top of the box itself, so I figured I'd be safe to grab it as long as I didn't stand around down there. I was not though, as the spikes apparently reach above the box, and it sent me plummeting to my doom.
I don't expect you to fix any of this, though. I wouldn't if I were you. This is already a fantastic level, and it's best to take the lessons learned onto future projects, rather than refining the same thing over and over again.
...That's all I have to say. Released. 10/10
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A dome structure? something that could be filled with diatomaceous earth?
The sky is the wrong dimensions too, and it stretches horizontally, making your stars oval shaped.
Do I even need to mention the cut in half clocks? I'm honestly not sure how you did that and didn't notice it. :p
Those are OpenGL issues. Both of those look right in Software. (The latter, at least the one instance of it I know of, was an accident in texture alignment that I liked enough to keep.)
I appreciate the rest of the criticism, though! I would eventually like to update it a bit, possibly if I put it into a level pack, to fix some of the issues and maybe add a bit more to the gameplay that I didn't have time to for the contest. Visual design is definitely my weak point, I will admit, and some areas of the level could really benefit from some custom texturework and general work on the geometry. The appearing/disappearing platforms were an effort to work around popup spikes not working properly on FOFs, and now that those work properly I could quite easily go through and add them where they'd benefit the gameplay.
Those are OpenGL issues. Both of those look right in Software. (The latter, at least the one instance of it I know of, was an accident in texture alignment that I liked enough to keep.)
Oh. My mad rig can't handle SRB2's ancient software, so I have no choice but to play in opengl. That being said, I've never seen it cause issues with midtextures before... and I'm making a level pretty much all midtextures used in weird ways. Never seen it stretch the sky either, but I'm sure that just has to do with the different way opengl handles sky images of different resolutions.
__________________
A dome structure? something that could be filled with diatomaceous earth?
Oh, maybe.
But, since that NeonSonik does not specified what was talking about, I take as reference the thread name, which is the zone name too.
But second thought, your idea makes more sense if we consider "title" and "zone name".
Right. I can get the more obvious things when someone's talking about something that can actually be a problem; the zone name isn't and shouldn't be the issue here, since it's just the level's name. He probably thought that the title screen wasn't fitting to him because it wasn't funny, lol
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Hanoi, Vietnam.
Think of your sensory inputs right now. You can see almost 180 degrees from left to right and from top to bottom. You can hear in two ears at once and have a few square meters of skin that report to your brain on texture, temperture, humidity and airflow. Are you cold? Are you sweating? Are your belt or shoes too tight?
Your nose, if it works properly, is pulling hundreds of scents from the air and trying to figure out what they are. Is that smell chocolate? A cup of coffee? A diesel truck driving by?
The senses don’t stop at the outside of your body. Past injuries still hurt and send signals to your brain: a torn muscle, a slipped disk and arthritis in your joints feed your brain information all the time. This is a constant flow of pain and it is easy to ignore — but tune into it and it still hurts. If you concentrate you can even feel your heart beating against your chest.
The senses don’t stop at the physical body. Imagine you’re sitting in a coffee shop. A friend sits down next to you and is unexpectedly wearing the same cut and color of Converse that your friend Sarah wore in highschool. Without wanting it your brain starts to drill into past memories with Sarah: the wind blowing on your face in a car with the sunroof down, scraping your knee climbing trees together in Golden Gate Park, clammy palms and the smell of mold as you snuck into a cheap movie theatre together.
Information is coming to our brain from physical senses and mixing with past memories and experience. The senses flow so quickly that it’s impossible to give each our full attention, our subconcious selects the bits that we can handle and presents them to our mind, our mind triggers a memory of a past experience and paints on top of what is happening right now with the ghosts of past sights, smells and thoughts.
As sit in a coffee shop in Vietnam writing this even a glimpse of a coffee cup from the corner of my eye brings to mind the taste of coffee and the feeling of a warm mug. For the instant those thoughts exist they feel entirely real, more real than what I feel with my two real hands or taste in my actual mouth.
My eyes don’t actually see that much, my ears don’t actually hear that much, but my brain is processing what they sense and painting all over it according to my past. That’s the lens through which I see the world.
So what are the biases of a traveler?
It’s not just that people in foreign countries speak limited English, it’s that their English skills only allow them to express basic thoughts. Over time this can wear me into thinking that foreign people aren’t intelligent. That’s bullshit, in their native language foreigners are having the same complicated conversations you and I have at home and are making the same complicated jokes.
I don’t read the news when traveling and I don’t pass friends in the office, so I don’t hear about TV shows and local fiascos. This can make me think that the world has stopped. The world hasn’t stopped, I’ve just tuned it out. Life in abroad isn’t necessarily more “in the moment” or slower than life at home — it’s just easier for us to tune out the distractions.
Interacting with people in the tourist industry can be frustrating, but people in the tourist industry are not representative of a culture. Think about your home country: are your brightest and nicest friends working at hotels and coffee shops? Does poor service represent how all people treat each other or just how service workers treat tourists?
Even being uncomfortable in an environment changes my perception. In Asia I’m often hot and have minor indigestion. I might think everyone in Asia feels like this all the time — they don’t. Home for them probably feels as comfortable as home does for me.
Few currencies are as valuable per unit as the US Dollar, the Euro, or the British Pound. So I sometimes can subconciously think “Our currency goes futher here, our currency is better.” This isn’t true, currency unit values are relics of the past. Most currencies these days are stable and the number of units of a currency from my home country needed to buy something has nothing to do with the intrinsic value of my country.
One of the least visible biases for me is that caucasians are almost universally considered to be richer, smarter, and better looking than other races. As a result as a caucasian traveler I will be more welcomed, treated with less suspicion, and assumed to have more money than other races. Over time this treatment could lead me to think I’m actually richer, smarter, and better looking than other people. I try to be aware of how people in foreign countries treat me and of the impact it could have on my perception of self.
What can I do about travelers bias? I can try to be aware that my own past is constantly altering reality. Another person will have a totally different experience than I will.
Maybe ask these questions:
How does what’s happening to me compare to what happens to a foreigner in my own country? How does where I am now compare to a similar tourist sight at home? Are people treating me differently because of my appearance? What’s the strangest thing in my home country and how would it look to someone from here? What’s going on around me that I can’t understand becuase I’m not fluent in the language? How is this familiar and how is it foreign? How would I feel if this were totally normal?
The most surprising fact of travel so far is that across countries most things are the same. People sit on chairs, drive cars and motorcycles, use smartphones, use Google and Facebook, and mostly wear western-style clothes. Urban people of the world like the same things: Levis, iPhones and button down shirts.
It’s not even clear that we can call these “western styles” anymore — there is as much Japanese and Chinese influence on the design of an iPhone 5s as there is European influence. It feels more accurate to call the prevailing style and tastes “monied world” tastes. As a traveler it’s so hard to escape the monied middle class world that much of the world feels just like home.
Travel photos try to show pristine views but this is far from reality. Every tourist sight I’ve been to swarms with people and getting a picture without another tourist is the exception rather than the rule.
There are two more travelers biases to mention: The first is that I can pull my ripcord at any minute. The GDP per capita of Vietnam is $1,600/year. At any minute I can pull out a credit card and buy a $3,000 flight to New York City. Locals don’t have this luxury, so they’re much more committed to this life than I can ever be.
The second bias is driven by information. I go where the airports are, where the metro takes me, and where Google recommends. When I travel somewhere and think “this doesn’t look like National Geographic” I remember that the truly local parts of the world, the undeveloped and “authentic” ones, aren’t going to show up on a map.
Locals might like it that way.
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Hi Mamas,
I am beside myself with a very beautiful, kind and shy DD who is 25 months. I have APed from the beginning and been a SAHM since her birth. I think in hindsight I should have introduced her and left her with more people, which I hardly did and now it feels even more difficult because of how she reacts.
We have a playgroup (have met about 7 times) and she doesn't interact with the other kids. She looks and smiles at the moms. No waving, no greetings. By the end of the time together she starts talking and having fun by herself - but until she has warmed up she is glued to me. Not unhappy just glued.
I have registered her for art, dance and swimming classes (all accompanied by me or DH). She seems to be enjoying the dance and swimming class and in the art class she would rather sit and watch the other kids than do art herself. I am supportive and loving about all of this BUT inside I am so upset and so frustrated that she is so SOCIALLY FROZEN.
That being said when she is with friends and family for long stratched of time, a whole day up to a couple weeks - she is interactive and loving. She plays with the other people - usually adults but also little babies and is genuinely comfortable. Interestingly she is much more comfortable with women than men.
Has anyone else experienced this? We have moved twice in her 2 years, across the country so her social network has been shifted and she has certainly felt sad about leaving her whole extended family behind - but that was 6 months ago.
We also go to the park with neighbourhood kids daily - so she is certainly seeing lots of people but I want her to interact a little more.
I would love ideas about ways to gently encourage that and to help bring her out of her shell. Book suggestions would be great too.
I feel like I have failed somehow and would really like to do something to help her feel more confident with people.
Boy, does your post strike a chord with me today! I had DD (22 mos.) at the play area in the mall and one of the other moms asked me if DD had any older siblings. I replied that she is my only child and the woman said, "yeah, you can really tell."
I wasn't offended or anything, but it really struck me how skittish DD is around other kids. We do playgroups and she has cousins her age, but she is easily overwhelmed by kids just being kids. I don't want to turn DD into someone she isn't, but I can see that I really need some guidance in building her confidence.
I hope you don't mind if I mooch off the answers to your post!
--Trish (mom to the Tiny Queen at home, but the Tiny Wallflower in public)
Now I see why you found my thread for sensitive children. I have been soul searching a lot about this. Our children (as mine is like yours) are very cautious. One thing I have learned with a little NLP practise is not to label. Only last week I said, "she is shy" and in this past week, have learned how limiting that is on her. Now I think, "she has behaved with shyness." which stops her past predisposing her future. One says - she IS shyness, and defines her as a person. The other says, - her actions have been a certain way, but won't necessarily stay that way. Leaves the options open.
Also, this cautious behaviour will have great benefits in the future, as she won't jump headlong into things without thoroughly checking it out first. Their behaviour is very intelligent, and beyond their years. It can hurt on many levels as a parent of such children. We all wish for our loved ones to be involved and reach out.
As long as they can eventually warm up, which you mentioned your dd does, then they are better than fine. They will choose wisely, including friends. Some people like myself and my dd are more happy with one good friendship, and one on one interaction. The occasional big loud get together or party is great to watch and be in, but in general, you may find she prefers smaller company.
What you are doing is perfect. Just giving her opportunities and standing by to support her through it is all she needs. Watch for signs of what she enjoys most, and duplicate those things. If she seems to like one particular person, get to know the mom and have play time. One on one is a much better way of developing social skills anyway.
Calm, that's a good point. I know that when I was young, my parents always told people "She's shy" and I just stayed that way my whole life. I try not to do the same with my daughter, who has shy tendancies. I tell people "she's going through a stranger anxiety phase", but encourage her to play with other kids at her own pace.
I bring her to a playgroup every once in awhile, and she just stand in the center of the room watching all of the other kids. She doesn't interact with them. Just watches.
When my husband decided to go back to school, and we had to look into childcare, we decided against a formal daycare because it just seemed like it would be too overwhelming for her. There were about 30 toddlers running around and she looked so frightened. We decided to ease her in, by taking her to a sitter first. Her sitter watches one other girl, who is a month or two older than her, and they get along great! It's really helped her get over her shy behavior. She even allowed my husbands aunt and uncle to hold her the other day, which is unheard of for her.
I really think that easing her into social situations was the best way to do it for her. She's much more outgoing now.
I know all kids are different, but maybe it is that age... my ds was the same way, I've sat at the mall play area with him for 20 minutes before he ventures out to play. And when he did play, he would stand aside until the toy was completely free of kids (about 2% of the time!) and then he'd go on it. He didn't interact often if we went to playgroup, finally warming up and not playing with just me by the very end.
Recently we went to a wedding and he was "shy" the whole time, until the band started up, then he danced for an hour! Two days ago at the mall, he rode the little train that is there, and really interacted with the kid in the caboose with us (I got to ride too, lucky me!), and then we went to the play area and he yelled "kids!!!" and ran right in there like the rest of them, playing on every toy like it was his last chance on earth. It was like a total shift in personality over the course of two weeks. I never pressured him to play or anything, he just decided to do it on his own. He is 33 months (he will be 3 in January).
mom of a "shy" 3.5 yr old here. i'll relate my experience and you can see what might resonate with you. dd1 was very shy even as a baby. we have not moved and have been involved in the same playgroup since she was about 5 mo old. she has never really played a whole lot with other kids, though. i think she just has a very cautious temperament. she hates people getting in her face or taking her stuff and y'know that's what a lot of little kids do. i've been reading "kids, parents, and power struggles" by kurcinka recently (highly recommend it) and she talks a lot about temperament in there. some folks just take longer to warm up than others. my dd1 is definitely one of those, but now she's starting to really open up.
we're taking a dance class this fall (moms stay and watch) and she loves it. she had developed the common 3 yr old girl ballerina obsession prior to class so it was an easy classs to decide to take. i guess we've been to 5 or so classes now and dd1 is really starting to participate. she was always dancing before, but was on the fringes and not joining in the circle, etc. last class she still did her own thing, but really listened to the teacher and followed instructions more than ever before. she even joined in the circle a time or two.
i think preschool would have been a disaster for us. maybe next year. dd1 is also _very_ oppositional. i have a post elsewhere (http://www.mothering.com/discussions...d.php?t=198122) where i mention a typical encounter with a well meaning person in the grocery store:
clerk: that's a very pretty dress
dd1: it's NOT a pretty dress!
clerk: what's your name?
dd1: i don't have a name!
this happens almost every time we're out. there have been a few tims recently where she's struck up a conversation with someone though not usually if they point out her dress orsomething. i can see glimmers, though, of her growing out of some of the opposition and defiance and shyness, too.
if you want my advice i would say do not push anything anymore than you would push her to walk or potty train or sleep through the night by herself. just be there for her and support her and when she's ready she'll let you know.
hth
Mama to two girl beans, Feb 2001 and Nov 2003 . DH , and two crazy . Running on biodiesel since 2004!"All you fascists are bound to lose" — Woody Guthrie
I really appreciated your suggestions and hearing about your little ones. I also really appreciated the positive reinforcement. I also try not to use the word "shy" and rather say that "DD just takes her time". But I find that other people are quick to label her "shy" and I am constantly correcting or ignoring.
Nikki, when your child was left with the sitter (that sounds like worked really wel!!)l - did she have a hard time adjusting?
My DD seems to be warming up to a great woman in the neighbourhood and I am thinking of suggesting that she spend time at their house with their DS ---after we spend more time together of course. I am wondering if you experienced resistance and to what extent you respected and listened and to what extent you pushed your childs boundaries a little. I would love to hear other mamas experiences here as well. Part of me could use the time away (professionally and academically) and part of wonders if it would just be good for her if I did.
It is really reassuring to read that some kids move past having such social isolation and join groups and enjoy themselves. I really wish that for my daughter.
Today was a funny day, she was super clingy, not wanting to do anything alone and wanting to nurse more than usual but she was happy in her dance class and with her dancing teacher and with this women I discussed above she was friendly, smailed brightly and blew them numerous kisses.
The degree of mystery to our actions and experiences always boggles my mind. The degree to which I understand does also.
I really appreciated your suggestions and hearing about your little ones. I also really appreciated the positive reinforcement. I also try not to use the word "shy" and rather say that "DD just takes her time". But I find that other people are quick to label her "shy" and I am constantly correcting or ignoring.
Nikki, when your child was left with the sitter (that sounds like worked really wel!!)l - did she have a hard time adjusting?
My DD seems to be warming up to a great woman in the neighbourhood and I am thinking of suggesting that she spend time at their house with their DS ---after we spend more time together of course. I am wondering if you experienced resistance and to what extent you respected and listened and to what extent you pushed your childs boundaries a little. I would love to hear other mamas experiences here as well. Part of me could use the time away (professionally and academically) and part of wonders if it would just be good for her if I did.
It is really reassuring to read that some kids move past having such social isolation and join groups and enjoy themselves. I really wish that for my daughter.
Today was a funny day, she was super clingy, not wanting to do anything alone and wanting to nurse more than usual but she was happy in her dance class and with her dancing teacher and with this women I discussed above she was friendly, smiled brightly and blew them numerous kisses.
The degree of mystery to our actions and experiences always boggles my mind. The degree to which I understand does also.
Hi all! My dd is 18 months & has been cautious about social interaction (especially men ) since the age of 4 MONTHS!!! amazing! she's still cautious- in all things. We figure it's just her personality. She gets tons of social interaction- she goes to work with dh 2-3 times a week, we live downstairs from my parents, my 2 oldest friends had babies within 3 weeks of us ( & we go to a music/sign language class on fridays. she has a warm up time in every single situation- even going to my best friend's house with her 3 fun kids!
ITA about the cautiousness helping her in later life. she doesn't run headlong into anything. she examines, contemplates, observes. She's a lot like my dh & i'm grateful. I'm very impulsive & quick to judge & am learning from the 2 of them
lovetomom, it sounds like you're doing great things to encourage socialization while respecting the warm-up & need to be close. your dd will be very secure & confident!
beanma: I didn't know I was raising your daughter! That is my 2.5 year old to the letter.
It is very reassuring to know that there are other toddlers like my oldest. It seems like social situations just drain all of the energy out of her. It is genuinely hard for her to be around large groups. The problem with this is that I would love to involve us in a couple of activities, but I don't want her to spend the whole time begging to go home (she does). What do all of you think: should we just keep trying or take a month off and try again?
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Q:
Shutdown Mechanism in Unity 3D for Therapeutic Video Game
I am working on a therapeutic video game in Unity 3D which can only be played by a patient for 2 hours per day. After the two hour mark is reached, the game should not be able to be played until a full 24 hours later.
What is the best way to go about this in C# using any built in features of Unity3D? I am looking to use PlayerPrefs, Time.time (for the time the game has been on) and can't find anything in the documentation or forum that would let me access the current date.
My pseudo code is as follows:
variable that stores current date
variable that stores total play time
variable that stores the time that the 2 hour mark was reached
if it has been 24 hours past the last 2 hour mark:
game turns on
else
Application.Quit() - turn off the game
current total play time variable += Time.time
A:
It's as simple as using System.DateTime to access the current system time and date. C# in Unity runs on the Mono Framework (or .NET under special circumstances) and you can use most of their functionality.
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In the last 24 hours, a computer simulation by a team of Belgian engineers that tracks the “spread droplets” and “slipstream” of the exhalations, coughs, and sneezes of people who are running, walking, or cycling has gone viral. Perhaps you have seen this gif on Twitter, Facebook, or NextDoor. Or, as some people on our staff have seen, perhaps write-ups of it have been texted to you by concerned friends or family:
Though this was not the specific goal of the simulation, it is currently being used on neighborhood groups and social media as scientific evidence that people who are jogging and biking are putting others at risk. If you are getting “droplets” or “globules” on you, the thinking goes, you are at risk of contracting coronavirus.
“People should read and not misread my tweets and texts,” Bert Blocken of Eindhoven University of Technology, the lead researcher on the simulation, wrote in an email to Motherboard. “I have never and nowhere discouraged people from walking, running, or cycling. Rather the opposite. Maybe people should read more, and react less.”
Blocken has yet to publish a peer-reviewed paper about the simulation. In fact, he hasn't even published a non-peer-reviewed study. Instead, he spoke to a reporter in Belgium about it, who wrote a news article, which has now been aggregated and shared widely by many publications. Given what Blocken has put into the world, taken at face value, some people are understandably concluding that it is impossible to run or cycle safely in many cities; he recommends a distance of 65 feet between bikers and other people, something that is impossible to do in cities. The issue with Blocken’s suggestion that we “read more, and react less” is that there is almost nothing to read, and there is no study to critique.
Blocken’s team took the extraordinary step of speaking to the media about his research before publishing anything about it. There is no written study to read or interpret. We do not know the specifics about how the study was done or how the simulation was run because the research team has not shared that information.
On Twitter, Blocken said the “crisis is urgent, so exceptionally we turned order upside down: (1) media, (2) today I submitted the proposal for funding (3) peer review article later. Public cannot wait months for peer review. I have a short text, I will post it on Linked In within the next hour.”
A day later, that LinkedIn post has not been published. What the team has published is something that it’s calling a “white paper,” but which is actually a Google-translated version of the Belgian newspaper article that was not written by Blocken or his team, but which quotes him. Ansys, the company that did the simulation in concert with Blocken, has also published a short but vague press release. In the meantime, this simulation has gone viral.
Studies like this are "not really useful. Not to epidemiologists anyway. The amount of transmission from this route even if it is possible will be dwarfed by that from others."
A Medium post written by Jurgen Thoelen, who describes himself as an “entrepreneur, building clouds in all forms and shapes and life-long athlete” has been shared thousands of times. On Facebook and Twitter, the article is being shared in neighborhood groups and is being used to spur a battle between pedestrians and runners and cyclists. A typical comment is something like this, shared in an Iowa City "Quarantine Survival" Facebook page: “Omg people keep doing this. Runners and bikers with zero regard for fellow pedestrians 🤬🤬” The simulation has also been written up by the Daily Mail, while gifs, stills, and memes of the simulation, shared with little or no context, have spread on their own.
This is all to say that we are unsure of the specifics of this study, what it actually shows, what its limitations might be, and how it was done. What it's suggesting could be accurate and useful, but we have no way of knowing that at the moment. And yet, this research is already being used to ask people to change their behavior and held up as definitive evidence that running and cycling are irresponsible during the pandemic.
Blocken said in an email that this was not his intention.
“Choice made in agreement with all the researchers involved and both university media agencies. The crisis is worldwide and the situation is urgent,” Blocken wrote. “We did not want to keep results behind closed doors until we have found the time to write down the full story. If I would have done the opposite, we would receive criticism about that. Never possible to do right for everybody. Given all the fuss I notice now, I will do an extra other late night effort and post the full story on Linked In tonight.”
“By the way this is aerodynamics work, not virology. Good luck with speeding up procedures in engineering journals," Blocken added. "COVID-19 will not wait months or until our paper is published."
Blocken is right: We face an urgent situation, and it’s important to get rigorous science out as quickly as possible. But hundreds of other scientists have managed to publish peer reviewed research about coronavirus in the last few weeks, on an expedited time scale. Thousands of others have published studies that are not peer reviewed, but that are at least studies in the way we usually think of them: Their methods and findings are explained in a rigorous way that can be critiqued. Even though this is a dire situation, scientific publishing safeguards exist for a reason, and we've already seen during this pandemic that a rushed process has led to bad, inaccurate science being published (that's not to suggest that Blocken's research is bad or inaccurate science, we simply have no way of knowing based on what's been published.)
Even if the simulations hold water and are accurate, virologists and experts should be the ones making public health recommendations, not random "entrepreneurs" on Medium, which is what has happened because these simulations were not published with the specifics of how they were done or what they mean. This type of research is of course important and should be done, but it should be released in a responsible way, with the caveats, limitations, and unknowns explained clearly. Then the research should be used by virologists and public health officials to make concrete recommendations to people.
I showed Blocken's research to William Hanage, an epidemiologist at Harvard's Center for Communicable Disease Dynamics. He said that the virality of Blocken's research is harmful, and that Blocken's suggestion in the white paper that this research is a "modest contribution" toward the fight against Covid-19 "makes my blood boil."
"Where the droplets are is much less relevant than the amount of transmission that occurs via this route"
Crucially, scientists are still unsure how well the coronavirus spreads in the air, and many have cautiously speculated that the overall risk of transmission appears to be less outdoors. Globules and droplets do likely carry the virus, but that doesn’t mean that anyone who gets a droplet on them from someone’s breath is going to be infected. Transmission depends on a host of factors; scientists believe an important one of these is “viral load,” which is a measure of how much of the virus is present.
"On the epidemiology side—where the droplets are is much less relevant than the amount of transmission that occurs via this route," Hanage said. "Advice on physical distancing is really about *reducing* the risk of transmission rather than eliminating it altogether."
He said studies like this are "not really useful. Not to epidemiologists anyway. The amount of transmission from this route even if it is possible will be dwarfed by that from others." He added "it's concerning" how fast the study has traveled … especially "when you consider I have had to write this email rather than putting the finishing touches to a model of nosocomial transmission [in hospitals]."
In a footnote on the white paper, Blocken admits “currently the subject of intensive debates between scientists world‐wide—is to what extent the residue of micro‐droplets with the virus, after evaporation, still carries an infection risk. Further virology research should shed more light on this issue.” Last week, the Atlantic's Ed Yong spoke to many virologists about this, and there currently is no consensus about how dangerous it is to exercise or be outside, but there is much research suggesting that the mental health benefits of exercising outdoors are important and should be taken seriously.
The issue of viral load and transmission is not addressed or mentioned in the Medium article nor in the Belgian newspaper Blocken spoke to.
When I asked if he was concerned about the fact his work had gone viral, especially in write-ups by non-experts, he said, “I am surprised by this question. You with your expertise should know that one can control the first line of media attention, and then people write stories of the stories, and it is impossible to control," Blocken wrote. "That would have happened equally if the full paper had already been published. This is not my first big media coverage, so I have been there, done that. There is free press."
Hanage said it's probably OK to exercise outside as long as you "apply common sense."
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Continuous flow plasma exchange in the treatment of homozygous familial hypercholesterolemia.
We report the clinical and laboratory effects of continuous-flow plasma exchange in two patients suffering from homozygous familial hypercholesterolemia. In one (Case 1) plasmapheresis was performed at fortnightly intervals over a period of 18 months; in the other (Case 2) the necessity for surgical relief of an associated supravalvular aortic stenosis resulted in premature termination of the trial. The plasma cholesterol levels in both patients fell by 35 per cent from the mean before study in the course of treatment. In Case 1 this was associated with marked regression of the patient's xanthomas, disappearance of the S-T segment depression seen on effort electrocardiograms obtained prior to the introduction of plasmapheresis, possible widening of the stenosis present at the origin of the left anterior descending coronary artery, and a marked increase in exercise tolerance and diminished frequency of anginal attacks. Cessation of cholestyramine and clofibrate administration during this study did not in any way reverse the reduction of plasma cholesterol achieved by means of plasmapheresis combined with drug therapy. We conclude that plasmapheresis has a role to play in the management of patients with homozygous familial hypercholesterolemia.
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By Marilyn Mehr
I don’t know why I started laughing. Nothing was funny, not really, not the two of us, Annie and I, lurching along the freeway at breakneck speed to reach a neglected neighborhood of West Los Angeles, to show up for an appointment with a doctor we had never met on a Saturday morning in September 1993. We were two middle-aged women who had lived together for nearly 20 years and were both scared. ‘Nothing funny about that. From our home in Mt. Washington, down the winding roads named after the Mexicans who had carved them—“Cazador,”” El Paso,” “Figueroa”—to the sprawling freeways, taking us to the edges of the old Culver City movie lots, we had barely spoken, not even smiled.
When we pulled up beside a white stucco apartment building, I was sure that Annie would tell me to drive away and find the nearest coffee shop. But no, she pushed open the car door and found her way unsteadily to her feet and began crossing the sidewalk in a crablike walk to a set of stairs.
“Wait! I’ll help you.” I called out, but she waved me away.
Not one to pay attention to those resisting my help, I hurried along behind her until she had reached midway to the second story. For a moment, I averted my eyes as she missed the next step, grabbed the wooden stairway and tumbled backward pell-mell into my arms as we spiraled downward onto the landing falling into a potted palm. The air was blue with expletives (“stupid, fucking palm tree!”) as Annie swore at the mulishness of the plant, brushed the dust and humiliation from her clothes and tried to stand up. That’s when I started laughing.
“What the hell is so damned funny?” she yelled at me.“Nothing,” I gasped, one hand over my mouth as I reached with the other to lift her up. “It is funny, too funny—the two of us falling into a potted palm in a godforsaken section of L.A. in search of a Chinese magic doctor.”
Annie stood up, straightening her tweed jacket, brushing off her gabardine pants, while glaring at me and squinting her eyes against the morning sun. She rubbed a bruised patch of skin on her cheek. Then, determined to navigate the steps alone, she said, “Come on. We’re going up there to meet this little lady and see what she knows. You can laugh like a fool in a funhouse, I’m going in.”
Starting up the stairs again, she held firmly to the railing, lifting each foot deliberately until she reached the top. Then, searching for the apartment number and, seeing that the door was open, she stepped inside.
Annie tells me that I laugh when nothing is funny because I’m tense and afraid to show how I really feel. How I felt at that moment was hysterical. Nothing could have kept me from laughing, although God knows, I tried. When I saw the tables lined up in what was meant to be the living room, I was just curious. When I noticed a middle-aged man who appeared to be Asian, his head barely visible above a mound of paper sacks and twigs, I wanted to laugh. I put my hand over my mouth, trying to muffle my giggles under the steady snap of breaking branches. No one noticed.
Hearing us enter, the man jumped up, bowed and offered us seats on folding chairs lined up against a wall. Now, I knew that we would leave. Annie would survey this twig factory, take in the odor of decaying trees, make an excuse and we would leave. Perhaps, we could drive out to Santa Monica for lunch. The ocean air would do us both some good. To my surprise, she sat down, folded her hands in her lap and waited. Taking her cue, I did the same.
Nobody said anything for about ten minutes, the only sound being the steady cracking of the twigs. To divert myself, I tried to count the snaps, “one, two, three,” then multiply them as though taking a pulse to get the numbers per minute. I tried to focus, just to get some control, but couldn’t.
Finally, a short woman in a white lab coat appeared at the door holding what looked like a patient’s chart. Wearing glasses and sensible shoes, she projected an air of quiet confidence. She smiled as she asked, “Which of you is the patient?”
Annie raised her hand and attempted to rise. Seeing her difficulty, the doctor came to her side, offering her arm as Annie rose unsteadily.
“You are Annie,” she said. “I am Dr. Chang.” She placed her arm around Annie’s waist and guided her to one of the rooms, leaving the door ajar as she began questioning her about her symptoms.
As the interview proceeded, I tried to settle myself on one of the foldout chairs lined up against the wall, tracing our path to this little office in the middle of an impoverished suburban park on the Westside of Los Angeles. How did we get here?
Six weeks ago, I had sat across from Annie at our breakfast table watching the clouds pass over the San Gabriel Mountains while Annie flipped through the newspaper. We were both dressed, ready to run off to our jobs as professors, she at a nearby university, where she taught in the Counseling Department, and I at a nearby graduate school of psychology.
Suddenly Annie put her hand to the side of her face, her eyes wide in alarm. “My face is so hot! Do you see anything? I’m burning up!”
I reached over the table, to put my hand on her forehead. Her temperature seemed normal, not feverish, and her complexion was its usual light olive color. I told her that I couldn’t feel anything.
“Well, then, it’s gone,” and she folded the paper. “No, there it goes again! It’s like a street light—on/off, walk/stop—and now, I can’t see out of my left eye!”
I tried to remain calm as I watched and measured her responses. For the next five minutes, the flashes of heat and blindness continued until I finally blurted out, “I’m going to call a doctor who’ll see you today. Something’s wrong!”
As a psychologist who had worked in hospitals, I knew many physicians in the area and soon set up an appointment with a well-known neurologist. We both cancelled our regular appointments and tried to while away the time until we saw her. We distracted each other by listening to music, sharing a sandwich and flipping through some old New Yorkers, pointing to cartoons and trying to laugh.
Finally, at 2:00, we met with Dr. Nielsen. A slight woman in a white coat, hair drawn into a knot behind her neck, she was efficient, thorough and brusque. After examining Annie, she sent her for an MRI and lab tests, indicating that there were several options, all of which scared us silly: brain tumor, compressed nerves in the spine, optical neuritis and MS, that is, “multiple sclerosis.” By the time we returned a week later, Annie’s vision had returned but the flushing continued as well as problems maintaining her balance.
“I just feel tipsy,” she complained.
The doctor sighed, reviewed the chart, threw the MRI onto a screen and pointed to the lesions scattered like spilled pepper over her brain.
“We don’t know for sure, but it looks like MS,” Dr. Nielsen said folding her arms as though to block out contagion.
Annie replied cautiously, trying not to push back too hard. “Well, O.K. What can be done to treat it?”
“Nothing really,” the doctor replied, pursing her lips. “It may get worse, it may get better. We can try a round of steroids if it returns and it probably will, but for now, nothing. We’ll just watch and wait.” And with that, she closed the chart and left.
For the next few weeks, Annie returned to work, balancing herself on doorways and desktops, trying to conceal her symptoms until one day, exasperated by the doctor’s curt brushoff, I marched down the hall of my school, seeking out a colleague who had a reputation for knowing about alternative medicines. Paula’s office was covered with the posters of 60’s idols—Janis Joplin, Ram Dass, the Grateful Dead—her sympathies were clear. She still lived in the 60’s, as her dress easily confirmed--the long blonde braid trailing down her back, the rows of beads, the colorful headband and the Indian cotton blouse—all expressed her love of the counter-culture.
Although I had lived in that time, too, I had become more traditional, accommodating to the scientific method to survive in graduate school culture. I was open to alternative medicine, but I didn’t advocate any of its methods. After all, my own mother had been an ardent student of “natural” treatments, schooling herself with the ministrations of chiropractors, nutritionists, iridologists and phrenologists. She insisted that we never touch white bread, always buying whole grains, which she would grind, then bake into huge round loaves, free of all contaminants and she always resisted antibiotics as they “killed off the good germs, too.”
Paula listened carefully, asked a few questions and gave me a slip of paper that said, Dr. Junjiang Chang, Chinese Medicine. “Try this,” she said. “I think you’ll like her.” As I left her office and walked down the hall, I had an uncontrollable urge to laugh and could hardly get to the door of my own office before I burst out giggling. Here I am, a Professor of Health Psychology, trained in the scientific method and I’m finding Annie a quack to cure her MS. Well, western medicine has nothing to offer, why not turn to the east? Anyhow, I reasoned, it doesn’t matter because Annie will never go.
Yet, she did. When I told her about my conversation with Paula, Annie was eager to set up the appointment. “There’s nothing else,” she had said stoically, as we got into the car this morning. I was even more surprised when she surveyed the building, looked around the modest neighborhood and walked up the stairs, straight into the twig-snapping factory of Dr. Chang.
Dr. Chang wasn’t a traditional doctor and didn’t observe traditional notions about privacy, either. She left her door open as she proceeded with Annie’s history so all could hear, including the man breaking up twigs, if he understood English. When Annie finished recounting her symptoms, Dr. Chang reviewed her notes and said gently, “Annie, I don’t think that this is MS. Have you traveled anywhere recently?”
“Yes, we were in Baja California six weeks ago. When I came back, I was very sick with a stomach virus which I thought I had gotten from the food.”“Hmm, I think what you have is a virus which has settled in your brain.”
Sure, I thought to myself, and next she’ll say that Annie has Mexican bees in her bonnet, but I kept listening.
“What you must do is to take these herbs—from China –boil them three times a day and drink the broth—drink it fast. Can you do that?”
Annie agreed that she could. I was certain she wouldn’t. She hated to cook and would never even open a can of soup for herself, preferring anything that didn’t require preparation—cereal, yogurt, dried apricots, licorice sticks. I knew she wouldn’t put twigs in a pot and drink the liquid. Still, my partner of twenty years had surprised me many times in the past few weeks. She might again.
“Come back in two weeks and we can talk about acupuncture,” Dr. Chang advised, putting her arm around Annie’s shoulder and guiding her gently to the door. “Annie is going to get better,” was all she said as we made our way out and down the steps.
For the next two weeks, Annie boiled the twigs and leaves filling the house with a foul brackish smell that seeped into the curtains, the carpets, the couches, even our clothing. Once, when Annie was at school, I sipped some of the broth and spit it out into the sink, gasping at the acrid taste, which was just as foul as the smell. Still, Annie drank vast quantities of it, never complaining.
On the Saturday of our next appointment, Annie looked across the breakfast table at me and said, “Just so you know, I can see now…no more blurring, no more fog... and the flushing has stopped.”
I didn’t laugh. I didn’t do anything, but stare at her and marvel. Someone I had known for twenty years had surprised me by her willingness to cross borders of training and culture, to try something new, anything, to get well.
Was it MS? Probably, but the symptoms didn’t return for eighteen years, time to grow her career, teach and write articles, see patients, travel, experience life fully. And then, one day, in 2005, she started to walk from the couch in our New York apartment to the television and fell. We dismissed it as clumsiness, but then she fell again. The next time she fell, we agreed that she should see a neurologist. At the Mt. Sinai MS Clinic, we met a warm Israeli doctor whose sense of humor was similar to Annie’s and mine. She too could laugh at the adversities of nature.
When Annie told her about Dr. Chang’s remedies, Dr. Leibowitz never scoffed at the story of a little Chinese doctor working out of the second story of an apartment building. She didn’t call us gullible or naïve, but marveled at the length of Annie’s recovery. This time, the new doctor had her own medicines to offer, but no real assurances. No one knew how the disease would progress, she cautioned, but she, too, took Annie’s arm, touched her gently and offered support for whatever lay ahead.
While they continued their visit, I remained in the waiting room. I had learned to stop laughing when I was scared. Now, I breathed in, breathed out and hummed a tune from the Grateful Dead, “Tell you what I’ll do, I’ll look out for you.”
Marilyn Mehr is a retired psychologist and Professor of Family Medicine who lives in New York City where she writes and engages in social protest. Her most recent publication was Such Charming Exiles: How Two Gay Women Learned to Live Openly and Love Fiercely. In addition, she has written The Courage to Achieve, with Betty A. Walker, and Holding the World Together, a novel about her Mormon ancestry. She lives with her wife, Betty Walker, whom she married at the Bronx County Courthouse in 2011, all thanks to the Great State of New York.
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A motor vehicle steering wheel is frequently provided with a signaling arrangement, e.g. for operating a horn of the vehicle, which can comprise a pair of spaced apart contacts, one of which is spring biased away from the other and is movable upwardly and downwardly during the making and breaking of the contact to operate a load such as the vehicle horn. To connect this movable contact or even the known movable contact in cases in which the steering wheel itself may be capable of upward and downward movement, e.g. in the case of an adjustable steering wheel, it is a practice to provide a slip ring which is engaged by a contact finger guided in an insulating sleeve mounted in the steering wheel hub. The contact finger is connected by a cable to one of the contacts of the signaling arrangement. The slip ring is connected to a source of electric current.
In such signaling devices it is known to provide the insulating sleeve with a rectangular cross section passage in which the contact finger is axially guided but prevented from twisting, the contact finger heretofore being constituted as a U-section sheet metal body within this rectangular cross section passage of the sleeve and having a tubular configuration at its head engageable with the slip ring. At the cable end of the contact finger, two tongues are bent from the sheet metal contact finger to form stops limiting the displacement of the finger toward the slip ring.
In this arrangement, the connection between the contact finger and the cable lies outside the insulating sleeve to permit the necessary degree of free movement for the stroke of the contact of the signaling arrangement. The connection between the contact finger and the cable is thus not fully reliable and, since the sheet metal contact finger cannot always be made to the desired tolerances and may be deformed by the upward and downward movement, even the operation of the contact finger is frequently unreliable.
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---
abstract: 'Hierarchical galaxy formation models predict the development of elliptical galaxies through a combination of the mergers and interactions of smaller galaxies. We are carrying out a study of Early-Type Galaxies (ETGs) using GAMA multi-wavelength and Herschel-ATLAS sub-mm data to understand their intrinsic dust properties. The dust in some ETGs may be a relic of past interactions and mergers of galaxies, or may be produced within the galaxies themselves. With this large dataset we will probe the properties of the dust and its relation to host galaxy properties. This paper presents our criteria for selecting ETGs and explores the usefulness of proxies for their morphology, including optical colour, Sersic index and Concentration index. We find that a combination of criteria including r band Concentration index, ellipticity and apparent sizes is needed to select a robust sample. Optical and sub-mm parameter diagnostics are examined for the selected ETG sample, and the sub-mm data are fitted with modified Planck functions giving initial estimates for the cold dust temperatures and masses.'
title: 'Using GAMA and H-ATLAS Data to Explore the Cold Dust Properties of Early-Type Galaxies'
---
Introduction
============
Early-Type Galaxies (ETGs) are thought to be a homogeneous class of E and S0 galaxies ([@Baldry2004 Baldry *et al.* 2004]). They are predominantly old and inert, have a high central surface brightness, and cover a wide range of luminosities ([@driver2006 Driver *et al.* 2006]). ETGs are classed as smooth, highly concentrated, and spheroidal systems with a lack of spiral arms. Their luminosity profiles tend to follow the de Vaucouleurs I(r)$\sim$r$^{1/4}$ law or a more general Sersic law distribution ([@donofrio2011 D’Onofrio *et al.* 2011]).
ETGs are thought to be quiescent at zero redshift, leading to the assumption that they are devoid of both cold gas and dust ([@bregman1992 Bregman, Hogg & Roberts 1992]). Recent detections of ETGs in infrared and sub-mm regimes have revealed largely unexpected amount of cold gas and dust (eg. [@leeuw2004 Leeuw *et al.* (2004)]). Our interests lie in exploring this dusty presence, with the aims of gaining further understanding about the processes which form ETGs.
![Optical Colour-Magnitude Diagram for our samples. The highly concentrated (C$\ge$2.86) optical GAMA sources are highlighted by green plus signs above a background of all the GAMA sources (black dots). On top of this is the sub-mm selected sources from the combined GAMA/H-ATLAS catalogues, shown as dark blue open triangles. The Red Sequence (RS) line ([@Bernardietal10 Bernardi *et al.* 2010]) is shown to indicate the regime above which we expect RS galaxies to sit. This plot shows that our sample of ETGs could not have been selected with a direct colour cut, as both blue and red ETGs are showing up. This is confirmed through visual classifications of the samples.[]{data-label="fig1"}](proceedingsplot.eps){width="5.0in"}
![\[Left\] The distribution of isothermal dust temperatures for the ETG sample with concentration index $\ge$ 2.86. Our mean temperature is the long dashed line and the short dashed line is the mean temperature from [@Amblardetal10 Amblard *et al.* (2010)] (for the H-ATLAS Science Demonstration Phase (SDP) sample). \[Right\] The distribution of dust masses fit by modified Planck functions, emphasising our mean dust mass (long dashed line) and that of the SDP ETG sample of [@Rowlandsetal11 Rowlands *et al.* (2011)] (short dashed line).[]{data-label="fig2"}](multiplot.eps "fig:"){width="60.00000%"} ![\[Left\] The distribution of isothermal dust temperatures for the ETG sample with concentration index $\ge$ 2.86. Our mean temperature is the long dashed line and the short dashed line is the mean temperature from [@Amblardetal10 Amblard *et al.* (2010)] (for the H-ATLAS Science Demonstration Phase (SDP) sample). \[Right\] The distribution of dust masses fit by modified Planck functions, emphasising our mean dust mass (long dashed line) and that of the SDP ETG sample of [@Rowlandsetal11 Rowlands *et al.* (2011)] (short dashed line).[]{data-label="fig2"}](multiplot2.eps "fig:"){width="60.00000%"}
Selection Techniques
====================
With the aim of creating a highly robust sample of sub-mm selected ETGs, we experimented with multiple proxies for morphology. Proxies tested were optical colour (eg. [@kaviraj2010 Kaviraj *et al.* 2010]), r band Sersic index (eg. [@leeuw2008 Leeuw *et al.* 2008]) and r band Concentration index (eg. [@blanton2010) Blanton 2003]. We used Sersic index values from the GAMA single Sersic fits to the objects, using GALFIT3 ([@Kelvininprep Kelvin *et al.* submitted]). The Concentration values are the ratio of SDSS Petrosian 90 radius to Petrosian 50 radius for each galaxy. We combined the GAMA I multi-wavelength survey with Herschel-ATLAS Phase 1 far infrared and sub-mm data to create a catalogue of sub-mm detected ETGs.
We examined the behaviour of the three proxies for both our matched multi-wavelength sample and an optical morphologically classified SDSS sample from [@NairAbe2010 Nair & Abraham (2010)]. From this we concluded that a straight colour cut is not a viable approach to take, as ETGs tend to be red, but not all red galaxies tend to be ETGs. For example, many red spirals were present in such a cut. Also, as our sub-mm approach picks out dusty galaxies, we may expect some blue ETGs to be present. A Sersic index cut at $\ge$3.5 seemed reasonable, but a concentration index cut of $\ge$2.86 gave us a sample which matched best with the optical visually classified ETGs in the Nair $\&$ Abraham control sample.
We have applied our concentration index criterion, with additional criteria for redshift, ellipticity and apparent effective radius, to our GAMA/H-ATLAS data. With the addition of visual classifications to root out any remaining late-type galaxies, this resulted in a condensed sample of 239 lenticular and elliptical galaxies, shown as open triangles in Fig.\[fig1\]. These sub-mm detected ETGs mostly have intermediate absolute magnitudes (-19.5$\sim$M$_{g}\sim$-21.5).
Sub-mm Single Temperature Fits
==============================
Modified Planck functions were fit to the sub-mm regime of our sample’s observed data. The resultant cold dust temperatures and masses are displayed in Fig.\[fig2\]. We find a mean dust temperature of 22.5$^{+10}_{-6}$K (for $\beta=$2.0) and a mean dust mass of 2.038$\times$10$^{8}$M$_{\odot}$. Fig.\[fig2\] shows that the sub-mm sample contains some ETGs with high dust masses and mainly low dust temperatures compared to these references. The low dust temperatures will need to be checked with additional flux points, including PACS data, covering the peak of the cool emission. Unlike the H-ATLAS galaxies which have been studied up to this point, our sample exhibits only intermediate stellar masses, not high stellar masses.
2010, *A&A*, 518, L9
2004, *ApJ*, 600, 681-694
2010, *MNRAS*, 2087-2122, 404
2003, *ApJ*, 594, 186-207
1992, *ApJ*, 387, 484-502
2011, *ApJ* (Letters), 727, L6+
2006, *MNRAS*, 368, 414-434
(2010)\][kaviraj2010]{} [Kaviraj, S., Schawinski, K., Silk, J., Shabala, S.]{} 2010, *ArXiv e-prints*, 1008.1583
2012, *ArXiv e-prints*, 1112.1956
2004, *ApJ*, 612, 837-847
2008, *ApJ*, 677, 249-261
2010, *VizieR Online Data Catalog*
2011, *ArXiv e-prints*, 1109.6274
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Q:
Integral of Gaussian Curvature over a Tube
I am working on a problem which I have come to a standstill on. The question reads:
Let $\alpha(s)$ be a closed regular curve in $\mathbb{R}^3$ parametrized by arc-length. Consider the tube:
$$X(s,v)=\alpha(s)+r(n(s)\cos(v)+b(s)\sin(v))$$
Where $n(s)$ and $b(s)$ represent the normal and binormal vectors of $\alpha(s)$. Integrate the Gaussain Curvature over the Tube.
Now I found the Gaussian Curvature (which was very tedious) and got the answer:
$$K(s,v) = -\frac{\kappa(s)\cos(v)}{r(1-r\kappa(s)\cos(v))}$$
Where $\kappa(s)$ denotes the curvature of $\alpha(s)$. Now, how am I supposed to Integrate this over anything? I'm assuming that I should use Stokes', as it is the only thing that makes sense to do. So should I just consider $dK(s,v)$? and then consider the tube $T=\partial M$ as the boundary of a solid Tube $M$?
A:
Use Gauss-Bonnet:
\begin{align*}
\int_M KdA = 2\pi\chi(M).
\end{align*}
Now the surface you discribe (somthing like a cylinder or torus) we have that $\chi(M)=0$. Thus $\int_M KdA =0$.
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Article content
A TTC employee was viciously beaten on Monday night after approaching a man who was hopping between moving subway cars.
Just before 9 p.m. on Monday night, a TTC employee spotted a man jumping the narrow space betwee two cars just as the train was pulling into Islington Station, the second-to-last stop on the western end of the Bloor Line.
The employee approached the man and was immediately kicked in the right leg, causing him to crumple to the floor of the train. His attacker then grabbed a compartment door and repeatedly slammed it into the face of the TTC worker. The attacker reportedly left the train briefly, only to return and resume kicking the door into the injured employee. The man fled the scene before transit police could arrive.
We apologize, but this video has failed to load.
tap here to see other videos from our team. Try refreshing your browser, or TTC employee viciously beaten Back to video
On Tuesday afternoon Toronto Police announced that they had charged Sover Aransibia, 26, with assault and two counts of mischief in connection to the incident.
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Q:
Fragment Tag vs pragmatically adding a Fragment to your layout
Final Update
Turns out that I had the problem all wrong. It was how I was using the Fragment tag. See my answer below to get a full explanation.
Update 3:
So I have continued messing with it for hours with no such luck. I have been able to get it to a point in which it does not crash. But now I believe I know what the issue might be. It was getting my calendarAdapter as null because it was null, but not by my intention. It seems onCreateView is running twice, once apparently before anything is sent in and after I have officially sent some parameters. This of course should not be happening... I think. I will provide the part in which my ActionBar is set at the bottom so you can let me know if I am doing something wrong for this behavior to be set the way it is.
I am having an issue with this specific thing for some reason, and I have no idea why. I have an Activity called MainDisplayActivity as well as a Fragment called MainDislpayFragment. As you can imagine, the Activity sets the Fragment on app start. Now this is the problem, I've been trying to sent as parameters into the newInstance constructor three numbers that came from a Calendar. Then I created the Calendar from within the constructor. But my app crashes. The weird part is when I keep the Calendar from being used by static fields the app works as expected, but trying to use the Calendar from anything that came from the constructor makes it crash. This is an example method:
private static Calendar cal;
static MainDisplayFragment newInstance(Context context, int y, int m, int d) {
c = context;
MainDisplayFragment f = new MainDisplayFragment();
//static global Calendar now
cal = Calendar.getInstance();
cal.set(y, m, d);
}//end of static newInstance method
Now its important to note that even if I tried taking Calendar out and putting it in onCreate and only having the static ints I am getting back as global, and setting them there, it still causes a crash. The notable error and the weird thing is that it seems to think Calendar is null for some reason. As you can tell right there it is definitely being created. But now I am starting to think there is an issue with Calendar and static fields. If it does not, then I may have an error, but I wanted to rule that out if I can. Thanks for anyones response.
Update: I should mention that I am using that Calendar info for nothing more than to pass into my MyCalendarAdapter. Regardless of whether I only send in numbers as a parameter or a Calendar itself, it crashes. The first null exception I see is where I am trying to set the MyCalendarAdapter.
Line 75 would be the last lin here:
public View onCreateView(LayoutInflater inflater, ViewGroup container, Bundle savedInstanceState) {
view = inflater.inflate(R.layout.main_fragment, container, false);
calendarAdapter = new MyCalendarAdapter(cal.get(Calendar.YEAR), cal.get(Calendar.MONTH), cal.get(Calendar.DAY_OF_MONTH));
UPDATE 2:
After more messing around I realized the issue is with MyCalendarAdapter, but its an issue that makes no sense. It seems if you instantiate a calendarAdapter with no arguments it works perfectly, but with arguments it crashes. These are the two constructors for MyCalendarAdapter so people can see that nothing looks wrong, which makes this worse because I have no friggen idea how to fix this. Its also important to know that this code that I have tried to move to a Fragment originally came from an Activity, which worked perfectly.
public MyCalendarAdapter() {
thisMonth = Calendar.getInstance();
now = new MonthDisplayHelper(thisMonth.get(Calendar.YEAR), thisMonth.get(Calendar.MONTH));//creating Helper for month display
currentDay = thisMonth.get(Calendar.DAY_OF_MONTH);//sets current day
...
}
public MyCalendarAdapter(int year, int month, int currentDay) {
thisMonth = Calendar.getInstance();
now = new MonthDisplayHelper(year, month);
this.currentDay = currentDay;
...
}
Complete List of Errors:
11-08 18:26:35.710: E/AndroidRuntime(7839): FATAL EXCEPTION: main
11-08 18:26:35.710: E/AndroidRuntime(7839): java.lang.RuntimeException: Unable to start
activity ComponentInfo{com.zeroe/com.zeroe.MainDisplayActivity}:
android.view.InflateException: Binary XML file line #9: Error inflating class fragment
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.ActivityThread.performLaunchActivity(ActivityThread.java:2059)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.ActivityThread.handleLaunchActivity(ActivityThread.java:2084)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.ActivityThread.access$600(ActivityThread.java:130)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.ActivityThread$H.handleMessage(ActivityThread.java:1195)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.os.Handler.dispatchMessage(Handler.java:99)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.os.Looper.loop(Looper.java:137)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.ActivityThread.main(ActivityThread.java:4745)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
java.lang.reflect.Method.invokeNative(Native Method)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
java.lang.reflect.Method.invoke(Method.java:511)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
com.android.internal.os.ZygoteInit$MethodAndArgsCaller.run(ZygoteInit.java:786)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
com.android.internal.os.ZygoteInit.main(ZygoteInit.java:553)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
dalvik.system.NativeStart.main(Native Method)
11-08 18:26:35.710: E/AndroidRuntime(7839): Caused by: android.view.InflateException:
Binary XML file line #9: Error inflating class fragment
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.view.LayoutInflater.createViewFromTag(LayoutInflater.java:704)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.view.LayoutInflater.rInflate(LayoutInflater.java:746)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.view.LayoutInflater.inflate(LayoutInflater.java:489)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.view.LayoutInflater.inflate(LayoutInflater.java:396)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.view.LayoutInflater.inflate(LayoutInflater.java:352)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
com.android.internal.policy.impl.PhoneWindow.setContentView(PhoneWindow.java:256)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.Activity.setContentView(Activity.java:1867)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
com.zeroe.MainDisplayActivity.onCreate(MainDisplayActivity.java:60)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.Activity.performCreate(Activity.java:5008)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.Instrumentation.callActivityOnCreate(Instrumentation.java:1079)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.ActivityThread.performLaunchActivity(ActivityThread.java:2023)
11-08 18:26:35.710: E/AndroidRuntime(7839): ... 11 more
11-08 18:26:35.710: E/AndroidRuntime(7839): Caused by: java.lang.NullPointerException
11-08 18:26:35.710: E/AndroidRuntime(7839): at
com.zeroe.MainDisplayFragment.onCreateView(MainDisplayFragment.java:80)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.FragmentManagerImpl.moveToState(FragmentManager.java:807)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.FragmentManagerImpl.moveToState(FragmentManager.java:1013)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.FragmentManagerImpl.addFragment(FragmentManager.java:1112)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.app.Activity.onCreateView(Activity.java:4664)
11-08 18:26:35.710: E/AndroidRuntime(7839): at
android.view.LayoutInflater.createViewFromTag(LayoutInflater.java:680)
11-08 18:26:35.710: E/AndroidRuntime(7839): ... 21 more
ActionBar part where Fragment is set
@Override
public boolean onNavigationItemSelected(int itemPosition, long itemId) {
Toast.makeText(this, "Position is "+itemPosition, Toast.LENGTH_SHORT).show();
if(itemPosition == 0) {
FragmentTransaction ft = getFragmentManager().beginTransaction();
//check to see if one already exists and remove it
Fragment prev = getFragmentManager().findFragmentByTag(MAIN_FRAGMENT);
if(prev!=null) {
ft.remove(prev);
Toast.makeText(this, "Fragment was found", Toast.LENGTH_SHORT).show();
}
MyCalendarAdapter calendarAdapter = new MyCalendarAdapter(year, month, day);
//Log.d("MAINDISPLAYACTIVTY", "calendarAdapter year is "+calendarAdapter.getCalendar().getYear());
currentFragment = MainDisplayFragment.newInstance(this, year, month, day, calendarAdapter);
ft.add((MainDisplayFragment)currentFragment, MAIN_FRAGMENT);
ft.setTransition(FragmentTransaction.TRANSIT_FRAGMENT_OPEN);
//ft.addToBackStack(null); //i do not want this behavior
ft.commit();
return true;
}
... continues on with the other cases
onCreate in Activity that sets the ActionBar
final ActionBar bar = getActionBar();
bar.setDisplayShowTitleEnabled(false);
bar.setNavigationMode(ActionBar.NAVIGATION_MODE_LIST);
bar.setListNavigationCallbacks(new ArrayAdapter<String>(bar.getThemedContext(), android.R.layout.simple_list_item_1, android.R.id.text1,
new String[]{"MONTH", "WEEK", "DAY"}), this);
if(savedInstanceState == null) {
bar.setSelectedNavigationItem(0);
} else {
bar.setSelectedNavigationItem(savedInstanceState.getInt(SELECTED_NAVIGATION_STATE));
}
A:
So I figured out what my issue was. It was something that was not too clear in the Android docs on Fragments. I was using the <fragment/> tag in my main.xml file. I was using it correctly, and when creating my Fragment, I was also setting it correctly. Unfortunately I was not getting the behavior I was hoping for.
It turns out that if you use the fragment tag in your xml then when setContentView runs it will also run your Fragment class immediately (at least when it encounters the tag as its going down the file). What this means is if your Fragment depends on information from the Activity you are launching it from, it will not have it, and essentially run twice; when setContentView runs, and when you actually run your Fragment pragmatically. This is important because I was under the assumption that you could choose either way to run your Fragment. But in reality the way you design your app or approach it actually dictates which way you should go, hardcode a Fragment or pragmatically on the fly.
Maybe this was obvious to the android experts, but I am new to Fragments, so I guess it was a rookie mistake. In either case, this should really be detailed in the Android docs. Hope this helps anyone else completely confused on the issue.
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Evidence-Based Facial Fracture Management.
With demands for an evidence-based approach to patient care, the management of facial fractures will come under increasing scrutiny because there is an overall deficiency in higher level clinical evidence. This article reviews the management of facial fractures, focusing on an evidence-based approach. It focuses on select areas of facial trauma in which there is controversy and presents randomized studies and meta-analysis to help define best practice. The article notes the many areas where the evidenced-based literature is weak and looks at the future of evidence-based facial trauma care.
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The tricky business of unblocking your brain
Don’t read this if you’ve got an aneurysm.
Spend 24 hours in the company of a couple of hundred brain surgeons and you’d have a sense of unease too. I’m at a conference where “minimally invasive neurological therapies” are being discussed. My take-home message? No one knows anything for sure. Until it’s too late, that is.
Not that they aren’t good at their job – they’re the best in the world at getting at blockages and other problems inside your brain. But they are here to discuss the things they don’t know. And those are conversations you’d rather not overhear.
The typical presentation goes like this. “So, we went to perform an angioplasty on patient A, who was suffering from acutely reduced vision” (I may be paraphrasing badly). “Here’s the imaging.”
On the screen appears a picture of some loopy, tangled-looking blood vessels. There are murmurs and sharp intakes of breath. A voice just behind me mutters “ay-ay-ay”.
I have no idea what I’m looking at. I’m only here to give a talk about more general issues in scientific research. But I have that sinking feeling, like in the first five minutes of an episode of Casualty, that something bad is about to happen.
“I’d like to know: what would you have done?” the presenter asks. She offers two options. The room votes. The split is even, an observation that makes me hugely uncomfortable. There is no consensus. Why is there no consensus? Surely there’s a right thing to do in any situation? The presenter goes on to explain what she did. There is another round of murmuring in the room. Clearly, many people – approximately half – think this was a very bad idea.
The next presenter describes a surgery that started to go wrong 4 hours into an operation. He talks like it’s Who Wants To Be A Millionaire. “What do you think?” he asks the audience. “Shall I go on or stop now?” A voice from the back shouts, “No, no, no. Stop. You have to stop!”
He did go on, as it happened. He describes how the procedure progressed, blow by blow. “No, no, don’t do that!” comes an anguished shout, like this is Surgery Live. It’s not: this all happened last year. “Yeah,” the presenter mutters. “Thanks, I know that now.”
The next presentation ends with, “Well, I’ll never do that again.” Then comes another: “So, I’d like your opinions – should I treat this? If so, how?” The audience is calling out answers like a classroom full of show-offs. The session chair asks for calm.
Not all the answers are helpful. “If you get bleeding there, that’s going to be catastrophic.” The presenter furrows his brow. “I know,” he says. “That’s why I’m asking.”
This one is not a done deal, as it turns out. “Thanks,” the presenter says as the deluge of conflicting answers abates. “I’m due to see her again in ten days, so that’s really helpful.”
Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman, and his most recent book is At The Edge of Uncertainty: 11 Discoveries Taking Science By Surprise.
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It’s Grand Final week, and we’re not just cheering for our favourite teams.
In early 2014, Newcastle Knights back-rower Alex McKinnon was critically injured in an NRL game against the Melbourne Storm. He suffered two fractured vertebrae after a tackle thew him head-first into the ground.
He was told he might never walk again.
But despite his ongoing struggles, McKinnon has refused to accept that he will forever be seated – and today, less than a year and a half after the shock diagnosis, the footballer walked a distance of 80 metres.
In an Instagram post, McKinnon celebrated being able to “lock out” his knees when walking in rehabilitation. With the help of trainers, who swung his legs, Alex was able to walk a distance of 80 metres.
You can watch his success here:
“Anybody that has had an injury be it big or small understands the frustration that comes with rehab and the feeling of it being out of your control,” McKinnon wrote.
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There is no way out whatsoever if we want to keep oblivious stock market prices, irrational growth of welfare rights, and peace altogether at the same time.
But I can suggest some ideas that could be implemented within a week (o tempora, o mores, o Narendra Modi), for a good start.
1) Go back to sound money
Replicate basic legislation according to the Peel Banking Act of 1844 but without exempting the demand deposits from the legal requirement of a 100-percent reserve which it did demand with respect to the issuance of paper money.
Yes that is killing commercial banking as we know it. Innovation please.
Open the mint to new metal discoveries.
Reinstate the 90 day real bill market.
Anchor national currency units to a metal value according to which, all debts can be extinguished upon ultimate payment in metal.
Central banking utter fraud is that they can print unlimited amounts of currency. But the are unable to print a single REAL cup of milk.
Of course this would disable oblivious P/E for all ! So ? Are we no longer able to withstand ever changing state of affairs, included poverty?
Of course this would create commodities wars at home too ! So ? Are we too lazy to fight wars at home all of a sudden ?
Of course this will kill the lunacy of financing long terms maturing goods (like a mortgage) with overnight phantom repos rehypothecated several times at the central bank as "eligible collateral".
Of course this would considerably shrink the current welfare state ! So ? Have we already been so badly sugar-coated that we feel no longer handsome eating without all our teeth?
2) Chop down welfare state
Welfare state is mandatory, meaning that whatever happens to the economic cycle, the government MUST provide by law a predetermined amount of goods and services for the needy.
An example would do great. In Spain, 2007 was base 100. By 2037, Spaniards must provide up to 30 GDP points in additional welfare state to that of 2007. Whatever happens ! No human nature can deliver such a compounded gains in productivity. But such is the rule of law.
Until central bankers print their first real glass of fresh milk, welfare state should be postponed.
Wars yes. Revolutions, welcome !
History is made of wars. Of are we just bringing conflict to a halt...? By currency printing ? That would be THE real first !!
When the needy couldn't stand it anymore, the rose up in revolutions to grab the assets of the dominant class, or they grabbed assets from their neighboring countries. So the needy, rise up from your sofa guys! Sponsored nap time over.
Provided sound money is reinstated and welfare state has been littered, the is a walk in the park.
4) Tax reform. Fairness and redistribution
- Eliminate by whatever means all tax havens.
- Replace all existing taxes by a minuscule transaction tax on all flow of capital. According to a well known study by Mark Chesney a 0.2% would pay for all currently existing taxes !
This has major redistributing effect, as the working class would be taxed 0.2% at receiving income and an additional 0.2% as they purchased goods, making taxation a maximum 0.4% grand total. Richer people would be taxed 0,2% every time they moved their zillions and so on.
----
Entire civilizations, much greater than ours, have collapsed due to abandonment of sound money, by mandatory spending, by overspending, and or by unfair overtaxing. This time is different right ?
I concur with all Mr. Shannon's remarks. My note is to say that a major addition to the problem is that the banking industry is a complex system; one of the primary approaches to reducing instability in a complex system is by increasing diversity in all dimensions. Instead, we have witnessed increasing concentration in the sector.
In addition, attempts to buffer prospective downward feedback loops by regulation have met with skepticism both on the part of some economists, especially the very influential Alan Greenspan, and so have been disabled and/or removed.
If, as seems likely, China's banking sector is inadequate, in whatever sense becomes relevant, to stem the acceleration towards disaster when the next cycle begins, who will remain as a still-productive and profitable outpost in the three major economic zones?
I hope to see more thoughtful articles on this topic such as this one, both here and elsewhere, in the near future.
Thank you for this great piece. It's a subject that doesn't get enough attention, and we know it's going to happen, therefore, it makes impeccable sense to begin to prepare for it now -- as your essay alludes.
I hope to see Per Kurowski's comment here in the coming days, he has thoroughly researched and written extensively on America's banking sector, especially in regards to how to enhance structural solvency for the banking sector. His thinking is one order of magnitude better than the present regulatory system.
I've written about China becoming the next real estate bubble and conceivably, it could be 4.5 to 6 times worse than the 2008 crash in the U.S. and it could come *as late as* 2022. But I'd expect it before then.
In early 2009, all standing banks were saved from the slaughterhouse by the magic of amending FASB rule number 157. Since that day onwards, bank's balance sheets statements are plain fiction, and any resemblance to reality is mere coincidence.
It is upon this fictional land that regulators have built "buffers" to withstand the coming economic seizure.
Ultra loose monetary policy has suppressed risk (yield) pricing from securities valuation, making the securities market an almost totally unproductive risk free space, as at least in theory every Treasury issued bond will be bid (indirectly) by outright central bank reserve creation.
Risk pricing experts will concentrate their skills in pricing the risks that influence a given currency bid. In all likelihood, the next big dislocation will come from a sudden removal of a given currency bid.
Imagine trust in the dollar vanished overnight. Dollar bid would disappear. And all dollar "risk-free" denominated securities would be prized at cents on the dollar.
Fictional accounting together with outlandish creation of dubious buffers have shifted risk pricing from the securities market to the currency markets. This tectonic shift will make the next crisis (whenever it comes) several orders of magnitude greater than the previous one.
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Mass protests over racial injustice, the COVID-19 pandemic, and a sharp economic downturn have plunged the United States into its deepest crisis in decades. Will the public embrace radical, systemic reforms, or will the specter of civil disorder provoke a conservative backlash?
For democratic countries like the United States, the COVID-19 crisis has opened up four possible political and socioeconomic trajectories. But only one path forward leads to a destination that most people would want to reach.
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Find Peace at Home with Paint
Are you painting a room in your house to achieve a peaceful atmosphere? Whether it be a bedroom or a living room, a foyer or a kitchen, your color choice can affect the mood you are trying to set.
Despite the environmental and cultural aspects of the way we perceive color and how it affects us, there are some universal notions we can rely on: warm colors (reds, yellows and oranges) can evoke feelings of warmth and safety, while cool colors (blues, purples and greens) bring on feelings of calm, peace and serenity.
To achieve a peaceful atmosphere you have a wide choice of hues to choose from inside the major cool colors, ranging from neutral blanc de chine to a bold blue via pastel purples. Depending on your own preference and affinities to different colors, there are different tools that can help you put together a nice palette to decorate your house with. One of my favorite is Seeds Design, which gives palettes based on photos. The Olympic collections has some nice inspiration palettes as well. I also like to visualize the different colors I pick using Colorjive to upload a photo of my room and change the color with a click.
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We help you make better business decisions by leveraging your data.
At Datalore, we combine data science expertise with contemporary tools and techniques to solve business challenges. We also augment the skills of your AI team and help build agile data-literate workforces
Data Science is a new rapidly evolving field that demands an array of skill sets in mathematics, statistics, machine learning, deep learning and software engineering. Individuals who can align these multi-functional competencies with their existing area of knowledge will prove indispensable for data-driven businesses.
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Our training programs are developed by data scientists and machine learning engineers, with years of experience and multi-functional competencies. Our training covers industry-relevant developments in the field of machine learning and deep learning. Through our workshops, you can gain the expertise necessary to become a competitive data scientist
Corporate:
We conduct tailored training/workshops aimed at sustained improvements to your workforce. By helping the staff, understand and utilize the data-driven platforms within the organization, they can more effectively address critical challenges within their functions.
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2016 Dauphine Route
The route for the 2016 Critérium du Dauphiné has been presented today, a ray of summer sunshine amid the clouds, mud and crosswinds that dominate the sport right now. The Dauphiné is a race that’s often seen in the shadow of the Tour de France because it borrow some of the same roads and also because it’s a dress rehearsal for July but this is always a good race in its own right, think Chris Froome’s final stage overhaul of Tejay van Garderen in last year or Andrew Talansky’s tactical masterpiece the year before. Here’s a closer look at this year’s route and more.
Things start with a novelty, an uphill prologue above the ski resort of Les Gets with 3.9km at an average of 9.7% but as the profile shows it’s got some much steeper sections. It’s novel to use such steep roads but not unique, you might remember the 2013 Tour de Romandie where Chris Froome beat Andrew Talansky in the 7.5km prologue, uphill but not as steep.
Stage 1 is for the sprinters as the race rides away from the Alps to the plains of the Ain for a likely sprint finish in St. Vulbas.
Stage 2 sees a ski station finish in Chalmazel but this isn’t a high altitude Alpine variety but a gentler arrival on the slopes of the Massif Central below the Col du Béal, where Chris Froome and Alberto Contador traded attacks in 2014. The exact location of the finish line isn’t clear yet but the approach roads are in the order of 4-5%.
Stage 3 has the riders heading for the Rhone valley and if the race goes above 1,000m above sea level it’s via some gentle, gradual climbs. There’s a sting in the tail, the race drops down to the Rhone before taking the climb to Sécheras, 3km at 7% and enough to eject many sprinters especially as there’s a false flat that goes on after the climb. A sprinter or two could hold on but only if they’re in peak condition for the summer.
Stage 4 is flat and for the sprinters with a finishing circuit around Belley, birthplace of Etixx-QS’s Maxime Bouet.
Stage 5 will matter to the overall classification with the summit finish in the ski station of Vaujany. It’d also be a good ride on its own as it takes the balcony road below the Belledonne mountains before dropping to Vizille and then taking the sapping valley road to Allemont. Vaujany is a short spin away from Alpe d’Huez and small village and ski station with a steep climb that’s got sustained sections of 8-10% and the finish is in the village rather than further up the road.
Stage 6 is the Queen Stage. It opens with the Col de Champ Laurent, an unsung but rewarding climb that’s steep but mercifully shaded for most of the way up if the sun is shining before heading onto the Grand Cucheron, passing the spot where David Moncoutié crashed out of the Tour de France and ended his career. The Madeleine is worthy of its HC status and many a rider will try to get in the breakaway here in order to win the mountains competition points on offer in the first hal before the long descent into the Tarentaise valley. After the balcony climb to Les Frasses the climb to the swanky ski town of Méribel is almost the easiest part of the day as after a 7% start it relaxes to 5-6% for most of the way.
Queen stage? a term applied to the biggest and most important stage of a race. It’s from the French, étape reine which is literally “queen stage”. The noun étape is feminine so has the matching feminine adjective or adjectival ending, reine meaning queen rather than roi or king. Normally in English it would be “King Stage” but the more literal translation seems to have been copied across. There’s no obvious royal connection, just something to suggest importance, size and perhaps the power to shape the GC or even crown the winner.
Finally Stage 7 is similar to the penultimate stage of the 2013 Dauphiné sharing the same start and finish although that included the Alpe d’Huez and Col de Sarenne recon in a nod to that year’s Tour de France route. This time it’s a more obvious route via a series of climbs before the breathtaking, in both senses, Col du Noyer (11.3km at 7.2% but with 10-11% percent kilometres near the top) and then the shorter climb to Superdévoluy, 4km at 5.7%.
Route Summary
One for the climbers. There’s only one time trial and that’s the uphill prologue with its double-digit gradients. But the other mountainous days have some steady climbs, the race never crosses above the 2,000m altitude and this might open the door to a wider cast of characters and maybe some different tactics. Certainly the race has seen some entrepreneurial moves in recent years, think Andrew Talansky’s 2014 win or last year’s deluged stage across the Vercors plateau where the GC riders like Vincenzo Nibali, Tejay van Garderen and Rui Costa were attacking on the first climb, this was one of the highlights of the year. It’s possible again, especially given the mountain stages are short with distances of under 150 km. The route offers a couple of chances for the sprinters, especially if they can cope with a hill or two, think Nacer Bouhanni or John Degenkolb.
Dauphiné vs. Switzerland
This race overlaps with the Tour de Suisse, the final weekend of the Dauphiné is the opening weekend of Suisse. Even if they didn’t clash most riders would opt for one or the other rather than combine both as one week’s racing is enough for those aiming for for the Tour de France. What’s new is that the Velon group of teams has its deal with the Infront sports agency, co-owners of the Tour de Suisse and they’ve announced a revenue sharing deal this week between the Swiss race and the cartel of teams, the idea being that the teams commit to send big name riders to Switzerland in order to try and make the race more valuable and therefore create more revenue to share. It’s a long term project given broadcast deals are multi-year and TV channels will watch and wait to see if it’s worth paying a premium for the Swiss race before renewing. But the immediate effect could see Velon member teams sending their better riders to Switzerland instead. We’ll see though as cyclingnews.com says “Froome and Contador set to clash” adding van Garderen is expected to start too, all ride for Velon members. Certainly the Dauphiné has been a pre-Tour ritual for these three in recent years they’ll need to balance earning income for their team managers with their sporting preparation before July. On sporting terms Suisse might be preferable for some because it contains an 18km time trial, a vital exercise given the Tour de France has two time trials for a change. Fortunately cycling fans don’t have to chose, they’re spoilt by two weeks of the finest Alpine racing possible.
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LAW OF THE LAND
Why 'gay' civil-rights claim is specious
Exclusive: Mychal Massie addresses 'same as blacks in 1960s' argument
Mychal Massie is the former chairman of the National Leadership Network of Black Conservatives-Project 21 – a conservative black think tank located in Washington, D.C. He was recognized as the 2008 Conservative Man of the Year by the Conservative Party of Suffolk County, N.Y. He is a nationally recognized political activist, pundit and columnist. He has appeared on Fox News Channel, CNN, MSNBC, C-SPAN, NBC, Comcast Cable and talk-radio programming nationwide. A former self-employed business owner of more than 30 years, Massie's website is mychal-massie.com.
Homosexuals try to pawn their perceived struggle for complete and total acceptance of their chosen lifestyle as being synonymous with what blacks went through to achieve civil rights. But their attempts to equate their radical agenda with discrimination as defined in The Civil Rights Act of 1964 is blatantly false and without merit.
Based on the color of their skin alone, blacks were prevented, in many domiciles because of prejudice and by codification of segregation, from voting, purchasing property where they chose, eating wherever they desired, attending events, ad nauseum.
The Civil Rights Act of 1964 specifically defines the act as same. It reads:
“An Act: To enforce the constitutional right to vote, to confer jurisdiction upon the district courts of the United States to provide injunctive relief against discrimination in public accommodations, to authorize the Attorney General to institute suits to protect constitutional rights in public facilities and public education, to extend the Commission on Civil Rights, to prevent discrimination in federally assisted programs, to establish a Commission on Equal Employment Opportunity, and for other purposes.”
The Act was intended to put “teeth,” if you will, into the 14th Amendment.
Homosexual activists are dishonest when they attempt to convince the public that rejection of homosexual marriage is tantamount to the culture of apartheid that opposed interracial dating and marriage. It is not.
The Civil Rights Act of 1964 was intended to end, prevent and address discrimination – discrimination against men and women regardless of their race and/or color.
While discrimination based on sexual preference is not specifically mentioned, I believe it is logically included in the Act. A woman cannot be discriminated against because she has a dating preference for gargantuanly obese men. A person cannot be discriminated against because he has an attraction to short people. A person’s choice of sexual interest, as long as it is legal, is protected, and it should be.
But I believe and argue that the Act is an appliance that guards against discrimination; it’s not to be used as cover for a malicious agenda that prescribes the redefining of our social construct. Christian organizations must hire homosexuals, and I find that fair (after all what better place for those in need of Christ). But being employed by a Christian organization doesn’t give the employee the constitutional right to change the policies and dogma of said employer. The employee had a choice to seek employment elsewhere.
There are logical exceptions. A morbidly obese person cannot favorably present the image of certain companies, but a person’s color does not affect same (allowing the employer isn’t the Ku Klux Klan). A church has the right to have strict, inflexible standards for ordination and for licensing of their clergy.
Homosexuals argue they are denied certain other entitlements that everyone else enjoys, and they cite the inability to make life-and-death decisions for those they cohabit with, etc. This, too, is a specious and fallacious argument. Heterosexuals cohabiting outside the bonds of marriage do not have the right to make such decisions, either.
That is why responsible people living outside the bonds of marriage make living wills. They take responsible measures to ensure their wishes are carried out and to allow for the person of their choice to make decisions for them in the event one or the other becomes unable to make the decision for himself.
Two men can purchase a property together; they can get health insurance and life insurance policies naming one another as beneficiaries, and so on. Their sexual preference doesn’t matter. Their ability to repay a mortgage, etc., isn’t affected by their sexual preference any more than that of heterosexuals.
I could go on, but suffice it to say the homosexual agenda isn’t about civil rights; it is about their desire to change the social construct by redefining marriage and family.
Legislating an employment and social environment that codifies the ability of an employee to present himself in ways that are detrimental to the welfare and health of the company is ludicrous. I speak specifically of cross-dressing and exhibiting inappropriate behavior.
It is maddening that a person can be discriminated against pursuant to employment and home renting because he smokes cigarettes. But nothing is said to address the fact that the Centers for Disease Control paint a horrifyingly high incidence for deadly disease with respect to the practice of homosexuality – including a staggeringly high rate of breast cancer incidences among lesbian women juxtaposed to heterosexual women.
According to the Centers for Disease Control report on HIV incidence, men who have sex with men accounted for 63 percent of the estimated new HIV infections in 2010. That rate means that as 1-4 percent of the total population, they’re as much as 86 times more likely to be diagnosed with HIV. Women with a history of sex with women may be a marker for increased risk of adverse sexual, reproductive and general health outcomes compared with women who reported sex exclusively with men (American Journal of Public Health).
I would submit it makes more sense for homosexual activists to tell people to flee the practice than it does to claim they are being denied civil rights.
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The desire and capability of Australian general practitioners to change their working hours.
To explore factors associated with general practitioners' desire to work less and their success in making that change. Waves 3 and 4 (conducted in 2010 and 2011) of a national longitudinal survey of Australian doctors in clinical practice (Medicine in Australia: Balancing Employment and Life). Of the broader group of medical practitioners in the survey, there were 3664 and 3436 GP completers in Waves 3 and 4, respectively. The association between the desire to reduce hours and doctor, job and geographic characteristics; the association between predictors of the capability to reduce hours and these same doctor, job and geographic characteristics. Over 40% of GPs stated a preference to reduce their working hours. Characteristics that predicted this preference were being middle-aged, being female, working ≥ 40 hours per week (all P < 0.01), and being on call (P = 0.03). Factors associated with not wanting to reduce working hours were being in excellent health, being satisfied or very satisfied with work (both P < 0.01), and not being a partner in a practice (P < 0.01 for a number of alternative options [ie, associates, contractors and locums]). Of those who wanted to reduce working hours, 26.8% successfully managed to do so in the subsequent year (where reduction was defined as reducing hours by at least 5 per week). Predictors of successfully reducing hours were being younger, female and working ≥ 40 hours per week (all P < 0.01). A number of factors appear to determine both the desire of GPs to reduce hours and their subsequent success in doing so. Declining working hours have contributed to the perceived shortage in GPs. Therefore, designing policies that address not just the absolute number of medical graduates but also their subsequent level of work may alleviate some of the pressures on the Australian primary health care system.
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LA Times Covered Up ‘Looting For Trayvon’ Riot
(INFOWARS) When a flash mob of around 40 teenagers went on a crime spree in Hollywood’s tourist center on Tuesday night, the LA Times reported that the riot was not related to the George Zimmerman verdict, contradicting a police official who later said the “protesters” were shouting “Let’s go mess up Hollywood for Trayvon,” as they conducted their rampage.
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The use of Complementary and Alternative Medicine by Asian women of Hawai'i in the treatment of breast cancer.
This qualitative investigation examined complementary and alternative medicine (CAM) by Hawai'i Asian women breast cancer survivors. The majority of participants felt that the conventional treatment they received was adequate in treating their cancer but was impersonal in nature leaving them feeling abandoned. Many sought CAM to improve their quality of life. Additional research on CAM and the patient-physician relationship is urgently needed.
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Check Out Richard Branson On 'Iconoclasts' Tonight
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A groundbreaking, celeb-filled new season of "ICONOCLASTS" premieres tonight, Oct. 16 at 10 PM ET/PT on Sundance Channel. The premiere episode features Desmond Tutu and Richard Branson, showing their love for life, deep friendship and some interesting lessons they both learn from each other.
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Q:
Как вызвать bat файл перед наполнением инсталлятора?
Мне нужно, чтобы файлы, помещающиеся в инсталлятор, сначала упаковывались upx'ом. Для этого у меня есть .\upx.bat. Что нужно сделать, чтобы перед помещением файлов в инсталлятор вызывался этот bat?
A:
Тут может быть, как минимум, 2 пути:
Написать еще один BAT-файл, который бы сначала вызывал ваш upx.bat, а затем бы вызывал InnoSetup с скриптом сборки инсталлятора, например, написав в этом BAT-файле так: C:\Path_to_InnoSetup\compil32 /cc "c:\isetup\my installer scips\my script.iss"
О параметрах командной строки компилятора можно почитать тут.
Использовать вызов BAT-файла upx.bat из Inno Setup непосредственно перед сборкой инсталлятора (полагаю, для вас это наиболее удобный вариант).
Реализуется этот способ написанием в вашем скрипте команды препроцессора Exec(), например, так: #expr Exec("c:\upx.bat"). В этом случае компилятор InnoSetup выполнит ваш BAT-файл, а на время его выполнения приостановит процесс компиляции.
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The invention concerns accessory tools of the type which are used with great frequency in association with a particular power tool and means for maintaining such accessory tools conveniently accessible while the power tool is in use.
Portable power tools sometimes incorporate a holder carrying the most frequently used accessory tool so that that tool is always conveniently available when needed. This is particularly true and useful for tools such as drills and circular saws where a cutting element (drill bit or saw blade) must be changed frequently. But often there is a secondary accessory tool also frequently, if not constantly, used for which no special provision is made. An example of the latter is a square with a graduated blade, used with a circular saw for marking on a workpiece the next intended cut such as a narrow rip.
Given that encumbering a power tool with even one accessory may be accepted with some reluctance by the tool designer because of space and weight constraints, second accessory tools are even less likely to find a home on the power tool. This is especially true if they are of awkward shape and relatively bulky, such as a square as typically used with a circular saw.
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After Obama
We can imagine what lies ahead in 2017 — no matter the result of either the 2014 midterm elections or the 2016 presidential outcome.
There will be no more $1 trillion deficits. About $10 trillion will have been added to the national debt during the Obama administration, on top of the more than $4 trillion from the prior eight-year George W. Bush administration. That staggering bipartisan sum will force the next president to be a deficit hawk, both fiscally and politically.
In addition, there will be no huge new federal spending programs — no third or fourth stimulus, no vast new entitlements. The debt is so large and voters so tired of massive borrowing that the next president will talk not of “investments” but of balancing the budget. In 2016, President Hillary Clinton or President Marco Rubio will tell us that cutting spending and living within our means is the new cool.
If eight years of borrowing, printing, spending, and lending vast sums of money at zero interest did not lead to economic recovery, then the antithesis of all that will be the explicit platform of Republicans and the implicit one of Democrats.
Obamacare may remain in name, but in fact most of its provisions will be discarded or amended. Its full implementation next year will result in almost everything that was not supposed to happen: higher health-care premiums, rationed care, scarcer doctors and fewer jobs. Obamacare will mostly go the way of the Defense of Marriage Act — officially the law of the land, but its enforcement simply ignored by the powers that be.
Despite an increase in carbon emissions since 2000, the planet did not heat up in the last 15 years. Scientists will continue to argue over global warming, but politicians will not talk much more of implementing costly cap-and-trade policies. They will still praise green energy as the way of the future, but they will not continue the massive subsidies to substitute it for far cheaper fossil fuels.
Instead, expect a renewal of federal oil and natural gas leases on public lands. There is too much newly discovered recoverable energy on federal property to continue to delay its full production — and too much of an upside in cheaper gas at the pump, more independence from Middle East autocracies, more jobs, more money and more economic growth.
Do not expect the same level of increases in disability and unemployment insurance and in food stamps. The trajectories of all those programs since 2009 are not sustainable. For all the talk that Social Security and Medicare are not in bad shape, Democrats and Republicans after Obama will be forced to save both programs by either upping the eligibility age, curbing some benefits or hiking payroll taxes — or all that and more.
The next president will jettison the sort of class warfare that has led only to short-term political gain and long-term polarization. Obama’s “fat cats” and “one percenters” will disappear from the presidential vocabulary. We will hear no more accusations that the successful really did not build their own businesses, or that they should have known when it was time not to profit because they had made quite enough money. Expect just the opposite: a Bill Clinton-like schmoozing of small businesses to please start buying, hiring and expanding again.
Aside from the partisan furor over whether the Obama policies have worked — Democrats will say that things would have been worse without them; Republicans will insist that a natural recovery was turned into long-term doldrums — we will not see them continued.
We are institutionalizing, in European style, huge government, high unemployment, sluggish GDP growth, serial annual deficits, ballooning aggregate national debt and massive dependency, along with near-zero interest rates. The two parties will disagree over the contours of this chronically weak economy, but not over the fact of its weakness — or soon, even its causes. Most Americans will not wish to continue down the road to Italy or Spain.
Barack Obama is a landmark figure: young, charismatic, seemingly post-national and supposedly post-racial. For those reasons alone, he enjoys a level of unshakeable political support not predicated on the actual record of his tenure as president — in the manner most remember fondly that he won the Nobel Prize but don’t quite know what he did to earn it.
Obama’s economic record will be dispassionately acknowledged to be similar to that of Jimmy Carter. But, unlike Carter, Obama will remain a mythical figure in liberal circles.
To borrow a line from a classic Western, “When the legend becomes fact, print the legend.” And so we will do just that.
(Victor Davis Hanson is a classicist and historian at the Hoover Institution, Stanford University. His new book, “The Savior Generals,” will appear this spring from Bloomsbury Press. You can reach him by e-mailing[email protected].)
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Cheer on Your Team With a Best of Times Bar
Football season is in full force.You and your teammates get together every week to watch the action as a crew. Sporting a jersey doesn't reveal the depth of your passion for the game. You're a player who loves to get together with friends and cheer on other teams. You need to check out a team-branded bar set called the Best of Times Bar.
The Best of Times Bar comprises an L-shaped bar, four stools, a side table and an umbrella. It instantly creates atmosphere for watching your team compete or hosting a team-building cookout the night before a game.
Football season is in full force.You and your teammates get together every week to watch the action as a crew. Sporting a jersey doesn't reveal the depth of your passion for the game. You're a player who loves to get together with friends and cheer on other teams. You need to check out a team-branded bar set called the Best of Times Bar.
The Best of Times Bar comprises an L-shaped bar, four stools, a side table and an umbrella. It instantly creates atmosphere for watching your team compete or hosting a team-building cookout the night before a game.
The BOT Bar features a UV-protected umbrella and table banner decked out in your choice of an NFL or college football team's logo and colors. More sports teams, including baseball and basketball teams, are currently being licensed and will likely be available in the near future. Neutral colors are available for off-season use.
We recently had a chance to test out a Dallas Cowboys Best of Times Bar. The set was surprisingly sturdy and fairly easy to assemble and tear down. The steel frame is solid but required no tools to assemble.
Although we set it up indoors, the bar and its accessories are made for outdoor use as well. In fact, the BOT Bar is designed to fit within the width of one parking spot (think "tailgating"). We liked how it became a central area to serve food and hang out, key aspects of team events.
The bar table has two shelves inside for storing snacks and soda, as well as a drop-down cooler, so if you're entertaining outside or tailgating, you don't have to bring an extra one.
If you're looking to showcase your colors and bond with teammates, the Best of Times Bar is worth a look. Visit bestoftimesbar.com for more information.
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Recombinant vaccinia virus: immunization against multiple pathogens.
The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.
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Email This
Your Name
Recipient's Email
Subject
The onus to prove negligence is on the complainant and not the doctor. The complainant has to make an allegation supported by evidence. Mere allegation has no value. It is for the complainant to produce expert opinion that the first reading was wrong and that the second reading was right.The defense is simple and should be based on expert opinion and literature evidence showing that blood pressure can show wide variations within the same day.
The onus to prove negligence is on the complainant and not the doctor. The complainant has to make an allegation supported by evidence. Mere allegation has no value. It is for the complainant to produce expert opinion that the first reading was wrong and that the second reading was right.
The defense is simple and should be based on expert opinion and literature evidence showing that blood pressure can show wide variations within the same day.
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Q:
Bypass a form in VBA and continue macro
When I open files in my macro I get a box popup saying "this workbook contains links to other data sources etc." and then prompts me to select "update" "don't update" or "help". I want the macro to select "don't update" close it or skip it. How would I do this using VBA. I just want to continue and not have this stop the macro.
A:
Running the Macro Recorder and checking the resulting code is helpful for things like this. If you do that I think you'll see something along the lines of:
workbooks.Open FileName:="MyWorkbook",UpdateLinks:=False
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Home
Image shows Lake Malawi cichlids that are just 13 days old. The fish are still attached to their yolk sacs. (Credit: Rob Felt, Georgia Tech)
Cichlid fishes from Lake Malawi offer the unprecedented opportunity to study the relationship between genotype and phenotype in wild vertebrates. Over the past ten years, with funding from the Alfred P. Sloan Foundation, NSF and NIH, we’ve pioneered genomic and molecular biology approaches in this natural system to solve problems difficult to address in traditional model organisms. Major projects include (i) tooth and taste bud patterning and regeneration; (ii) genomics of complex social behavior; and (iii) developmental diversification of the cranial neural crest, placodal plate and neural plate. We analyze and manipulate genomes and development in multiple species of Malawi cichlids, spanning divergence in embryonic/adult phenotypes and behavior – and translate our findings to zebrafish and mouse models.
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Showing that Medical Ethics Cases Can Miss the Point: Rewriting Short Stories as Cases.
I propose a new role for literature in medical ethics: rewriting short stories as ethics cases. This activity is instructive for its power to show that our standard ways of analyzing cases can overlook deeper ethical problems, such as those the short stories raise. To illustrate this claim, I begin by distilling Richard Selzer's story "Fetishes" to an ethics case. Then, using principle-based ethics as a representative analytical framework, I argue that a typical principlist's response to the "Fetishes" case misses the point, failing to address insidious issues like physician arrogance and patient mistrust. By comparing short story and case, we are led to wonder anew whether ethics cases that represent real events might also fail to probe to the heart of the matter. Thus, rewriting short stories as ethics cases can inculcate a healthy skepticism as to whether any case has succeeded in conveying what is most at stake.
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Introduction {#s1}
============
Stimulation of G-protein coupled receptors results in elevated amounts of the second messenger molecule cAMP, leading to the activation of protein kinase A (PKA). PKA is a holoenzyme composed of two regulatory (RI or RII) and two catalytic (C) subunits. Activated PKA phosphorylates key substrates (reviewed in [@pone.0046316-Pidoux1]). A-kinase anchoring proteins (AKAPs) anchor PKA, by means of its R subunit dimer, to subcellular structures [@pone.0046316-Scott1], forming microdomains (recently reviewed in [@pone.0046316-Diviani1]--[@pone.0046316-Mauban1]). This binding confers subcellular localization of PKA, and contributes to PKA specificity and the rapid and effective modulation of PKA-dependent signaling [@pone.0046316-Scott2].
More than 70 AKAPs have been described to date in multiple cell types and cellular compartments. [@pone.0046316-Pidoux1], [@pone.0046316-Diviani1], [@pone.0046316-Skroblin1] Defects in AKAP anchoring or expression have been linked to pathologies such as cardiac arrhythmia [@pone.0046316-Nicolas1]--[@pone.0046316-Kurokawa1], hypertrophy [@pone.0046316-Carnegie1]--[@pone.0046316-AppertCollin1], and the progression to heart failure [@pone.0046316-Zakhary1], [@pone.0046316-Ruehr1]--[@pone.0046316-Aye1]. Our studies, and those of others, have demonstrated that PKA target phosphorylation is decreased in failing hearts. [@pone.0046316-Zakhary1], [@pone.0046316-Zakhary2], [@pone.0046316-Manni1] The importance of proper PKA signaling in failing myocardium is also linked to transcription, as treatment of patients by use of β-blockers reverses the fetal gene switch between α-myosin heavy chain and β-myosin heavy chain. [@pone.0046316-Pandya1]--[@pone.0046316-Rajabi1] Taken together, the scaffolds formed by AKAPs represent a powerful mechanism for mediating PKA signaling in the cell, and impaired AKAP:PKA interaction has serious implications for development of cardiac disease.
AKAPs are a functionally diverse family with little sequence similarity, except that they share a characteristic amphipathic α-helical domain that is approximately 14--18 amino acids (aa) in length. [@pone.0046316-Carr1] This helix has a hydrophobic face that fits in a groove formed by the amino-termini of the R dimer. [@pone.0046316-Carr1]--[@pone.0046316-Vijayaraghavan1] In binding PKA as well as PKA substrates and regulators, AKAPs create a scaffold to effectively localize and modulate signaling through PKA. Given the importance of AKAPs in cardiac function, we used a T7 phage display assay to identify PKA binding proteins. [@pone.0046316-Russell1] When we screened a cDNA library derived from human heart, we isolated multiple clones of chromodomain helicase binding protein 8 (Chd8), suggesting that Chd8 could also act as an AKAP.
In studies conducted to date, Chd8 has been primarily characterized as a nuclear protein [@pone.0046316-Yamashina1] that regulates chromatin dynamics [@pone.0046316-Ishihara1]--[@pone.0046316-Thompson1], transcription [@pone.0046316-Yates1], [@pone.0046316-RodriguezParedes1], [@pone.0046316-Yuan1], and cell survival [@pone.0046316-Rodenberg1]--[@pone.0046316-Nishiyama2]. Chd8 was first identified as a 749aa nuclear protein named "duplin," [@pone.0046316-Sakamoto1], [@pone.0046316-Kobayashi1] and found to inhibit the Wnt signaling pathway. [@pone.0046316-Sakamoto1] In humans, Chd8 predominantly exists in two larger isoforms, Chd8-L1 (2582aa) and Chd8-L2 (2301aa). [@pone.0046316-Ishihara1], [@pone.0046316-RodriguezParedes1], [@pone.0046316-Yuan1] ([***Figure 1***](#pone-0046316-g001){ref-type="fig"}) Chd8 contains binding sites for and negatively regulates β-catenin [@pone.0046316-Thompson1], [@pone.0046316-Nishiyama1], [@pone.0046316-Sakamoto1], [@pone.0046316-Kobayashi1] and p53 [@pone.0046316-Nishiyama2] by means of its ability to bind histone H1. Loss of Chd8 in knockout mice resulted in an embryonic lethal phenotype at embryonic day (E) 8.5 resulting from increased p53-dependent apoptosis. [@pone.0046316-Nishiyama2], [@pone.0046316-Nishiyama3] Double knockout of Chd8 and p53 extended embryo survival to E10.5, at which point embryos failed to form mesoderm and exhibited massive hemorrhaging characteristic of cardiovascular defects. [@pone.0046316-Nishiyama2] Chd8 binds other components of transcription, including di−/trimethylated lysine 4 on the histone H3 subunit (H3K4) [@pone.0046316-Yates1], [@pone.0046316-RodriguezParedes1], [@pone.0046316-Menon1], RNA polymerase II [@pone.0046316-RodriguezParedes1], and members of the Mixed Lineage Leukemia (MLL) complex, WRD5, ASH2L, and RbBP5 [@pone.0046316-Menon1]. Considering the diversity of its binding partners, Chd8 is likely a component of the scaffold of several large protein complexes, each with a distinct function [@pone.0046316-Menon1].
![Three isoforms have been identified for Chd8.\
Chd8-S arises from passage of transcription through the end of exon 9 into intron 10, where it terminates. [@pone.0046316-RodriguezParedes1], [@pone.0046316-Rodenberg1] Chd8-S contains a single chromodomain (C1). Two longer isoforms contain the tandem chromodomains (C1 and C2) and helicase domain characteristic to Chd proteins [@pone.0046316-Marfella1], [@pone.0046316-Hall1]. Chd8-L1 and Chd8-L2 result from two different start sites for transcription [@pone.0046316-RodriguezParedes1], with the Chd8-L1 transcript encoding an amino terminus extension that encompasses a p53 binding domain [@pone.0046316-Nishiyama2]. All three isoforms contain a series of five nuclear localization signals (NLS) [@pone.0046316-Kobayashi1] and a β-catenin binding domain (β) [@pone.0046316-Thompson1], [@pone.0046316-Sakamoto1], [@pone.0046316-Nishiyama3] that is also required for histone H1 [@pone.0046316-Nishiyama1] binding [@pone.0046316-Nishiyama1] and STAT3 [@pone.0046316-Yamashina1] binding. Chd8-L1 and Chd8-L2 also contain a pair of BRK domains, which mediate chromatin interaction via the histones and is required for interaction with CTCF [@pone.0046316-Ishihara1]. All three isoforms contain the AKAP domain (RII) characterized in this study.](pone.0046316.g001){#pone-0046316-g001}
Given the evidence for a nuclear microdomain of PKA [@pone.0046316-Sample1], [@pone.0046316-Zippin1], we hypothesized that Chd8 is a novel nuclear AKAP. Also, as Chd8 has largely been studied as a nuclear protein and in the context of development [@pone.0046316-Sakamoto1]--[@pone.0046316-Nishiyama3], we investigated subcellular localization and expression of Chd8 during cardiac development and in non-cardiac cells. Our findings demonstrate a novel localization of Chd8 to a discrete perinuclear microdomain, as well as to the nucleus, and define a new link between PKA-dependent signaling and proteins responsible for chromatin remodeling.
Materials and Methods {#s2}
=====================
Ethics Statement {#s2a}
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All animal studies were conducted in compliance with the Animal Welfare Act, Public Health Service Policy on Humane care and Use of Laboratory Animals, and in compliance with the *Guide for the Care and Use of Laboratory Animals,* published by the National Institutes of Health (NIH publication No. 85--23, revised 1996). All animal work was performed under protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland, School of Medicine.
Antibodies, Reagents {#s2b}
--------------------
Commercial Chd8 antibodies were obtained from Bethyl Laboratories and used for Western blotting and immunoprecipitation (Bethyl Laboratories, Montgomery, TX), and immunofluorescence (Bethyl). Anti-myc-epitope (Cell Signaling Technology, Danvers, Massachusetts), RIIα/β (EMD Millipore, Billerica, Massachusettes), RIIα (BD, Franklin Lakes, New Jersey), RIIß (BD), and anti-Golgi apparatus (EMD) antibodies were used for western blotting of immunoprecipitates or immunofluorescence, as described. GAPDH (Life Technologies/Ambion, Grand Island, New York) was used for loading control.
Cell Culture {#s2c}
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Pregnant Sprague-Dawley female rats were ordered from Harlan Labs (Frederick, Maryland). Primary neonatal cardiomycytes (NCMs) were harvested from pups at post-natal day 1 and cultured as previously described. [@pone.0046316-Russell1], [@pone.0046316-Wright1] CHO cells (American Type Culture Collection, Manassas, VA) were cultured in Ham's F12 media with 10% FBS. HeLa cells (ATCC) and HEK cells (ATCC) were cultured in DMEM media (high glucose) with 10% FBS. All transfections were carried out with Lipofectamine-2000 (Life Technologies/Invitrogen).
Plasmids {#s2d}
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A plasmid for the 'duplin' isoform of Chd8 (which we refer to as Chd8-S) was kindly provided by Dr Akira Kikuchi, Hiroshima University, Japan. The QuikChange XL Site-Directed Mutagenesis Kit (Agilent/Stratagene, Santa Clara, California) was used to introduce point mutations of key constructs, according to the manufacturer instructions. RIIα, RIIα-SA, and RIIα-SD mutants were created as described [@pone.0046316-Manni1] and cloned into peGFP-C1 (Clontech Laboratories, Mountain View, California), in which a CFP was substituted for GFP, for creation of CHO cell lines.
Phage Display Screening {#s2e}
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Phage display screening was performed using a human heart cDNA library as previously described. [@pone.0046316-Russell1] Briefly, a 96-well dish was coated with recombinant RIIα purified from *E. coli* expressing RIIα-pET11d. 106 clones from a T7-select Phage Display Library (EMD) specific for human heart cDNA were screened. Phage-specific primers were then used for PCR amplification of RII binding peptides, which were sequenced (DNA Sequencing Core Facility, Lerner Research Institute, Cleveland Clinic Foundation) and analyzed with Lasergene software (DNASTAR) and BLAST programs (NCBI, National Institutes of Health). Three clones were isolated and identified by BLASTn search as Chd8.
Western Blotting {#s2f}
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For protein extraction of transfected cells, cells were lysed 48 hours after transfection with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, Illinois) with protease inhibitor cocktail (Sigma-Aldritch). NCMs were harvested for protein extraction four days after isolation using a buffer containing M-PER, 40 mM EDTA, 300 mM NaCl, and protease inhibitor cocktail (Sigma-Aldritch), as described. [@pone.0046316-Russell1] Micro-BCA was used to determine protein concentration (Thermo Scientific). Lysate was boiled with 4×SDS loading buffer (with DTT), separated by SDS-PAGE, and transferred to PVDF. Western blots used 50 micrograms of total protein per lane, unless noted. The blot was blocked with 5% milk-Tween solution. Blots were incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific), and positive bands detected by chemiluminescence. Primary antibodies were used at a dilution of 1∶2000, and secondary antibodies at a concentration of 1∶10,000, except where otherwise noted in the figure legend.
RII Overlay {#s2g}
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PCR primers were used to generate cDNA encoding the RII binding site of rat Chd8 (Chd8^RII^). This cDNA was cloned into the pTrcHis2/TOPO vector (Invitrogen). Colonies were selected based on antibiotic resistance and sequenced by the Genomics Core Facility (University of Maryland, Baltimore). Bacteria were grown to an OD600 value of 0.6, and isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce expression according to manufacturer's instructions. A control vector containing LacZ was transformed and used to verify expression. Samples were taken hourly, prepared with 1× Laemli loading buffer with β-mercaptoethanol, separated by electrophoresis and transferred to nitrocellulose for Western blotting. For RIIα overlays, 5 mL of bacterial culture suspended in LB was collected at hour 3, spun down, and lysed in 1× SDS loading buffer. Bacterial lysate separated on a 12% Tris-HCl gel and transferred to nitrocellulose membrane. The control expression protein (LacZ) as a negative control. Purified recombinant RIIα (5 µg/mL in 10% BSA/TBS), was incubated alone, or with either 50 uM Ht31 or Ht31P peptides (generated by Biopolymer Core, University of Maryland, Baltimore). After blocking for 1 hour, separate blots were incubated overnight with the RIIα solutions. Each blot was washed thoroughly and developed using antibody against RIIα/β (EMD).
Co-immunoprecipitation {#s2h}
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Immunoprecipitations of tagged protein constructs were conducted with the myc-tag co-immunoprecipitation kit (Thermo Scientific) according to manufacturer's instructions.
Immunostaining and Inverted Fluorescent Microscopy {#s2i}
--------------------------------------------------
Cells were grown on glass coverslips and fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature, then permeabilized with 0.1% Triton X/PBS for 10 minutes. Cells were blocked with 3% BSA/PBS, and then incubated with primary antibody at the following dilutions: myc antibody (CST, 1∶250), Chd8 (Bethyl, 1∶200 or 1∶50), RIIα/β (EMD, 1∶80), Golgi apparatus (EMD, 1∶100), α-actinin (Sigma-Aldritch, 1∶250). For [Figure S4](#pone.0046316.s004){ref-type="supplementary-material"}, cells were immunolabeled with Chd8-Sigma, a polyclonal antibody purified from anti-sera from New Zealand White rabbits inoculated with 77--90aa of Chd8 (Sigma-Aldrich Genosys, St Louis, Missouri). Sera was purified, eluted from the purification column, and used for immunofluorescence at a dilution of 1∶250 (HeLa) or 1∶150 (primary rat neonatal cardiac cells). Cells were washed for 1 hour with 1% BSA/PBS, incubated with Alexafluor conjugated antibodies (Life Technologies/Invitrogen, Grand Island, New York) as indicated at 1∶500, and washed again. Coverslips were mounted and nuclei were stained using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingam, California). All antibody incubations occurred for 1 hour at room temperature, except for myc and Chd8-Sigma, which occurred at 4°C overnight. Immunostaining was visualized with a Nikon TE2000-U inverted fluorescent microscope (Nikon Instruments, Melville, New York) and images were obtained with a Spot digital camera (Diagnostic Instruments, Inc., Sterling Heights, Michigan) with Spot Advanced 4.0.2 software.
Confocal Microscopy {#s2j}
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A Zeiss 5 Live slit scanning confocal microscope was used to image HeLa cells that were colabeled with antibodies for Golgi apparatus and Chd8. Imaging was performed with a 63X, 1.4 NA oil immersion objective, and data acquisition was set at .21 µm/pixel, 12 bit. The Alexafluor goat anti-mouse 568 antibody used to detect Golgi signal was excited at 561 nm and emission was long pass filtered at 575 nm. Excitation of Chd8 signal, detected by Alexafluor goat anti-rabbit 488 was at 489 nm and emission was bandpass filtered at 495--555 nm.
Taqman Quantitative PCR and Analysis {#s2k}
------------------------------------
Rat hearts were harvested for RNA extraction with the Quick Prep Total RNA Extraction kit (GE Life Sciences, Piscataway, New Jersey) at embryonic days (16 and 19), post natal days (1, 3, 7, and 21), and six months. RNA was converted into cDNA with the iScript kit (Bio-Rad, Hercules, California). 20 ng of cDNA was used per assay. Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described. [@pone.0046316-Lund1] Each sample was assayed in triplicate, and the average of the three values was normalized to GAPDH (internal reference). Relative quantitation of mRNA was calculated using 2^−ΔΔCt^ method, as described elsewhere [@pone.0046316-Schmittgen1], with embryonic day 16 set to "1".
Results {#s3}
=======
Identification of Chd8 as a Novel Binding Partner for RIIα {#s3a}
----------------------------------------------------------
The T7 phage display assay was used to screen a human heart cDNA expression library to identify novel cardiac AKAPs. This approach identified several known RIIα binding proteins that served as positive controls and verified our method. These included RIIα [@pone.0046316-Lohmann1] and two known cardiac AKAPs, mAKAP [@pone.0046316-Kapiloff1] and AKAP-Lbc [@pone.0046316-Carnegie1], [@pone.0046316-Diviani2]. We isolated clones that contained fragments of seven previously unidentified RIIα binding proteins. One of those proteins, synemin, is an intermediate filament protein that we characterized as a novel AKAP that associates with the Z-discs and cell junctions in cardiac myocytes [@pone.0046316-Russell1], [@pone.0046316-Lund1].
Among the proteins we identified as binding partners for RII, three clones contained peptide fragments of Chd8. Using BLAST analysis of the peptide fragments, we found that the three clones overlapped with the amino terminus of Chd8 and encoded 391--545aa of the Chd8-L1 isoform. This sequence is present in all three isoforms of Chd8 and is located immediately upstream of a nuclear localization signal (NLS). ([***Figure 1***](#pone-0046316-g001){ref-type="fig"}) Because of the known role for Chd8 in regulating transcription and cell survival, we focused our investigations on the potential role of Chd8 as an AKAP.
Given the importance of an amphipathic α-helix to the RII:AKAP interaction [@pone.0046316-Carr1], we used bioinformatics to analyze the predicted secondary structure of Chd8 to identify putative amphipathic helices. We translated the Chd8 cDNA and used this amino acid sequence to predict regions of α-helical domains within the Chd8 sequence. ([***Figure 2A***](#pone-0046316-g002){ref-type="fig"}) A search was conducted using GeneiousPro, a bioinformatic platform that includes a tool for generating secondary structure predictions via the EMBOSS Garnier algorithm. [@pone.0046316-Garnier1], [@pone.0046316-Robson1] A second tool, JPRED, predicts secondary structure and utilizes position-specific scoring matrices, Hidden Markov Model profiles, and structures stored in databases like UniProt and PDB to predict protein structure and accessibility of amino acid residues. [@pone.0046316-Cole1] Both algorithms predicted an α-helix within the Chd8 peptide that was isolated by the phage display, generating a targeted prediction of an AKAP domain. ([***Figure 2A, 2B***](#pone-0046316-g002){ref-type="fig"}).
{#pone-0046316-g002}
A ClustalW alignment [@pone.0046316-Larkin1] was performed with the Chd8 fragment and the RII binding domains of several known AKAPs. ([***Figure 2C***](#pone-0046316-g002){ref-type="fig"}) The AKAP domains aligned with amino acids within the Chd8 α-helix (455--473aa of Chd8-L1). Hydrophobic profiles of aligned sequences were also similar. ([***Figure 2C***](#pone-0046316-g002){ref-type="fig"}) A two dimensional helical wheel plot was generated of the proposed Chd8 RIIα binding site (KKQEKANRIVAEAIARAR). The 2D plot shows clustering of hydrophobic residues on one side of the helix, consistent with RII binding domains of other AKAPs. [@pone.0046316-Fischer1] ([***Figure 2D***](#pone-0046316-g002){ref-type="fig"}) This region of the Chd8 sequence is very highly conserved across species. ([***Figure 2E***](#pone-0046316-g002){ref-type="fig"}) Thus, using a large scale screening method in conjunction with bioinformatics-based approaches, we identified an amphipathic α-helical structure within the Chd8 peptide fragment that associated with RIIα in our phage display assay. This α-helical region displayed similar amino acid properties as other known AKAPs.
Chd8 Binds to RIIα *in vitro* {#s3b}
-----------------------------
RII overlay was used next to determine the binding capability of RIIα to this fragment of Chd8. RII overlay is frequently used to identify novel PKA binding proteins [@pone.0046316-Lohmann1], [@pone.0046316-Carr3] and exploits the ability of RII to bind AKAP protein on a Western blot. We cloned a 150 amino acid fragment of Chd8 (Chd8^RII^, corresponding to 390--530aa of Chd8-L1). This peptide was then expressed as an inducible His/myc fusion protein in *E. coli*. ([***Figure 3A***](#pone-0046316-g003){ref-type="fig"}) Mutation of a hydrophobic residue within the amino acid sequence of an AKAP to a Pro residue is sufficient to abolish α-helical structure and disrupt PKA binding [@pone.0046316-Carr2], [@pone.0046316-Carr4], [@pone.0046316-Hausken1]; therefore we used site-directed mutagenesis to introduce a Pro mutation at Ile 464 (Chd8^RII^-P). Based on the bioinformatics analysis of the predicted RIIα binding domain, we predicted that this mutation would eliminate RIIα binding. Immunoblotting methods were used to demonstrate expression of each construct (∼22 kDa MW). ([***Figure 3B***](#pone-0046316-g003){ref-type="fig"}).
{#pone-0046316-g003}
In RII overlay assays, purified recombinant RIIα bound to a 22 kDa band in protein extracted from bacteria expressing Chd8^RII^, indicative of a 22 kDa RIIα binding protein. ([***Figure 3C***](#pone-0046316-g003){ref-type="fig"}, top, center lane) In contrast, no corresponding bound RIIα was detected in lysate from bacteria expressing Chd8^RII^-P, indicating a loss of RIIα binding ability in the fragment carrying the I464P mutation. We then pre-incubated RIIα with Ht31 or Ht31P. Ht31 is a peptide derived from the AKAP domain of AKAP-Lbc that is commonly used to inhibit AKAP:PKA interaction and Ht31P is the same peptide containing a Pro mutation that disrupts the structure of the AKAP domain and prevents association of the peptide with the RII dimer. [@pone.0046316-Carr1], [@pone.0046316-Carr2], [@pone.0046316-Herberg1], [@pone.0046316-Alto1] Pre-incubation of RIIα with Ht31 peptide prevented the binding of RIIα to Chd8^RII^ in RII overlay, whereas the pre-incubation of RIIα with Ht31P had no effect. ([***Figure 3C***](#pone-0046316-g003){ref-type="fig"}, center and bottom) We concluded that, like other known AKAPs, RIIα binds Chd8^RII^, but not Chd8^RII^-P, in RII overlay assays.
We then investigated whether Chd8 and RIIα interact in intact cells. CHO cells were transfected with either Chd8-S or Chd8-S-P, the latter of which contained the same I464P mutation that was sufficient to prevent binding of RIIα to Chd8^RII^-P in the RII overlay assays. Immunofluorescence and Western blotting methods showed that the I464P mutation did not interfere with localization or expression of myc-tagged Chd8-S. ([***Figure 4A, 4B***](#pone-0046316-g004){ref-type="fig"}) CHO cells were cotransfected with RIIα and with either Chd8-S or Chd8-S-P. RIIα co-immunoprecipitated with Chd8-S, but not with Chd8-S-P, demonstrating that the I464P mutation in the AKAP domain of Chd8-S resulted in loss of binding to RIIα. ([***Figure 4C***](#pone-0046316-g004){ref-type="fig"}) No RIIα was detected in immunoprecipitation of single transfections. We concluded that Chd8-S and RIIα coimmunoprecipiate when coexpressed in CHO cells, but that mutation of the RIIα binding domain in Chd8-S leads to loss of interaction. As I464P was sufficient to prevent coimmunoprecipitation of Chd8-S and RIIα, we also concluded that the RIIα binding domain which we identified by bioinformatics and RIIα overlay is the only RIIα binding domain in Chd8-S.
{#pone-0046316-g004}
As experimental and modeling evidence has defined a nuclear microdomain of AKAP-bound PKA in the nuclei of HEK cells [@pone.0046316-Sample1], we used cAMP-coupled agarose beads to pull down cAMP-binding proteins in HEK cell lysate. RIIα and RIIβ were detected in proteins that eluted with cAMP agarose. Addition of 8-Br-cAMP, a non-hydrolyzable analogue of cAMP, competed with the cAMP agarose for cAMP binding and reduced the amount of RIIα and RIIß pulled down by the beads. A high molecular weight band corresponding with Chd8-L1 was also detected in pulldown assays with cAMP beads, but not in samples incubated with 8-Br-cAMP. ([***Figure 5***](#pone-0046316-g005){ref-type="fig"}) We concluded that Chd8 co-elutes with the cAMP-binding proteins RIIα and RIIβ.
{#pone-0046316-g005}
Phosphorylation of RIIα at Serine 96 Inhibits PKA Anchoring by Chd8-S {#s3c}
---------------------------------------------------------------------
Autophosphorylation of RIIα by the C subunit at a Ser (Ser96) in the inhibitory domain of RIIα promotes the activation of C for target phosphorylation. [@pone.0046316-Taylor1]--[@pone.0046316-Erlichman1] Our recent findings demonstrate that dephosphorylation of Ser96 promotes reassembly of the PKA holoenzyme and reduces binding of RIIα to AKAP15/18. [@pone.0046316-Zakhary2], [@pone.0046316-Manni1] Thus, autophosphorylation of RIIα at Ser96 plays a key role in modulation of PKA activity and localization of the holoenzyme. We next investigated whether RIIα regulation via autophosphorylation affects the interaction between Chd8-S and RIIα.
We created three CHO cell lines stably expressing RIIα constructs in which Ser96 was not altered (RII), or was mutated to Ala, in order to mimic constitutively dephosphorylated RIIα (RII-SA). RIIa was also mutated to Asp to mimic constitutively phosphorylated RIIα (RII-SD). Expression of each RIIα construct was verified by immunofluorescence, using a CFP tag ([***Figure 6A***](#pone-0046316-g006){ref-type="fig"}), and by Western blot analysis ([***Figure 6B***](#pone-0046316-g006){ref-type="fig"}). Each cell line was transiently transfected with Chd8-S. ([***Figure 6B***](#pone-0046316-g006){ref-type="fig"}) Chd8-S was immunoprecipitated from all transfected cultures. Western blot analysis of the Chd8-S immunoprecipitate identified RII and RII-SD, but not RII-SA. ([***Figure 6C***](#pone-0046316-g006){ref-type="fig"}) As this result was consistent with the anchoring dynamics of other AKAPs [@pone.0046316-Zakhary2], [@pone.0046316-Manni1], we concluded that dephosphorylation of Ser96 (RII-SA) eliminates the interaction of RIIα and Chd8-S, whereas autophosphorylation of RIIα at Ser96 promotes binding of Chd8 and RIIα.
{#pone-0046316-g006}
Subcellular Localization of RII and Chd8 {#s3d}
----------------------------------------
Consistent with the first study of Chd8-S [@pone.0046316-Sakamoto1], [@pone.0046316-Kobayashi1], our localization studies of cells overexpressing the Chd8-S isoform show that it is restricted to the nucleus ([***Figure 4A***](#pone-0046316-g004){ref-type="fig"}). [@pone.0046316-Sakamoto1], [@pone.0046316-Kobayashi1] However, since those studies were published, additional isoforms of Chd8 (Chd8-L1 and Chd8-L2) have been described. Other AKAP genes, including those for AKAP350 [@pone.0046316-Shanks1] and AKAP-Lbc [@pone.0046316-Rogers1], [@pone.0046316-Mayers1] encode multiple isoforms with different patterns of subcellular localization. To determine if the longer Chd8 isoforms exhibit the same subcellular localization pattern, we examined the endogenous localization of Chd8.
We immunostained HeLa human adenocarcinoma cells, which have been previously used to identify Chd8 binding partners [@pone.0046316-Thompson1], with a polyclonal antibody raised against a 50 amino acid fragment of the carboxy terminus of Chd8-L1 and Chd8-L2. Our immunostaining of endogenous Chd8 revealed nuclear staining ([***Figure 7A***](#pone-0046316-g007){ref-type="fig"}, arrowhead; [Figure S2A](#pone.0046316.s002){ref-type="supplementary-material"}), but, interestingly, we also found a perinuclear pattern of immunofluorescence. ([***Figure 7A***](#pone-0046316-g007){ref-type="fig"} ***, arrows***) This staining pattern was reproduced using an alternate antibody raised against the amino terminus of Chd8. ([Figure S4A](#pone.0046316.s004){ref-type="supplementary-material"}) Because of the distinctive perinuclear pattern, we costained HeLa cells with an antibody raised against the Golgi fraction of human cells. We observed perinuclear immune-labeling of Chd8 in the region of the Golgi apparatus. ([***Figure 7A***](#pone-0046316-g007){ref-type="fig"}) Confocal microscopy of HeLa cells co-labeled with antibodies raised against Chd8 and Golgi showed similar plot profiles of the immunofluorescence patterns in the same focal plane. ([***Figure 7B***](#pone-0046316-g007){ref-type="fig"}) Addition of the immunogen against which the Chd8 immunostaining antibody was raised resulted in loss of both the nuclear and perinuclear immune-labeling. ([Figure S2](#pone.0046316.s002){ref-type="supplementary-material"}) Costaining of HeLa cells with antibodies against Chd8 and PKA RIIα/β demonstrated perinuclear staining for both proteins. Overlay of images demonstrated subcellular colocalization of these signals, particularly in the perinuclear domain. ([***Figure 7C***](#pone-0046316-g007){ref-type="fig"} ***,*** arrows).
{#pone-0046316-g007}
Chd8 is Expressed in Rat Cardiac Development {#s3e}
--------------------------------------------
Chd8 was isolated in our screen for novel cardiac AKAPs. [@pone.0046316-Russell1] As Chd8 has been implicated in embryonic development [@pone.0046316-Nishiyama2], [@pone.0046316-Sakamoto1], [@pone.0046316-Nishiyama3], we examined the expression of Chd8 in developing rat cardiac tissue. In a study of mouse embryos, Chd8 mRNA is expressed at high levels early in embryogenesis and lower levels found in adult mice. [@pone.0046316-Sakamoto1], [@pone.0046316-Nishiyama3] Given Chd8's role as a negative regulator of p53, differential regulation of Chd8 may contribute to p53-dependent apoptosis required in organogenesis [@pone.0046316-Nishiyama2], or to the selective regulation of the Wnt/β-catenin pathway during development [@pone.0046316-Thompson1], [@pone.0046316-Nishiyama1].
Two sets of Taqman probes were used to detect Chd8 mRNA in developing cardiac muscle. ([***Figure 8A***](#pone-0046316-g008){ref-type="fig"}) Probe 1, which spans exons 2--3 and encompasses the AKAP binding domain of Chd8, recognizes all three isoforms transcripts of Chd8. Probe 2 recognizes Chd8-L1 and Chd8-L2 and spans exons 12 and 13, which encode the helicase domain. A probe against the mRNA transcript for RIIα (PKAR2A) was used to measure RIIα mRNA. We detected high levels of cardiac CHD8 mRNA at PN7, followed by a decrease to low but detectable levels in 6 month-old rat heart. ([***Figure 8B***](#pone-0046316-g008){ref-type="fig"}).
{#pone-0046316-g008}
We next utilized rat neonatal cardiac myocytes to study expression of Chd8 protein in the heart. Western blot analysis of myocyte protein extract identified both the Chd8-L1 and Chd8-L2 isoforms. ([***Figure 8C***](#pone-0046316-g008){ref-type="fig"}) Immunofluorescence of fixed cultures showed Chd8 expression in both myocytes ([***Figure 8D***](#pone-0046316-g008){ref-type="fig"}, arrows) and fibroblasts ([***Figure 8E***](#pone-0046316-g008){ref-type="fig"}, arrowheads). As we previously reported above for HeLa cells, the antibody to Chd8 detected nuclear immunostaining, as well as a perinuclear pool of Chd8, in both cell types. ([***Figure 8D***](#pone-0046316-g008){ref-type="fig"}) This staining pattern was reproduced using an alternate antibody raised against the amino terminus of Chd8. ([Figure S4B](#pone.0046316.s004){ref-type="supplementary-material"}) In contrast, we observed only perinuclear staining of RIIα/β in myocytes and fibroblasts. ([***Figure 8E***](#pone-0046316-g008){ref-type="fig"}) These findings indicated that Chd8 is expressed in cardiac cells, where it localizes to both nuclear and perinuclear domains, similar to the immunostaining pattern seen in HeLa cells. ([Figure S2B](#pone.0046316.s002){ref-type="supplementary-material"}).
Discussion {#s4}
==========
We have shown that Chd8, previously identified as a chromatin binding protein, also binds RIIα in intact cells and is thus a new member of the AKAP family. Similar to our previous findings that phosporylated RIIα has a higher affinity for AKAPs than dephosphorylated RIIα [@pone.0046316-Zakhary2], [@pone.0046316-Manni1], we demonstrated that phosphorylation of RIIα is required for Chd8:PKA association. Furthermore, in addition to the identified nuclear localization of Chd8, we also demonstrated a novel perinuclear localization of Chd8 in close proximity to the Golgi apparatus.
Chd8 Contains an AKAP Domain in its Amino Terminus {#s4a}
--------------------------------------------------
Having established RIIα binding to Chd8 by phage display, we used bioinformatics, including the prediction of secondary structure, predictive modeling, alignments, to identify the AKAP domain of Chd8. ([***Figure 2***](#pone-0046316-g002){ref-type="fig"}) Bioinformatics has been previously used to study R:AKAP interactions, and is a powerful tool when paired with experimental approaches. [@pone.0046316-Alto1], [@pone.0046316-McLaughlin1], [@pone.0046316-Means1] A study by McLaughlin *et al*, published while this manuscript was in progress, also identified a 24 amino acid peptide of Chd8 that overlaps with the Chd8 PKA binding domain. This domain was identified using our own bioinformatics-based approach. McLaughlin *et al* used a panel of Ala mutations within the Sphingosine kinase interacting protein (SKIP) to show that SKIP is a dual AKAP. [@pone.0046316-McLaughlin1] In contrast, another recent study used molecular modeling together with site-directed mutagenesis and immunoprecipitation, to show that SKIP is exclusively an RI-specific AKAP that does not bind RII. [@pone.0046316-Means1] These divergent findings highlight the importance of pairing experimental manipulation with bioinformatics approaches.
Our findings that dephosphorylation of RIIα at Ser96 prevents the binding of Chd8 and RIIα suggest a mechanism by which anchoring of PKA to Chd8 may be regulated within nuclear and/or perinuclear microdomains. ([***Figure 5***](#pone-0046316-g005){ref-type="fig"}) RIIα phosphorylation at different sites modulates the affinity of the RIIα dimer for AKAPs within the same subcellular compartment. [@pone.0046316-Chen2], [@pone.0046316-Landsverk1] Our previous work shows that PKA autophosphorylation of RIIα in the inhibitory domain at Ser96 increases the affinity of RIIα for AKAP15/18. [@pone.0046316-Manni1] Past work from our lab also demonstrated that the relative decrease in affinity of RIIα for AKAPs upon dephosphorylation at Ser96 varies between AKAPs. For example, the decrease in binding affinity between dephosphorylated RIIα and AKAP15/18 is more than 600 times greater than the decrease in binding affinity for dephosphorylated RIIα and AKAP-Lbc, as compared to phosphorylated RIIα. [@pone.0046316-Ruehr1], [@pone.0046316-Zakhary2] Our studies likewise demonstrate that phosphorylation of RIIα at Ser96 increases the probability of RIIα association with Chd8. In unstimulated cells, low levels of RIIα phosphorylation have been observed, under baseline conditions, and exogenously expressed RIIα can be phosphorylated and dephosphorylated at Ser96. [@pone.0046316-Manni1] Since we observed immunoprecipitation of Chd8-S with RII-SD but not RII-SA, it is likely that the RIIα that is immunoprecipitated in our assay is phosphorylated. We also observed a perinuclear distribution of RII and RII-SD. ([***Figure 6B***](#pone-0046316-g006){ref-type="fig"}, arrows) We hypothesize that phosphorylation of RIIα at Ser96 may serve as a molecular switch that increases binding affinity for Chd8 versus RIIa binding to other AKAPs in the same compartment.
AKAPs target other proteins that regulate stability of cAMP and the phosphorylation state of R. [@pone.0046316-DodgeKafka2] Based on our findings, we propose that Chd8 anchors PKA in close proximity to p53, histone H1, or β-catenin in the nucleus following activation of the PKA signaling pathway. ([***Figure 1***](#pone-0046316-g001){ref-type="fig"}) Other AKAPs bind phosphodiesterases or protein phosphatases, which attenuate the PKA pathway after the elevation of cAMP. [@pone.0046316-Diviani1], [@pone.0046316-DodgeKafka2] Recent work characterizing a nuclear PKA microdomain identified candidate binding proteins for nuclear AKAPs, including soluble adenylyl cyclase (AC). Soluble AC could participate in nuclear PKA signaling. [@pone.0046316-Zippin1], [@pone.0046316-Zippin2] One intriguing result arose from modeling the activation of a nuclear microdomain of cAMP and PKA. [@pone.0046316-Sample1] This study reported a nuclear microdomain of PKA that permitted rapid activation kinetics of PKA in the nucleus following activation of sAC. Introduction of a hypothetical nuclear AKAP into this kinetic model likewise implicated the importance of PKA and phosphodiesterase anchoring in the nucleus, although no specific AKAP was manipulated experimentally. [@pone.0046316-Sample1] Chd8 is a possible candidate for this unknown AKAP.
Chd8 Exists within Nuclear and Perinuclear Microdomains {#s4b}
-------------------------------------------------------
Few AKAPs have been reported to reside in the nucleus. Despite a longstanding model in which C subunits of PKA translocate to the nucleus following elevation of cAMP, several reports indicate that a nuclear microdomain of PKA [@pone.0046316-Sample1], [@pone.0046316-Zippin1], [@pone.0046316-Patel1]--[@pone.0046316-Mednieks1] and cAMP [@pone.0046316-Zippin1], [@pone.0046316-Zippin2], [@pone.0046316-Zippin3] does exist, possibly governed by sAC. [@pone.0046316-Sample1], [@pone.0046316-Zippin1], [@pone.0046316-Zippin2], [@pone.0046316-Chen3] The nuclear distribution of PKA regulatory subunits has been reported in multiple cell lines and tissues [@pone.0046316-Sample1], [@pone.0046316-Patel1], [@pone.0046316-Miller1], [@pone.0046316-Mednieks1], [@pone.0046316-Zhang1]--[@pone.0046316-SquintoSPJungmann1], including HeLa cells [@pone.0046316-Zippin1]. Localization of AKAPs to the nucleus permits rapid and effective signal transduction in the nuclear compartment. [@pone.0046316-Sample1] To date, nuclear AKAP95 has been best characterized: PKA anchoring via AKAP95 is required for proper chromatin condensation during mitosis. [@pone.0046316-Landsverk1], [@pone.0046316-Eide1]--[@pone.0046316-Eide2] AKAP7 [@pone.0046316-Brown1] and nAKAP150 [@pone.0046316-Zhang1] localize to the nucleus during development, whereas the splicing factor SFRS17A is a dual AKAP that regulates pre-mRNA splicing. [@pone.0046316-Jarnaess1] Our identification of an AKAP domain in Chd8 expands the understanding of the roles of nuclear AKAPs.
Some AKAPs, e.g. AKAP350, AKAP13, exhibit alternative subcellular localization of different isoforms. [@pone.0046316-Shanks1]--[@pone.0046316-Mayers1] Our results indicate that Chd8 exists in at least two microdomains, one nuclear and one perinuclear. ([***Figure 7***](#pone-0046316-g007){ref-type="fig"}) It remains to be determined if the two pools of Chd8 contain different isoforms, or if Chd8-L1 and Chd8-L2 are present in both. Given the functional diversity of AKAPs, isoforms of Chd8 may play differential role in anchoring PKA to different subcellular microdomains.
Similar to its subcellular distribution in HeLa cells, we showed that Chd8 exhibits nuclear and perinuclear localization in cardiac cells. ([***Figure 8D***](#pone-0046316-g008){ref-type="fig"}) The distribution of the perinuclear immunostaining in cardiac myocytes differed from the distribution in fibroblasts. Interestingly, in myocytes, connexin-43, which is trafficked from the Golgi apparatus to cell junctions by anterograde vesicular transport, exhibits a similar compact perinuclear immunostaining pattern, attributed to its localization in the Golgi. [@pone.0046316-Smyth1] The similarity of staining patterns suggests colocalization of perinuclear Chd8 with the Golgi apparatus in myocytes. Our immunostaining of RII in myocytes did not show detectable RII in the nuclei of myocytes or cardiac fibroblasts.([***Figure 8E***](#pone-0046316-g008){ref-type="fig"}) As analysis of mouse heart protein has shown expression of all four isoforms of R subunit [@pone.0046316-Scholten1], it is possible that nuclear localization of R varies between cell types, or that rat heart expresses PKA isoforms in a different pattern than in murine cardiac tissue. Alternatively, the nuclear microdomain of PKA may be more easily detectable in other cell lines. HeLa cells, among other cell types, have been shown to contain nuclear PKA holoenzyme. [@pone.0046316-Sample1], [@pone.0046316-Zippin1], [@pone.0046316-Trinczek1] Thus, Chd8 may act as an AKAP in the nucleus and in the perinuclear domain of HeLa cells, whereas in cardiac myocytes, PKA anchoring by Chd8 may be restricted to the perinuclear domain.
The mechanism by which Chd8 localizes to the perinuclear region remains to be determined. The phosphorylation of residues within an NLS is one mechanism to regulate nuclear localization of a protein. [@pone.0046316-Zhang2] Analysis of the Chd8 NLS with PKAps, a program designed to predict PKA phosphorylation sites, identifies several potential PKA targets in the Chd8 NLS. [@pone.0046316-Neuberger1] ([Table S1](#pone.0046316.s005){ref-type="supplementary-material"}) Given the close proximity of anchored PKA to the NLS, one function of anchored PKA may be the phosphorylation of Chd8 itself.
Chd8 is Expressed at High Levels in Post-natal Heart {#s4c}
----------------------------------------------------
We demonstrated that Chd8 is expressed during embryonic and post-natal cardiac development and also in myocytes and fibroblasts from post-natal rat hearts. ([***Figure 8***](#pone-0046316-g008){ref-type="fig"}) Previous reports described peak levels of Chd8 mRNA in whole mouse embryos, with a decline of Chd8 mRNA in newborn mice. [@pone.0046316-Sakamoto1] In contrast, our results indicate that, in rat heart, a high level of Chd8 mRNA is detected for at least a week after birth. To date, Chd8, a regulator of cell cycle genes and apoptosis, has been studied in cancer cell lines and in vascular smooth muscle cells, capable of division in culture. In contrast, cardiac myocytes grow in three phases: a fetal period characterized by proliferative hyperplasia, a perinatal phase between birth and weaning that is characterized by hypertrophic growth and binucleation, and a third phase that spans weaning through adulthood, where myocytes grow primarily by hypertrophy. [@pone.0046316-vandenHoff1] The immediate postnatal period is a time of intense cardiac remodeling. [@pone.0046316-vandenHoff1], [@pone.0046316-Smolich1] A study of sheep heart reported that right ventricle mass is greater than the left *in utero*, This imbalance was reversed in the weeks following birth as left ventricular cardiac myocytes grew larger. [@pone.0046316-Smolich1] A large scale analysis of rat cardiac DNA, RNA, and protein in the three stages of development also showed an oscillation of ventricular DNA in the perinatal period, with the highest recorded time point at PN7. [@pone.0046316-vandenHoff1] Given the role of Chd8 in regulating genes that correspond with cell growth and survival, the high levels of Chd8 mRNA expression observed at PN7 raise the possibility that elevated Chd8 protein plays a role in these transcriptional events.
Conclusions {#s5}
===========
In summary, we demonstrated that Chd8 contains an amino terminal RIIα binding domain, between residues 455 and 473, and that this domain is required for RIIα binding to Chd8. Immunofluorescence indicates a non-nuclear pool of Chd8 that appears to colocalize with RII and in proximity to markers against the Golgi apparatus. Nuclear and perinuclear microdomains of Chd8 were also identified in HeLa cells and in isolated rat NCMs. Moreover, dephosphorylation of RIIα at Ser96 eliminates binding of RIIα to Chd8-S, whereas RIIα subunits psuedophosphorylated at Ser96 bind Chd8-S. These results indicate that Chd8 is a novel AKAP and demonstrate roles for Chd8 beyond its regulation of development, transcription, and cell survival.
Supporting Information {#s6}
======================
######
**Negative control immunostaining with secondary antibodies in CHO and HeLa cells.** A) CHO cells were incubated with Alexafluor Goat anti-Mouse 568 and imaged in conjunction with Chd8-S/Chd8-S-P transfections in [Figure 4](#pone-0046316-g004){ref-type="fig"}. Cells were imaged with inverted fluorescent microscopy at a magnification of 60X. Scale bar represents 38 µm. B) HeLa cells were incubated with Alexafluor Goat anti-Rabbit 488 and Alexafluor Goat anti-Mouse 568 and imaged with inverted fluorescent microscopy at a magnification of 90X. Pane label indicates 488 or 568 channels. Scale bars represent 25 µm. C) HeLa cells were incubated with Alexafluor Donkey anti-Goat 568 and Alexafluor Donkey anti-Mouse 488 and imaged with inverted fluorescent microscopy at a magnification of 90X. Scale bars represent 25 µm. Pane label indicates 488 or 568 channels. D) HeLa cells were incubated with Alexafluor Donkey anti-Rabbit 568 and Alexafluor Donkey anti-Goat 488 and imaged with inverted fluorescent microscopy at a magnification of 90X. Scale bars represent 25 µm. Pane label indicates 488 or 568 channels.
(TIF)
######
Click here for additional data file.
######
**Specificity of Chd8 and RIIα/β antibodies in immunofluorescence.** A) *Upper panels:* Unblocked immunofluorescence of Chd8. *Lower panels:* Immunofluorescence of endogenous Chd8 in HeLa cells with antibody preincubated for 1 hour with a three-fold excess of the peptide encompassing the antibody epitopes. Insets show immunofluorescence with secondary antibody (Alexafluor Donkey anti-Rabbit 568) alone. B) *Upper panels:* Unblocked immunofluorescence of RIIα/β. *Lower panels:* Immunofluorescence of endogenous RIIα/β in HeLa cells with antibody preincubated for 1 hour with a three-fold excess of purified recombinant RIIα. Insets show immunofluorescence with secondary antibody (Alexafluor Donkey anti-Goat 488) alone. All cells were imaged with inverted fluorescent microscopy at a magnification of 90X. Scale bars represent 25 µm.
(TIF)
######
Click here for additional data file.
######
**Negative control immunostaining with secondary antibodies in NCMs.** A) Isolated rat cardiac cells were incubated with Alexafluor Goat anti-Mouse 568 and Alexafluor Goat anti-Rabbit 488 and imaged with inverted fluorescent microscopy at a magnification of 90X. B) Isolated rat cardiac cells were incubated with Alexafluor Donkey anti-Goat 568 and Alexafluor Donkey anti-Mouse 488 and imaged with inverted fluorescent microscopy at a magnification of 90X. Scale bars represent 25 µm.
(TIF)
######
Click here for additional data file.
######
**Immunofluorescence of HeLa cells and NCM with an alternate Chd8 antibody.** A) *Top Row*: HeLa cells were incubated with Chd8-Sigma antibody and Alexafluor Goat anti-Rabbit 488, and imaged with inverted fluorescent microscopy. *Bottom Row*: HeLa cells were incubated with secondary antibody alone. Scale bars represent 25 µm. B) NCMs were fixed at four days in culture and stained for α-actinin (red) and Chd8-Sigma (green), detected with Alexafluor Goat anti-mouse 568 and Alexafluor Goat anti-rabbit 488, respectively.Short arrows indicate myocytes, while long arrows indicate fibroblasts. Cells were imaged with inverted fluorescence microscopy. Scale bar represents 25 µm.
(TIF)
######
Click here for additional data file.
######
**Prediction of phosphorylation sites on the amino terminus of Chd8.** The prediction program PKAps was used to generate predictions of PKA phosphorylation targets in the first 800 amino acids of Chd8 [@pone.0046316-Neuberger1]. Phosphorylation sites that occur within a known domain of Chd8 are marked. Phosphorylation sites that fall within regions. The phosphorylated residue is bolded and underlined.
(DOC)
######
Click here for additional data file.
We would like to thank Dr. Akira Kikuchi (Hiroshima University, Japan) for the gift of the rat Chd8-S plasmid, and Yinghua Zhang (University of Maryland, Baltimore) for the cardiac embryonic timecourse tissue.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: MOS LL SM MR JM MB. Performed the experiments: MOS LL SM MR JM. Analyzed the data: MOS JM. Contributed reagents/materials/analysis tools: MOS SM MR JM MB. Wrote the paper: MOS LL MB. Editing: MOS LL MB.
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