on denominator of -17/1458 and 113/325116?
26334396
Calculate the smallest common multiple of 16701013 and 7511.
617937481
Find the common denominator of -131/120 and 44/2599209.
103968360
Calculate the common denominator of -151/2335236 and 35/18.
7005708
Calculate the lowest common multiple of 810920 and 106700.
4054600
Calculate the lowest common multiple of 25194 and 66396.
278796804
Calculate the lowest common multiple of 33466872 and 10.
167334360
Find the common denominator of -53/28326240 and -31/540.
28326240
Calculate the common denominator of 43/15145 and 39/30410.
92111890
Calculate the lowest common multiple of 8 and 5651818.
22607272
What is the lowest common multiple of 44 and 3166064?
3166064
What is the common denominator of 29/4447630 and 49/1819485?
40028670
Calculate the common denominator of 197/826500 and -7/377000.
21489000
What is the common denominator of -193/1267320 and 41/85920?
5069280
Calculate the common denominator of 37/13962984 and -119/1308.
1521965256
Calculate the smallest common multiple of 90 and 113589940.
1022309460
Calculate the common denominator of -25/13472 and 47/255968.
255968
Find the common denominator of 17/500136 and 1/312.
500136
Calculate the least common multiple of 22624 and 858601.
192326624
What is the least common multiple of 1535 and 171175?
52550725
What is the common denominator of -27/67463600 and -53/101195400?
202390800
What is the lowest common multiple of 654604 and 44?
7200644
Calculate the common denominator of -43/57258 and 97/25823358.
25823358
What is the lowest common multiple of 27565350 and 150?
27565350
Find the common denominator of -167/1750770 and 31/39267270.
274870890
What is the common denominator of 95/28261532 and -62/91?
197830724
What is the least common multiple of 824920 and 738?
7424280
Find the common denominator of -77/7360 and 23/666464.
153286720
Calculate the smallest common multiple of 153988500 and 27717930.
1385896500
What is the lowest common multiple of 10302541 and 76?
41210164
What is the least common multiple of 6016 and 24246360?
387941760
Calculate the least common multiple of 693014 and 833966.
49203994
Calculate the smallest common multiple of 2908364 and 7270910.
14541820
What is the common denominator of 60/1199341 and 7/3146?
26385502
What is the lowest common multiple of 199477215 and 3?
199477215
Find the common denominator of 39/3162400 and -17/2017800.
540770400
What is the lowest common multiple of 1780174 and 566419?
12461218
What is the least common multiple of 1397840 and 19030?
15376240
What is the smallest common multiple of 15587349 and 1005?
77936745
Calculate the common denominator of -41/8333118 and 1/48.
66664944
Calculate the smallest common multiple of 1004986 and 474050.
25124650
Calculate the lowest common multiple of 142702197 and 102003.
142702197
What is the common denominator of 59/140318832 and 43/2776092?
1543507152
Find the common denominator of -75/4869728 and 115/186.
14609184
Find the common denominator of -151/1256088 and 139/4044.
423301656
What is the lowest common multiple of 715 and 6741020?
6741020
Calculate the lowest common multiple of 4641819 and 102018.
9283638
Find the common denominator of -19/10735974 and -55/329072.
85887792
Calculate the lowest common multiple of 439842 and 273.
3078894
Find the common denominator of 117/14 and -1/12824256.
89769792
Find the common denominator of -35/152896 and 55/84895504.
339582016
Calculate the smallest common multiple of 1417839 and 145.
7089195
Calculate the least common multiple of 19446 and 936186.
6553302
Find the common denominator of -57/209776 and 89/5379256.
75309584
Find the common denominator of -101/6 and -13/32085582.
32085582
Calculate the common denominator of 40/6835549 and -17/735.
102533235
What is the lowest common multiple of 1431540 and 3354720?
332117280
Find the common denominator of -109/28803480 and -83/26403190.
316838280
What is the common denominator of 22/45293845 and -51/45293845?
45293845
Calculate the common denominator of -199/17796240 and 23/46224.
17796240
What is the least common multiple of 84448 and 1282554?
20520864
What is the least common multiple of 14580 and 4925340?
132984180
What is the least common multiple of 356 and 35656?
3173384
What is the least common multiple of 6360820 and 25?
31804100
Find the common denominator of -50/22782069 and 11/12.
91128276
Calculate the smallest common multiple of 1832692 and 112252385.
449009540
Find the common denominator of 13/3 and 83/103463540.
310390620
Calculate the common denominator of 47/259404880 and -79/4.
259404880
What is the common denominator of -51/1561 and -23/25837895?
180865265
What is the smallest common multiple of 3850 and 272195?
2721950
Calculate the least common multiple of 1044963 and 135.
15674445
Calculate the lowest common multiple of 233728 and 189904.
3038464
What is the smallest common multiple of 763236 and 151848?
29002968
What is the common denominator of -9/3949972 and 111/40?
39499720
What is the lowest common multiple of 240 and 8585616?
42928080
Calculate the least common multiple of 40 and 1553360.
1553360
Find the common denominator of -31/139623984 and -95/18.
139623984
Find the common denominator of -17/20511000 and 61/18576.
123066000
What is the smallest common multiple of 1372911 and 41187330?
41187330
Find the common denominator of 181/17700 and 7/13600.
2407200
What is the smallest common multiple of 52872954 and 9?
158618862
Calculate the common denominator of -34/7533 and 131/47503098.
47503098
Find the common denominator of -83/4 and -37/654724.
654724
What is the lowest common multiple of 78995 and 677100?
4739700
Calculate the smallest common multiple of 11880 and 33156.
3647160
Calculate the least common multiple of 113124 and 1187802.
2375604
What is the smallest common multiple of 3472 and 4239312?
4239312
What is the smallest common multiple of 271286730 and 226072275?
1356433650
Calculate the smallest common multiple of 132 and 289140.
3180540
Find the common denominator of -47/17933248 and -97/80699616.
161399232
Calculate the common denominator of 38/247321 and -85/18152.
1978568
Find the common denominator of -109/3045 and 29/15189.
15416835
Calculate the common denominator of -123/2454920 and -139/16450.
4038343400
What is the least common multiple of 12 and 49692850?
298157100
Calculate the common denominator of -139/108 and -1/31640958.
189845748
Find the common denominator of -59/9 and 27/41022605.
369203445
What is the lowest common multiple of 763 and 28224?
3076416
What is the common denominator of 37/121402 and 47/61302?
6191502
What is the lowest common multiple of 5337294 and 16?
42698352
Calculate the common denominator of -37/491391 and -22/45.
2456955
What is the lowest common multiple of 759240 and 384408?
213346440
What is the smallest common multiple of 980 and 8292760?
8292760
Find the common denominator of -109/166416 and 83/19552.
203360352
Calculate the lowest common multiple of 15738 and 5083008.
218569344
What is the smallest common multiple of 7112772 and 61317?
7112772
Find the common denominator of 51/4 and 3/98108060.
98108060
Calculate the least common multiple of 1780251 and 3398661.
37385271
What is the lowest common multiple of 22 and 1031875838?
11350634218
What is the common denominator of -65/1230278 and -47/56417034?
394919238
Calculate the smallest common multiple of 34903158 and 28719.
104709474
What is the least common multiple of 50328 and 8343?
15551352
What is the common denominator of -45/8852 and -53/6128?
13561264
What is the common denominator of 145/66 and 181/16929066?
16929066
Calculate the common denominator of 8/9663355 and -1/23786720.
309227360
What is the common denominator of 56/15717843 and 121/616386?
31435686
What is the common denominator of 47/6011026 and 25/65262568?
456837976
Calculate the common denominator of 61/3876084 and 11/4824.
7752168
What is the lowest common multiple of 149332 and 12786048?
473083776
What is the lowest common multiple of 292048 and 152?
5548912
What is the common denominator of -53/737667 and 169/270?
7376670
Find the common denominator of 17/73430 and -31/29606976.
148034880
Calculate the common denominator of 77/8891450 and -2


Q:

How to sqrt a number and if its a decimal, give a different output

I'm trying to write some python code where it performs a sqrt on a give number.  Realizing that some situations might simply spit out a decimal (which I don't want).  How do I check or what code should I add to determine whether the result is a whole number or a decimal in the code before it decides what to do with it?
This question is different as it is not a list and that question was how to do so in a list, not how to ensure the accuracy of the square root function.

A:

Why don't you check if int(num) == num:. That should work.
from decimal import *
getcontext().prec = 20 #It represents the precision. Increase it if you need.
num = Decimal(num).sqrt()
if int(num) == num:
    print 'whole number'
else:
    pass


, -5/3, 2/7?
-5/3
Let x = -1926 - -1929. Which is the nearest to x?  (a) -0.04  (b) -1  (c) 0  (d) -0.1
c
Let p = 1787/6 + -297. Let y = -51.7 - -61.7. Which is the closest to 1?  (a) p  (b) -4  (c) y
a
Let r = 24.1 + -28.1. Let h(f) = 3*f. Let p be h(1). Let w = -2 + 1.7. Which is the closest to 3?  (a) p  (b) w  (c) r
a
Let l be 0 + 1 + 20/15*-3. Which is the closest to -1?  (a) 0  (b) -1/4  (c) l
b
Let k = -8.61 - -0.81. Let m = 12.8 + k. Which is the closest to -12?  (a) m  (b) -1/4  (c) -5
c
Let u(c) = 73*c**2 - c - 7. Let s be u(0). Let a = 136/5 + -27. What is the closest to a in -1/2, 0, s?
0
Let j be 3*(2 + 80/(-36)). Let u = j + 16/15. Let x = 0 - -0.1. Which is the closest to u?  (a) 3/7  (b) -4  (c) x
a
Let v = 0 + 0.2. Let j = -0.8 - v. Let o = 15.9 - 13.9. What is the closest to -1/3 in o, j, -2?
j
Let y = -9.36 - -9. Let o = -0.46 - y. What is the closest to -4 in -0.2, o, -4?
-4
Let u = -24 + 24. Suppose u = 3*a - 2*a. What is the nearest to -0.02 in 2/11, a, 3?
a
Let r = -1.5 - 0.5. Let z be (-14)/49 - 3/231*-8. What is the nearest to 1 in r, -4/9, z?
z
Let y = -0.13 + 1.13. Suppose -11*q + p = -12*q, -15 = -5*p. Which is the nearest to y?  (a) 2  (b) 6  (c) q
a
Let t = -3.04 - -0.04. Let i = 8552/5 - 1710. Which is the nearest to i?  (a) t  (b) 2/3  (c) -5
b
Let j = 6.6 + -7. Let h = -50 + 49. What is the closest to h in 1/6, j, -2?
j
Let f = 0.1 - 0. Let j = -5.06821 + 0.06821. Which is the nearest to f?  (a) j  (b) 5  (c) -0.3
c
Let h = -11 - -8. Let w(z) = -z**3 - 10*z**2 - 2*z - 21. Let n be w(-10). Which is the closest to 1?  (a) h  (b) 2/7  (c) n
b
Let z = -335 - -335. Which is the closest to -1?  (a) -0.1  (b) z  (c) -2/11
c
Let g = -0.3 - -4.3. Let f be (6 - 4/2)/(34 - 8). Which is the closest to f?  (a) 0.3  (b) g  (c) 2
a
Let k = -1698.9 - -1797. Let h = k + -110. Let j = h + 12. Which is the nearest to -1?  (a) j  (b) 4  (c) -0.05
c
Let v = 16 - 15.6. Let c(x) = -3*x + 31. Let n be c(11). What is the nearest to -2/5 in n, v, -4/5?
-4/5
Suppose -3*s = 0, -4*s - 17 = -3*t + 10. Let g = -12 - t. Let w be (-2)/6 - g/27. Which is the closest to 1?  (a) w  (b) 2  (c) -2
a
Let j = -136 - -131. Which is the closest to 3?  (a) 1  (b) j  (c) 0.1
a
Let x = -143 - -205. Let i = -62 + x. Let h = 3 - 2.8. What is the nearest to -1/2 in h, -5, i?
i
Let i = -8/221 + -189/884. What is the closest to 0 in 3, 149, i, -5?
i
Let h = -2.522 + 2.022. Which is the closest to 2?  (a) h  (b) 1/5  (c) 0.55
c
Let s = -2009/3 - -669. Let x(h) = -h - 1. Let o be x(-1). Which is the closest to o?  (a) 0.5  (b) s  (c) 2/9
c
Let l = -8.869 - -5.886. Let y = 0.017 - l. Which is the closest to -2?  (a) -2/3  (b) y  (c) -3
c
Let s = 0.14 + 0.86. Let u be (1 - 1) + 1/(-2). Let o = 2849 + -2849. Which is the closest to o?  (a) s  (b) u  (c) -5
b
Let c be (-6)/4*(-26)/(-13). What is the closest to 0 in c, -20, -2?
-2
Let c = -6 - -6.07. Let l = -0.37 + c. What is the nearest to 2/5 in 1/7, -5/6, l?
1/7
Let h = 47 - 69. Let j = 27 + h. Let s(z) = -z**3 + 8*z**2 + 8*z + 7. Let w be s(9). Which is the closest to 0?  (a) w  (b) 1  (c) j
b
Let p = -308.1 + 308. Which is the nearest to p?  (a) 1  (b) 2/3  (c) 3/4
b
Let k = 6 + -3. Let d = 74 + -73.5. What is the nearest to -1 in d, k, 1?
d
Let n = -52.1 - -53.09. Which is the closest to 1?  (a) 0.2  (b) 2/11  (c) n
c
Let o = -9 + 6. Let p = -392 + 391.8. Which is the closest to o?  (a) 0.1  (b) p  (c) 2/7
b
Let f be 6/(-2)*2/(-3). Let p be ((2/(-4))/(-1))/(-117 - -116). What is the nearest to 2/9 in -3/5, f, p?
p
Let m = 0.114 - -0.886. Let a = -0.07 - 0.13. What is the nearest to 1/4 in m, a, -5?
a
Let x = -0.2 - -1.2. Let q = 57 - 57.03. Which is the nearest to x?  (a) -5  (b) 0  (c) q
b
Let n = 1364.1 - 1364. Which is the closest to 0.1?  (a) n  (b) 3  (c) -0.2  (d) 0.01
a
Let m = -1.045 - -1.025. Which is the nearest to -1?  (a) m  (b) 0.06  (c) -2/3
c
Suppose 3*l - 5*y = 13 - 3, 2*l - 11 = -y. What is the closest to l in 3, -0.3, -1/6, 2?
3
Let d = 0.088 - -0.382. Let u = -0.47 + d. Which is the closest to u?  (a) -5  (b) -0.5  (c) 0.3
c
Let m = -0.2 - -0.1. Let f = 0.7 + 0.3. Let l = 1907 - 1906. Which is the nearest to l?  (a) 4  (b) m  (c) f
c
Let t = -0.071 - 0.04. Let d = 0.889 - t. Which is the closest to 4?  (a) -0.4  (b) -1  (c) d
c
Let s(j) = j**3 - 12*j**2 + 12*j - 10. Let y be s(11). Suppose 8 = -8*x - 24. Which is the closest to 1/4?  (a) 1/10  (b) y  (c) x
a
Let p = 0.081 + 3.919. Which is the closest to 1?  (a) -7  (b) 0.1  (c) p
b
Let g = -323 - -322. What is the closest to -1 in 8/7, g, -2/13?
g
Let x = -42 - -73.5. Let z = x - 32. Which is the closest to 0?  (a) -2/13  (b) z  (c) -3
a
Let s = 816.06 - 816. Which is the closest to s?  (a) -1/3  (b) -5  (c) -2  (d) 8
a
Let i = -110.27 + -0.73. Let d = i + 111.04. What is the closest to d in 0, -3, 4/5?
0
Let k = -2185/272 - -144/17. Which is the closest to -1?  (a) 4  (b) 3  (c) k
c
Let r = 1/391 + -1571/2737. What is the nearest to 1/8 in r, 0, 1.4?
0
Let w = 248 - 250. What is the nearest to 2/5 in -3, w, 4/5?
4/5
Let s = -4/33 - -5/11. Let w = 1 + -1. Let m = 0.32 - 2.32. What is the nearest to s in -0.5, w, m?
w
Let b = -11959/271138 - -15/362. Let c = b - -65918/2247. Let l = c - 29. What is the closest to -2/5 in 5, l, 2?
l
Let c = 5.5 - 5. What is the nearest to 0.1 in -3, -11, c, 0.4?
0.4
Let o = -10 + 15. Let c = -6 + o. Let j be 3*((-29)/6 + 2 + 3). What is the closest to -2/5 in -2, j, c?
c
Let y = -167.2 + 167. Suppose 5*d = -4*i - 21, -2*d - 7*i - 22 = -2*i. What is the closest to y in 0.8, d, -2?
d
Let t = 0 - -1. Let f = -0.465 + 0.165. Which is the closest to t?  (a) -3  (b) f  (c) 0.5
c
Let k = 0.16 - 0.44. Let n = k + -0.22. What is the closest to 4 in -5, -2, n?
n
Let o = 1/189 - -373/945. What is the closest to 1/7 in o, -0.1, 6/5?
-0.1
Let v = 814 + -5696/7. What is the nearest to -0.1 in -2/41, -0.2, -1, v?
-2/41
Let s = -448.6 + 449. What is the closest to 1/2 in s, -1/4, 150?
s
Let i = 1 + -1.1. Let g = 29.5 + -29.3. Let o = -190 + 190.4. What is the closest to g in i, o, -3?
o
Let m = -1 + 1.1. Let o be 1/(24/70 + (-2)/10). Let d = o + -8. Which is the closest to m?  (a) 0.4  (b) d  (c) -4
a
Let k = -129 + 78. Let r = k + 50. Let f be (-2)/3 - 1/(-3). Which is the closest to r?  (a) 1  (b) f  (c) 3
b
Let o = 12 + -9. Let p be ((-4)/(-18))/((-1 + 4)/(-9)). What is the nearest to 0 in o, p, 0.5?
0.5
Let t be (1/(-4))/(9/12). Let s be -5 + 3 + ((-15)/(-4) - 2). What is the nearest to t in -1, s, -0.5?
s
Let r = -322 + 2579/8. Let f = 0.051 + -2.051. What is the nearest to -0.2 in f, r, -5?
r
Let w = -8 - -6. Let g = 269/28 + -62/7. What is the nearest to w in g, 5, 2/11?
2/11
Let y = -0.1 - -0.2. Let n = 1.2 - 3.8. Let h = n + 3.1. What is the closest to y in 2, 0, h?
0
Let m = 2.11 - 0.11. Let g = 9.15 + -0.15. Let c = -13 + g. What is the nearest to 1 in -1, m, c?
m
Let f = 262 - 261.9. Which is the closest to 1?  (a) -32  (b) f  (c) 2
b
Let u = 7714/3 + -2571. What is the nearest to -0.31 in u, -1, 5?
u
Suppose 5*a - 5 = -2*y, 2*a = a - 1. Suppose -10*i + 35*i + 75 = 0. What is the nearest to -1 in y, 1/7, i?
1/7
Let r = 0.32 - 0.12. Suppose -45*d + 36 + 54 = 0. Which is the nearest to d?  (a) r  (b) 1.7  (c) 2/11
b
Suppose -5*y = -3*c + 136, 3*c + 2*y - 135 - 29 = 0. Let n be 2/c*4*-1. Let u = 16.4 - 16. What is the closest to -0.1 in n, 5, u?
n
Let g = 36/19 - 233/133. Which is the closest to 0?  (a) g  (b) -3  (c) 0.1
c
Let y = 27.7 - 26.7. What is the nearest to y in -3, 2/3, 10?
2/3
Let p = 29 + -30. Let r = -3 - p. What is the nearest to r in 0, -0.05, -0.1?
-0.1
Let c = -23 + 22.2. Let t = 1.7 - c. Let r = t - 1.8. What is the nearest to 1 in -5/2, -0.3, r?
r
Let x = -131.7 - -131.6. Which is the nearest to -1?  (a) -1  (b) x  (c) 2  (d) -6.2
a
Let q be 0 - 3*1/6*2. What is the nearest to q in -0.2, 13, -0.06, 0?
-0.2
Suppose -2 = -5*k - 35*w + 31*w, 0 = 3*k + w + 3. Let z = -5 + 7. What is the closest to k in -3/5, 4, z?
-3/5
Let i = -16 - -36. Let u = i + -22. What is the nearest to u in 5, -0.5, 1/2?
-0.5
Let v = 1080 - 1081. Which is the closest to v?  (a) 9  (b) -3  (c) -1/4
c
Suppose 5*y = -3*x - 5, 41 = -6*y + 2*y + 5*x. Let w = 6 + y. Let d = -14 + 14. What is the nearest to d in -1, w, -0.1?
-0


Q:

How to link avatar and nickname to profile

On the single post page I want to link the avatar function to the author profile, but I tried
<a href="<?php the_author_posts_link(); ?>">
<img src="<?php echo get_avatar($post->post_author, '64', $avatar); ?>">

but that doesn't work.
Also, how can I display the nickname (display name) and still link to the profile the following code gives a strange link and 404s
<a href="<?php echo get_author_posts_url(); ?>"><?php the_author_nickname(); ?>

some help would be greatly appreciated.
Thanks

A:

This a short compilation of the multiple comments above, so that future visitors don't have to read each and every one of them.
First of all, the_author_posts_link() is a deprecated function since version 2.1, so get_author_posts_url() or the_author_posts_url() should be used instead http://codex.wordpress.org/Function_Reference/get_author_posts_url
The the/get_author_posts_url() takes an argument that requires "ID of the author whose URL should be retrieved.", so the_author_posts_url( get_the_author_meta( 'ID' ) ) will work, and if you decide to use the get_author_post_url() instead of the_author_posts_url() don't forget to echo it out.


Structure-activity relationships of dermorphin tetrapeptides carrying a ketomethylene linkage.
The synthesis and in vitro pharmacological tests of 5 ketomethylene tetrapeptide analogues of the opioid heptapeptide dermorphin H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 are described in this study. The substitution of the original Phe-Gly peptide bond (-CO-NH-) by the ketomethylene linkage (-CO-CH2-) provided analogues with reduced opioid activity.


Scottish Secretary Michael Moore made a statement to MPs on the legal status of a future referendum on Scottish independence.

During his address, news came through that the Scottish National Party had named its preferred time period for the poll - Autumn 2014.


Cult leader Dave McKay, founder of the self-proclaimed “Jesus Christians” is apparently back in business using the new name “End Time Survivors” preaching on YouTube.

Dave McKay, now in his seventies, is originally from Rochester, New York. McKay married and moved to Australia in 1968 where he joined the cult “Children of God (COG).” COG became infamous for child sexual abuse. McKay later decided to leave COG and become leader of the so-called “Jesus Christians” in 1982.

Like Moses Berg, the founder of COG, McKay became notorious for the total control that he exercises over his followers. Now on YouTube he is “Brother Dave.” Claiming divine revelation McKay says, “When I tell people that God talks to me I can see the wheels turning, their thinking ‘psycho,’ ‘psycho,’ this guy needs professional help.” Maybe McKay is a psycho. Apparently if you disagree with Dave you disagree with God.

McKay’s group was dubbed the “kidney cult” because reportedly more than half of his followers donated a kidney in what many saw as a ploy for media attention.

Brother Dave seems to be adept at “brainwashing” his disciples according to numerous press reports. And after new recruits join his group they frequently cut off virtually all communication with family and old friends. The group has also garnered controversy for recruiting minor children.

Complaints from estranged families and bad press followed Dave McKay wherever he went and made his group appear toxic. No wonder McKay has chosen to rebrand his group online with a new name. Brother Dave even blurs his face and his followers are masked to obscure their real identity on YouTube.

YouTube is a popular venue for some groups called “cults,” who use it for recruitment and fund raising. McKay now has more than 3,000 YouTube subscribers.

One of the End Time Survivors videos states, “The information on this channel is coming from a lot of people and most of them have been able to hide their identity they do it for the express purpose of getting you to think about what is being said rather than making you depend upon some guru who will do all your thinking for you.”

However, this is exactly the reason that families and others concerned complained about the undue influence of Dave McKay, a religious guru who they said was thinking for their loved ones.

Don’t be fooled by this “kidney cult” or trust in Brother Dave’s supposedly divine revelations.

Instead, recognize the familiar pattern of a con man and cult leader feeding off of his followers to fulfill his ego.

Dave McKay expects his devotees to give up their identity, family, friends, virtually everything and anything upon demand, even at times their vital organs.

Beware of Dave McKay and his band of masked “End Time Survivors.”


[Update] English Trophies have surfaced online now. You can check them out here.

[Original Story] Dark Souls 3, the new entry of the popular action role playing game series developed by From Software, is going to be released in just a few days in Japan on PlayStation 4 and Xbox One, and new details on the game have started coming in today. A few hours ago, the game’s full Trophy List has emerged online, revealing a few new details on the highly anticipated role playing game.

The Dark Souls 3 Trophy List includes 43 Trophies – 26 Bronze Trophies, 13 Silver Trophies, 3 Gold Trophies and the Platinum one. The list, which is only available in Russian as of now, includes some very heavy spoilers on bosses and Covenants, so avoid looking at it if you want the game to be a complete surprise for you when it launches next month.

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Last week, we were able to see more of the PC version of Dark Souls 3 thanks to a new IGN livestream, which has revealed the game’s graphics and keybinding options. The game really looks great when running at High settings, so those with powerful enough machines will be able to enjoy the game with much better graphics. The PC version of Dark Souls 3 will also run at 60 FPS, and not 30 as it was being rumored a couple of weeks back.

Last week, new Dark Souls 3 gameplay footage has surfaced online from SXSW Gaming 2016. The new footage, which is commented by a Bandai Namco rep, revealed some new details on luck scaling weapons and spells, lore, and other gameplay mechanics.


PathEngine's tiled 3D content process is based on the application of a horizontal tiling to the existing 3D content processing functionality. The set of source data elements in each tile is processed independantly, with the resulting ground pieces then combined back together for run-time pathfinding.

This ensures that things like process complexity and peak memory load are completely decoupled from overall world complexity, and that total processing times scale essentially linearly with world size.

But the biggest advantage is the possibility for incremental world updates, with processing only required for tiles local to changed content, and with the possibility also for per tile processing to be distributed across threads (to take advantage of multicore CPUs) or even across multiple machines, for the fastest possible update times.

Tiled result pieces can be combined either into a single ground mesh, or into a set of federated overlapping tiles (for use with the SDK's existing 'mesh federation functionality), which then has the effect of also decoupling things like pathfinding preprocess generation and run-time query times from total world size.

Demo applications are included in the PathEngine evaluation SDK, with source code, for tiled 3D content processing with cached per tile results, and also for the construction of federated meshes from tiled ground pieces.

Please see the following page (in the online PathEngine documentation) for more details:

PathEngine was founded in 2000 based on experience providing pathfinding and AI to the games industry for more than 10 years.

The PathEngine SDK is built around an advanced implementation of points-of-visibility pathfinding on three-dimensional ground meshes. The approach enables PathEngine to provide both pathfinding and collision in tight integration against a single, sophisticated, agent movement model that takes agent shape into account and seamlessly supports overlapping geometry, with dynamic obstacles directly integrated into this movement model.

PathEngine features in the genre-breaking MMORPG 'Granado Espada' (by IMC Games) (released in the west as 'Sword of the New World'), 'Mobile Suit Gundam: Operation Troy' (by Dimps Corporation), 'Titan Quest' (by Ironlore Entertainment), 'Pirates of the Burning Sea' (by Flying Lab Games) and many other triple-A titles.

For a full list of developers and titles already using the PathEngine SDK please see:

Games Press is the leading online resource for games journalists. Used daily by magazines, newspapers, TV, radio, online media and retailers worldwide, it offers a vast, constantly updated archive of press releases and assets, and is the simplest and most cost-effective way for PR professionals to reach the widest possible audience. Registration for the site and the Games Press email digest is available, to the trade only, at www.gamespress.com


Q:

Qual a diferença entre usar ou não um "constraint" no "foreign key"?

Ao criar uma tabela no MySQL usei na criação de campos CONSTRAINT FOREIGN KEY como está a seguir para criar a chave estrangeira:
CREATE TABLE socio
(
  id_socio      INTEGER NOT NULL,
  nome          VARCHAR(256) NOT NULL,
  cpf           VARCHAR(11) NOT NULL,
  email         VARCHAR(256),
  id_situacao   INTEGER,
  CONSTRAINT pk_id_socio PRIMARY KEY (id_socio),
  CONSTRAINT un_socio_cpf UNIQUE (cpf),
  CONSTRAINT socio_id_situacao_fk_ref FOREIGN KEY (id_situacao) REFERENCES situacao (id_situacao)
);

Qual a diferença de na criação de tabelas, usar apenas o FOREIGN KEY ou o FOREIGN KEY com CONSTRAINT para criar chaves estrangeiras?

A:

A cláusula CONSTRAINT é usada para dar nome a este restrição de integridade. Só isto, dar um nome da sua escolha.
Mesmo sem ela ainda será uma restrição imposta à tabela, porém o nome será o mesmo do índice associado em FOREIGN KEY, e no caso de não ter um índice indicado ele cria um nome baseado no nome da coluna. Neste caso como não tem esse índice sem a CONSTRAINT o nome seria gerado a partir da coluna. No seu exemplo o nome dessas restrições serão pk_id_socio, un_socio_cpf, socio_id_situacao_fk_ref.
Documentação.


708 A.2d 861 (1998)
TOWNSHIP OF PENN, York County, Pennsylvania, Appellant,
v.
Bradley V. SEYMOUR.
Commonwealth Court of Pennsylvania.
Argued December 9, 1997.
Decided February 24, 1998.
*862 Walter A. Tilley, III, York, for appellant.
Judith Koper Morris, Hanover, for appellee.
Before DOYLE and LEADBETTER, JJ., and RODGERS, Senior Judge.
DOYLE, Judge.
The Township of Penn appeals an order of the Court of Common Pleas of York County, which vacated an order of a District Justice entering judgment against Bradley Seymour for violating the Township's zoning ordinance.
Seymour owns four Rottweiler dogs which he houses in a fenced area located in the backyard of his residence. He breeds two of the dogs once each year and sells the puppies when they are eight weeks old. Seymour's residence is located in the Township's R-15 Suburban Residential zoning district. Single family homes and group homes are permitted by right in the R-15 zone; various other uses, primarily personal service, professional, and public service uses, are permitted by special exception. Section 203.2 of the Penn Township Zoning Ordinance (Ordinance).
The Township received complaints that Seymour had an excessive number of dogs on his property and was breeding dogs. On May 21, 1996, the Township's zoning officer issued a notice of violation to Seymour for operating a kennel in a residential zone. The Township's zoning ordinance defines the term "kennel" in part as a building or land used for the boarding or breeding of four or more dogs. Section 103.3 of the Ordinance. It is undisputed that kennels are not a permitted use in the R-15 zoning district.
On July 11, 1996, Seymour filed an application with the Township's Zoning Hearing Board (ZHB) for a variance to allow him to maintain a kennel on his property. The ZHB denied the application on August 14, 1996. Seymour, however, continued to operate the kennel, and on November 21, 1996, the zoning officer issued a second notice of violation for operating a kennel in a residential zone. The notice included, inter alia, the following language:
FAILURE TO COMPLY WITH THIS NOTICE WITHIN THE TIME SPECIFIED CONSTITUTES A VIOLATION. EACH DAY THAT A VIOLATION CONTINUES WILL CONSTITUTE A SEPARATE VIOLATION.... YOU HAVE THE RIGHT TO APPEAL TO THE ZONING HEARING BOARD. SAID APPLICATION FOR APPEAL SHALL BE FILED WITH THE TOWNSHIP ZONING OFFICER BY THE DATE CORRECTIVE ACTION WAS TO HAVE BEEN COMPLETED. (Emphasis added.)
Seymour did not correct the violation, nor did he appeal the notice of violation to the ZHB.
On December 27, 1996, the Township filed a zoning enforcement complaint against Seymour with the District Justice. After a hearing, the District Justice entered a judgment against Seymour in the amount of $750.00 plus costs. The District Justice further ordered that, if the zoning violation continued after March 2, 1997, judgment in the amount of $250.00 per day would be imposed.
Seymour appealed the District Justice's order to the Common Pleas Court, and, in accord with Pa. R.C.P.D.J. No. 1004(B),[1] a rule was entered directing the Township to file a complaint. The Township filed a complaint seeking both a penalty and fine as well as an injunction directing Seymour to cease and desist from operating a kennel. In response, Seymour filed preliminary objections challenging the constitutionality of the Ordinance, *863 the validity of the enforcement action, and asserting that the Ordinance was preempted by state statute. The Township filed preliminary objections to Seymour's preliminary objections, in which it argued that the Common Pleas Court did not have subject matter jurisdiction over the issues raised by Seymour because he never appealed the November 21, 1996 violation notice to the ZHB.
Common Pleas determined that the violation notice issued by the zoning officer did not fully inform Seymour of his right to appeal to the ZHB and, for that reason, vacated the District Justice's order. The court noted that, when zoning enforcement actions are litigated, the zoning hearing board determines whether a zoning violation has occurred, and the penalty is decided and imposed by a district justice. Johnston v. Upper Macungie Township, 162 Pa.Cmwlth. 170, 638 A.2d 408 (1994). The Court explained:
[I]t is the Court's opinion that this [violation] notice is not sufficient as the law has been modified by Johnston..... A notice merely stating that a defendant has the right to file an appeal to the Zoning Hearing Board does not begin to inform a defendant that the failure to do so results in a binding determination that he has committed a violation. In fact, the use of the term "appeal" itself is misleading. It would be more appropriate to inform a defendant that he is entitled to request a hearing as to whether the defendant is in violation. More important[ ], the notice should state that the failure to request a hearing creates a binding determination that the defendant is in violation. Since the revised procedure established by Johnston... is little known to lawyers and municipal officials, let alone the general public, most defendants would be deprived of their day in court unless this hybrid procedure ... is adequately explained to them.
....
Since the Court has determined that the Township's notice was defective, the Court hereby finds that the District Justice had no jurisdiction to hear this enforcement action. Even though the defendant did not raise that objection, jurisdiction is a matter that can be addressed by the Court on its own motion. Therefore, the Court finds that the District Justice had no jurisdiction to hear the Complaint because of the lack of a proper notice, and therefore the proceedings of the District Justice should be vacated.
(Common Pleas Court Opinion at 4-6.)
Further, the Common Pleas Court determined that the procedure for filing a complaint under Pa. R.C.P.D.J. No. 1004(B) was not applicable in this matter. The Court concluded that, because the issues in zoning enforcement appeals are limited to whether the violation has been discontinued and the amount of the civil penalty, there is no need for pleadings, and the matter may be treated in a manner similar to a summary criminal appeal. This appeal by the Township followed.
The Township contends that (1) the notice of violation was legally and factually sufficient to inform Seymour of his appeal rights, (2) Seymour's failure to appeal to the ZHB waived his challenges to the constitutionality, validity, and enforceability of the Ordinance, and (3) the Common Pleas Court erred in concluding that Pa. R.C.P.D.J. No. 1004(B) was not applicable to Seymour's appeal from the District Justice. We agree with each of the Township's contentions.
Formerly, municipalities enforced zoning ordinances by instituting a summary criminal action before a district justice. Plains Township v. Krasner, 7 Pa.Cmwlth. 56, 298 A.2d 627 (1972); Commonwealth v. Joki, 330 Pa. Superior Ct. 406, 479 A.2d 616 (1984). The district justice's decision could be appealed to a common pleas court, which heard the case de novo. Township of Vanport v. Brobeck, 22 Pa.Cmwlth. 523, 349 A.2d 523 (1975). In 1988, however, the General Assembly changed this procedure. Zoning enforcement proceedings are now commenced by a municipality sending an enforcement notice to the landowner in accordance with Section 616.1 of the Pennsylvania Municipalities *864 Planning Code (MPC).[2] When a landowner has been given notice of a zoning violation under Section 616.1 of the MPC:
[the] landowner can contest the asserted violations only by way of appeal to the municipality's zoning hearing board and cannot merely defend the charge when the municipality seeks ordinance violation fines before a district justice. Johnston v. Upper Macungie Township, [162 Pa.Cmwlth. 170] 638 A.2d 408 (Pa.Cmwlth.1994). In Johnston, we explained that zoning hearing boards have exclusive jurisdiction over ordinance violation determinations, and, therefore, a landowner's failure to appeal a Section 616.1 zoning violation notice to the zoning hearing board is fatal and results in a conclusive determination of guilt for which a district justice may impose sanctions under Section 617.2 of the MPC. For this reason, we held that a district justice may not conduct a de novo review of the merits of a violation notice where the landowner has not gone first to the zoning hearing board; rather in that situation, upon the municipality's showing that no appeal was taken by the landowner, a district justice is limited to imposing a fine pursuant to Section 617.2 of the MPC [, 53 P.S. § 10617.2].
City of Erie v. Freitus, 681 A.2d 840, 842 (Pa.Cmwlth.1996), petition for allowance of appeal denied, 547 Pa. 738, 690 A.2d 238 (1997) (emphasis in original).
The Township first contends that the Common Pleas Court erred in holding that the notice of violation was insufficient as a matter of law to advise Seymour of his rights. Specifically, the Township argues that, contrary to the MPC, the Court erroneously concluded that the notice of violation had to explain the "hybrid" procedure outlined in Freitus and Johnston and that the failure to appeal the notice to the ZHB, would result in a binding determination that the Ordinance was violated. This argument is well taken.
Section 616.1(5) of the MPC requires a municipality to include in a violation notice a statement that "the recipient of the notice has the right to appeal to the zoning hearing board within a prescribed period of time in accordance with procedures set forth in the ordinance." Nothing in Section 616.1 requires a municipality to explain to a landowner the consequences of failing to file such an appeal or to outline the procedures articulated in Johnston and Freitus.
The Township's November 21, 1996 notice of violation was in compliance with Section 616.1 of the MPC. The notice informed Seymour that he had a right to appeal to the ZHB, and no further explanation of Seymour's appeal rights was required by the MPC. Although the information mandated by the Common Pleas Court might be valuable to a defendant/landowner, a court may not graft additional provisions onto a statute which the General Assembly did not see fit to include. Commonwealth v. Rieck Investment Corp., 419 Pa. 52, 213 A.2d 277 (1965). Therefore, Common Pleas erred in holding that the notice of violation was insufficient.
Next, the Township contends that Seymour waived his right to raise issues regarding the constitutionality and enforceability of the Ordinance because he failed to appeal the notice of violation to the ZHB. Again, we agree.
If a landowner does not appeal a zoning violation notice to the zoning hearing board, the failure to appeal renders the violation notice unassailable. Freitus. Therefore, in the event a landowner does not appeal to the zoning hearing board and the municipality files an enforcement action with a district justice, neither the district justice nor a common pleas court may conduct a de *865 novo review of the question of whether the landowner violated the zoning ordinance. Id. In that circumstance, the only question before the district justice and the Common Pleas Court is whether the penalty imposed for the violation was proper. Id.
Applying these principles to the instant case, we observe that Seymour filed preliminary objections to the Township's complaint challenging the constitutionality, validity and enforceability of the Ordinance. However, Seymour's failure to appeal the notice of violation to the ZHB precluded any inquiry into those issues, and, accordingly, those issues were waived.
Finally, the Township argues that the Common Pleas Court erred in concluding that the Township should not have followed the appeal procedure under Pa. R.C.P.D.J. No. 1004(B). The court concluded that the procedure under Pa. R.C.P.D.J. No. 1004(B), which requires the appellee to file a complaint where the appellant was the defendant in the district justice action, was inapplicable in zoning enforcement cases. Specifically, the court stated that no pleadings were necessary and that it had adopted procedures for hearing zoning violation appeals that mirror those used in summary criminal proceedings. We disagree with Common Pleas' analysis.
The General Assembly, in the Act of December 21, 1988, P.L. 1329, repealed Section 616 of the MPC, 53 P.S. § 10616, which provided that zoning ordinances were to be enforced through summary criminal proceedings. Under Section 616, a violator could be imprisoned for a maximum of 60 days for failing to pay a fine.
By repealing Section 616, the General Assembly decriminalized zoning enforcement actions. Now, Section 617.2 of the MPC, 53 P.S. § 10617.2, provides for the following enforcement remedies:
Any person ... who ... has violated ... the provisions of any zoning ordinance ... shall, upon being found liable therefor in a civil enforcement proceeding commenced by a municipality, pay a judgment of not more than $500 plus all court costs.... If the defendant neither pays nor timely appeals the judgment, the municipality may enforce the judgment pursuant to the applicable rules of civil procedure. ... (Emphasis added.)
Imprisonment is not an available remedy under Section 617.2 of the MPC. In addition, our review of the Ordinance reveals that imprisonment is not a possibility for the violation of its provisions, and that the penalties imposed under the Ordinance are substantially the same as Section 617.2 of the MPC.
The procedure favored by the Common Pleas Court is similar to the summary appeal standards in the Rules of Criminal Procedure. The rule governing appeals from summary judgments, Pa. R.Crim. P. 86(a), provides:
When an appeal is authorized by law in a summary proceeding, including a prosecution for a violation of a municipal ordinance which provides for imprisonment upon conviction or upon failure to pay a fine, an appeal shall be perfected by filing a notice of appeal within 30 days after the conviction ... and by appearing in the court of common pleas for a trial de novo. The notice of appeal shall be filed with the clerk of courts. (Emphasis added.)
In light of Section 617.2 of the MPC, the appeal procedure in Pa. R.Crim. P. 86(a) is inapplicable to zoning enforcement matters, because the MPC's civil enforcement procedure does not allow imprisonment as a sanction in any circumstance. Cf. City of Philadelphia v. Pennrose Management Co., 142 Pa.Cmwlth. 627, 598 A.2d 105 (1991) (when violation of a municipal ordinance may result in a fine or imprisonment, the enforcement of such ordinances must follow the Rules of Criminal Procedure, regardless of the fact that the action is technically civil), petition for allowance of appeal denied, 530 Pa. 661, 609 A.2d 169 (1992).
When a party takes an appeal from an order of a district justice involving the violation of a local ordinance, and the ordinance does not provide for incarceration, the appeal must be perfected under the Rules of Civil Procedure Governing Actions and Proceedings Before District Justices, and not the summary criminal appeal provisions of the *866 Rules of Criminal Procedure. Commonwealth v. Carter, 36 Pa.Cmwlth. 569, 377 A.2d 831 (1977), modified on reargument, 36 Pa.Cmwlth. 569, 389 A.2d 241 (1978);[3]City of Easton v. Marra, 230 Pa. Superior Ct. 352, 326 A.2d 637 (1974).
Therefore, for all of the above reasons, we conclude that civil appeal procedures in Pa. R.C.P.D.J. 1004 are applicable to a zoning enforcement appeal from an order of a district justice. The Common Pleas Court erred in determining that the Township should not have filed a complaint as required by Pa. R.C.P.D.J. 1004(B), and, instead, used a summary procedure analogous to Pa. R.Crim. P. 86.[4]
Accordingly, the Common Pleas Court's order is reversed.

ORDER
NOW, February 24, 1998, the order of the Court of Common Pleas of York County in the above-captioned matter is hereby reversed.
NOTES
[1]  Rule 1004(B) provides as follows:

If the appellant was the defendant in the action before the district justice, he shall file with his notice of appeal a praecipe requesting the prothonotary to enter a rule as of course upon the appellee to file a complaint within twenty (20) days after service of the rule or suffer entry of judgment non pros.
[2]  Act of July 31, 1968, P.L. 805, as amended, 53 P.S. § 10616.1. Under Section 616.1 of the MCP, an enforcement notice must contain the following information: (1) the name of the owner of record of the parcel where the zoning violation occurred; (2) the location of the property; (3) the specific violation and a description of the requirements that have not been met; (4) the date when compliance must be completed; (5) that the recipient of the notice has the right to appeal to the zoning hearing board, and (6) that failure to comply with the violation notice constitutes a violation and will result in sanctions. Section 616.1 was added to the MPC by Section 62 of the Act of December 21, 1988, P.L. 1329.
[3]  Although our order was modified on reargument, this Court, sitting en banc, agreed with the prior panel's conclusion that the appeal was civil in nature. Carter, 389 A.2d at 242; see also Borough of McAdoo v. Lawn Specialties, 120 Pa. Cmwlth. 120, 547 A.2d 1297 (1988).
[4]  In Commonwealth v. Harchelroad, 154 Pa. Cmwlth. 259, 623 A.2d 878, petition for allowance of appeal denied, 535 Pa. 649, 633 A.2d 153 (1993), we determined that, although the proceeding filed with the district justice by the code enforcement officer following a notice of violation issued by the township was technically denominated a "criminal complaint," there was no prejudice to the appellant/property owner because the case was "simply an appeal from a summary conviction for the violation of [the] illegal commercial use of property zoned R-2 single family Residential .... no more, no less." Id. 623 A.2d at 881. We held that, because Harchelroad had ignored the Notice of Violation and had failed to appeal to the Zoning Hearing Board, which had exclusive jurisdiction under the procedures set out in the MPC, he became subject to the enforcement proceeding where his equitable defenses (laches) were not available. Id. at 881, n. 5. Any interpretation of the dicta in the Harchelroad opinion to the contrary is simply incorrect.


Attorney General Jeff Sessions has offered to testify Tuesday in front of the Senate Intelligence Committee to answer accusations from former FBI Director James Comey.

"In light of reports regarding Mr. Comey's recent testimony before the Senate Select Committee on Intelligence, it is important that I have an opportunity to address these matters in the appropriate forum," Sessions wrote in a letter to Sen. Richard Shelby, chairman of the Senate Appropriations Subcommittee on Commerce, Justice, Science and Related Agencies.

"The Senate Intelligence Committee is the most appropriate forum for such matters, as it has been conducting an investigation and has access to relevant, classified information," he wrote.

Comey testified in front of the Senate Intelligence Committee in closed session that Sessions may have had a third, unreported meeting with Russian Ambassador Sergei Kislyak. Sessions omitted information about meetings with Kislyak as a senator during his confirmation process, and following the ensuing controversy he recused himself from the investigation into Russian meddling in the 2016 election.

Despite the recusal, Sessions was involved in the decision to fire Comey. Senators have questioned why Sessions was involved in Comey's dismissal since he recused himself from the Russia investigation and Comey was leading that investigation.

Sessions has been under siege in recent weeks after reports surfaced that President Trump remains furious over his decision to recuse himself from the investigation. Trump has repeatedly called the reports of Russian interference in the 2016 election a hoax that's been generated by Democrats to excuse their loss in the election.

Sessions said he made the decision to testify in front of the Senate Intelligence Committee instead of the appropriations panel after hearing that committee's members were planning to focus their questions on Comey's testimony.

"I previously accepted an invitation to testify on behalf of the Department of Justice before the House and Senate Appropriations Subcommittees about the Department's FY18 budget, which funds the Department's efforts in several important areas such as combatting the opioid crisis and violent crime," he wrote.

"Some members have publicly stated their intention to focus their questions on issues related to the investigation into Russian interference in the 2016 election, from which I have recused, and for which the Deputy Attorney General appointed a Special Counsel."

Sessions is planning to send Deputy Attorney General Rod Rosenstein to testify to appropriations committees in the House and Senate in his stead.


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Single-Center Case Series of Donor-Related Malignancies: Rare Cases With Tremendous Impact.
Donor-related malignancy is a rare complication of organ transplantation. In this case series, we discuss three cases of donor-related cancers in kidney transplant recipients who were registered in our center between 1979 and 2015. They account for an incidence of 0.29% of donor-related malignancies of a total of 1015 transplanted kidney grafts (deceased and living donors). The three cases that we describe presented in different ways and with different severity, although the response to the initiated treatment was comparable. All three patients not only survived their cancer episode but also had a complete oncological remission and underwent successful second kidney transplantation, accounting for a 100% survival rate in our small cohort. Despite the very low incidence of this complication, transplant clinicians must be aware of the occurrence of donor-related malignancies when selecting a donor and should be able to diagnose and treat a case of donor-related cancer.


1. Like Jane Eyre, my “organ of veneration” is enormous and I would do an excellent job swearing fealty to someone God had rightly set above me in the natural order. This would free me both from the burden of emotional responsibility and decision-making while also unleashing my natural propensity for viciousness towards anyone who has offended the people I love.

2. I am already a landowner, I possess roughly 2400 feet of mostly concrete in the hills of California and will gladly quarter as many of the king’s horses as is physically practical upon my modest estate (I think probably two in the backyard and one in the carport).

3. My hatred for the thrice-damned Merovingians is equalled only by my hatred for paintings that demonstrate an accurate use of perspective, and the market economy.

4. I would have no problem allowing peasants use some of my property to set their pigs a-grazing on, as long as I could keep one out of every ten or so pigs.

5. I oppose both the spread of guilds and the Dutch.

6. I am prepared already to join either the noblesse d’épée or the noblesse de robe, as I already own both a pretty fair fencing épée and a wizard’s robe that I got off the Internet.

7. I know a reasonable amount of Latin (mostly from the Wiccan scenes on Buffy, studying Latin roots in high school English, and a brief fascination with High Anglicanism during a semester at Oxford); certainly enough to get through an hommage service.

8. I would very much enjoy escaping a life of physical labor and periodic famines.

9. I am willing to oppose social mobility in all forms, excepting my own, and even then only in an upwardly direction.

10. I rode English-style for a few years during junior high and have a fairly steady seat, so I would stand a reasonable chance of making it through a calvalry charge.

11. I’m happy to invade Scotland or give away my sons to the Scots, or whatever.

12. My greatest fears are Lutherans, mercantilism, and a united France.


Q:

cpu utilization over 100% in linux system

In one of our production Linux servers, CPU utilization is crossing 100 % and sometimes it is 250%.
How can I find which process is using more CPU utilization and where can I find CPU utilization logs?
OS : RHEL 5.5

A:

CPU utilization is measured relative to a single CPU. The maximum is 100% for each CPU, so a four-CPU system would have a maximum CPU utilization of 400%.

A:

Try using the top command
man top for more info

A:

The sar utility collects some statistics about your system if it is switched on.  The simplest way to access them is to type sar on the command line.
A far better option is to install a monitoring system such as Cacti, Munin or Zabbix.  (Although Nagios can do graphing, this is not its forte.)  There are plenty of questions on ServerFault already about which monitoring system is the best.  Once you install one of them, you will have trouble remembering how you lived without it.
Generally, these will not tell you which process was using all the CPU at any given time.  For that, top or htop at the time is your best bet.  Bear in mind that 25 Apache processes using 10% CPU each shows up differently in top to a single MySQL process using 250% CPU.
The other key metric to watch is the "load average".  This is an indication of how many processes are currently waiting to get on a processor.  This is a very good indicator of how slow your system will feel to use.
Another note on CPU utilisation, virtual machines can dynamically alter the CPU capacity in response to the needs of the VM.  I have seen a single process showing up as using 9999% CPU in top from within a VM.  The advice given by VMWare about this is "Don't trust metrics measured from within the VM".


INTRODUCTION
============

Bt (*Bacillus thuringiensis*) is a Gram-positive soil bacterium that produces insecticidal proteins called Cry toxins during sporulation. Cry toxins are specific to insects and do not affect humans and domestic animals. In addition, these toxins are environmentally friendly because they are proteinaceous and easily degrade in the soil and on plant surfaces. Therefore Bt formulations represent pesticides that possess a low impact on human health and the environment. For this reason, Cry toxin genes have been used in the development of GMOs (genetically modified organisms) \[[@B1],[@B2]\]. However, acquisition of Cry toxins with high insecticidal activity against coleopteran, hemipteran, dipteran and hymenopteran insects from the soil is difficult. The development of a method for engineering Cry toxins with improved insecticidal activity against target pests and possessing a broader target pest spectrum is desired. To establish protein-engineering methods for the Cry toxin, the mode of action by which Cry toxin binds to the cadherin receptor must be characterized.

One hypothesis for the insecticidal mechanism of Cry toxins is the pore-forming model \[[@B3]\]. A recent version of the pore-formation model proposed the following sequence of events: protease-activated Cry toxin monomers bind to GPI (glycosylphosphatidylinositol)-anchored proteins, such as APN (aminopeptidase N) and ALP (alkaline phosphatase). This interaction promotes the binding of toxin monomers to the cadherin BtR (Bt receptor), which facilitates protease cleavage of the toxin\'s N-terminus, including helix α1 of domain I, and induces oligomerization of the toxin. Then toxin oligomers bind with increased affinity to GPI-anchored receptors and create pores in the midgut membrane, leading to osmotic shock and cell death. In contrast, the signalling model hypothesises that an adenylate cyclase/PKA (protein kinase A) signalling pathway is activated by BtR-bound monomer toxins, inducing programmed cell death \[[@B4]\]. Thus, BtR is an important receptor, although the correct hypothesis remains unknown. The generation of various Cry toxin-resistant insect strains has been attributed to a mutation or deletion in BtR \[[@B5]--[@B8]\]. In addition, there have been reports that cultured BtR-expressing insect or human cells are susceptible to Cry1A toxins, thus validating this hypothesis \[[@B9]--[@B12]\].

Seven 3D (three-dimensional) Cry toxin structures have been determined by X-ray crystallography \[[@B13]--[@B19]\]. Similar 3D structures consisting of three domains (domains I, II and III of the N-terminus) were confirmed to be associated with these toxins. Domain I is composed of a seven-α-helix bundle with a central helix and six surrounding helices, domain II is a β-prism consisting of three antiparallel β-sheets and loops and domain III is a β-sandwich consisting of two antiparallel β-sheets \[[@B13],[@B14],[@B18]\]. Loops α8, 1, 2 and 3 of domain II are thought to be BtR-binding regions \[[@B20]--[@B26]\]. However, it is unclear whether all regions are required for binding to BtRs.

A high-efficacy medicine is defined as a compound that can bind to and act on receptors at low concentrations. Accordingly, a major goal for the improvement of a lead medicinal compound is receptor-affinity maturation. Based on this concept, we anticipate that mutant toxins with higher BtR-binding affinities will likely also exhibit higher insecticidal activity. The insecticidal activity of Cry1A toxins is correlated with the binding affinity of Cry1A toxins for BBMV (blush-border membrane vesicles) prepared from insects \[[@B27]\]. Furthermore, binding to BtR has been determined to be indispensable to the insecticidal activity of Cry toxins \[[@B28]\]. Thus, we have developed a genetic-engineering method for improving the binding affinity of Cry toxin for BtR. Indeed, when a Cry1Aa loop suspected to be a BtR-binding site was replaced with that of Cry4Ba, the engineered Cry1Aa lost activity against *Manduca sexta* and obtained activity against *Culex pipiens* \[[@B29]\]. Similarly, when Cry4Ba loop 3 was replaced with a Cry4Aa-loop 3-mimic peptide, the engineered Cry4Ba gained activity against *C. pipiens*, which was originally resistant to Cry4Ba \[[@B30]\].

Cry1Aa, whose 3D structure has been determined, is active against the easy-bleeding insect *Bombyx mori* \[[@B14]\]. We have attempted to develop an affinity maturation process for Cry1Aa activity against *B*. *mori* BtR (BtR175). This involved applying a directed evolution-based method for the improvement of insecticidal activity using phage display and bio-panning, as described elsewhere \[[@B31]\]. However, as with other toxins, the only information known about this interaction is that the BtR175 binding site of Cry1Aa is near loops 1, 2 and 3 \[[@B25],[@B26]\]. To identify the critical binding sites, we performed a comprehensive binding assay that utilized mutants containing four random amino acid mutations spanning nearly all of the loop regions. Specifically, four serial amino acid residues were replaced with random amino acids at sites 275--278, 278--281 and 280--283 of loop α8; 310--313 of loop 1; 365--370, 367--370, 371--374 and 375--378 of loop 2; and 435--438, 439--442 and 443--446 of loop 3. This resulted in the generation of a total of 30 mutant proteins whose binding affinities for BtR175 were assessed by Biacore. Surprisingly, no critical binding site on CRY1Aa was found in the loop regions, leading us to propose a flexible, multi-binding model for BtR175 binding by Cry1Aa.

EXPERIMENTAL
============

Bacterial and insect strains
----------------------------

Wild-type T7 phage and Cry toxin displaying phages were propagated and titrated using *Escherichia coli* BLT-gene10 as described previously \[[@B31]\]. *E. coli* BL21 was used for the production of the wild-type or mutant Cry1Aa protoxin. Kinshu × Showa, a hybrid race of the silkworm, *B. mori* was reared as described previously \[[@B31]\].

Construction of phage libraries
-------------------------------

To create Cry1Aa mutants-displaying-phage libraries the regions encoding regions spanning domains II loops to domain III were amplified by PCR using 5′-acgaacccagtattagaaNNNNNNNNNNNNagttttcgtggaatggctcag-3′, 5′-agaaaattttgatNNNNNNNNNNNNggaatggctcag-3′, 5′-tttgatggtagtNNNNNNNNNNNNgctcagagaatag-3′, 5′-tttatactgatgtgNNNNNNNNNNNNaattattggtcagg-3′, 5′-ttatcttcacctNNNNNNNNNNNNattatacttggttc-3′, 5′-tcttcacctttatatNNNNNNNNNNNNcttggttcaggc3′, 5′-gaagaattataNNNNNNNNNNNNccaaataatcagg-3′, 5′-cttggttcaggcNNNNNNNNNNNNgaactgtttgtc-3′, 5′-ttgagtcatgttNNNNNNNNNNNNcaagcagctggag-3′, 5′-caatgctgagcNNNNNNNNNNNNgcagtttacacct-3′, 5′-gccaagcagctggaNNNNNNNNNNNNttgagagctccaac-3′ as sense primers, respectively, for libraries with respect to mutations at 275--278, 278--281, 280--283 of loop α8; 310--313 of loop 1; 365--370, 367--370, 371--374 and 375--378 of loop 2; and 435--438, 439--442 and 443--446 of loop 3 and 5′-agctcattctcgagtgcggccgctctttctaaatcatattctgcctcaa-3′ as an antisense primer. Next, the regions encoding domains I--III of Cry1Aa toxin were amplified by PCR using above-described DNA fragments as sense mega-primers and 5′-agaagtattaggtggggatccaatagaaactggttacaccccaa-3′ as an antisense primer, digested by restriction enzymes and inserted between BamHI and XhoI sites of T7Select10-3b DNA (Novagen) as described previously \[[@B31]\].

Preparation of wild-type and mutant toxins
------------------------------------------

Wild-type and mutant toxins were prepared as described previously. Briefly, DNAs from toxins displayed on the phage were inserted between SpeI and SacI sites of GST (glutathione transferase)-Cry1Aa protoxin fusion-protein expressing vector, *pB9* and *E. coli* BL21 was transformed with each resulting vector \[[@B31]\]. Fusion proteins were harvested as inclusion bodies and solubilized, and then solution of each fusion protein was applied to a DEAE column (Shodex IEC DEAE-825) connected to an HPLC system (Waters 600), and protoxins were activated by 0.5 mg/ml trypsin for 2 h at 37°C in the column \[[@B25]\]. Activated toxins were eluted using a linear gradient of Tris/HCl buffer and protein concentrations were determined by densitometry using BSA as a standard.

Binding kinetics analysis with SPR (surface plasmon resonance)
--------------------------------------------------------------

Cry1Aa toxin-binding region of BtR175 (BtR175-TBR) was prepared as described previously \[[@B32]\] and immobilized on a CM5 Biacore sensor chip using the amine-coupling method. Eight different concentrations (3.125--200 nM) of mutant Cry toxins diluted in PBST (PBS with 0.005% Tween 20, pH 7.4) were applied to the surface of the BtR175-TBR-immobilized CM5 sensor chip for 120 s. For dissociation, toxin flow was replaced by PBST, and the response was recorded for at least 240 s. The response curves were fit to a 1:1 Langmuir binding model and analysed using global fitting. Rate constants for association (*k*~a~) and dissociation (*k*~d~) were determined. The sensor chip was regenerated using 30 μl of 10 mM NaOH.

Bioassay using insect larvae
----------------------------

Solubilized GST--protoxin fusion proteins were mixed with artificial diet, Silk Mate PM, resulting in a final concentration of 1.3 μg/g diet. The diets (2.5 g) were placed in 9 cm dishes and then 25 third-instar larvae were reared on each diet at 25°C. Dead larvae were confirmed after 48 and 72 h.

Activation of toxins with the midgut juice
------------------------------------------

Activation profile of toxins by the silkworm midgut juice was assessed as described previously \[[@B31]\]. Briefly, midgut juice was prepared from fifth-instar larvae and diluted to be 25% (v/v) with deionized water. The inclusions of GST-protoxin fusion proteins were mixed with the diluted midgut juice to a final concentration of 1.3 μg/μl. Samples were incubated at 25°C for 0.5, 1, 2 and 4 h, and subjected to SDS/PAGE.

Preparation of two-site mutants
-------------------------------

The region encoding loop 1 to mutated loop 2 was amplified using R^375^GPD^378^ mutant DNA as a template and 5′-ttgagtgaatttgttcccggt-3′ and 5′-gaaaactccgttccatcaaggacaa-3′, respectively, as sense and anti-sense primers. The region encoding loop 3 was amplified using Q^439^PRG^442^, H^439^MPR^442^ or R^443^LGR^446^ mutant DNA as templates and 5′-ctgtttgtccttgatggaacg-3′ and 5′-aagaaaacgttggagctctca-3′, respectively, as sense and anti-sense primers. Both regions were connected by overlap PCR and resulting DNA fragments were digested by restriction enzymes and then inserted between *Spe*I and *Sac*I sites of GST-Cry1Aa protoxin fusion-protein-expressing vector, *pB9*, and *E. coli* BL21 was transformed with each resulting vector.

RESULTS
=======

Assessment of BtR175 binding affinity of Cry1Aa loop mutants
------------------------------------------------------------

We have attempted to establish a model system for directed evolution to acquire mutant toxins with higher BtR-binding affinity and higher insecticidal activity using a combination of *B*. *mori* and Cry1Aa (Y. Fujii, S. Tanaka, M. Otsuki, Y. Hoshino, H. Endo and R. Sato, unpublished work). As part of this trial, a mutant library was constructed by replacing four serial amino-acid residues with random amino acids at various sites, as described above ([Figure 1](#F1){ref-type="fig"}). To analyse the role of each of the four loops in the BtR175-binding mechanism of Cry1Aa, more than four phage-displayed Cry1Aa loop-mutants were randomly selected from each library (from each replacement site) and produced in *E*. *coli* as inclusions using the fusion protein connected to GST. The mutants were activated by the removal of GST in an anion-exchange column and purified, which resulted in successful acquisition of 30 trypsin-tolerant loop-mutants. BtR175-binding affinities were assessed by Biacore ([Figure 2](#F2){ref-type="fig"}). Global fitting using a 1:1 Langmuir binding model was used to calculate the association rate constant \[*k*~a~ (M^−1^·s^−1^)\] and dissociation rate constant \[*k*~d~ (s^−1^)\] ([Table 1](#T1){ref-type="table"}). Then the dissociation constant \[*K*~D~ (M)\], a primary parameter for binding affinity, was calculated according to the formula *K*~D~=*k*~d~/*k*~a~. All loop-mutants indicated high binding affinities for BtR175. The highest *K*~D~ was that of T^367^LGP^370^ and the lowest *K*~D~ was that of R^375^GPD^378^, resulting in an affinity range that spanned from 1.6 times lower to 4.2 times higher than that of Cry1Aa for all 30 mutants ([Table 1](#T1){ref-type="table"}, [Figure 2](#F2){ref-type="fig"}). All mutants contained different amino acid sequences with respect to the corresponding loop region and no general trend was observed in the amino acid sequence composition compared with the wild-type toxin ([Figures 1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}).

![Mutation sites introduced in loops α8, 1, 2 and 3 of Cry1Aa and amino acid sequences of the mutants used in this experiment\
Cry1Aa mutant toxins containing four serial amino acid residue replacements in three regions of loop α8, one region of loop 1, four regions of loop 2 and three regions of loop 3 were expressed on the phage surface to construct 11 classes of phage libraries. Thirty mutants were prepared as activated toxins. The mutated sequence of each mutant is shown compared with wild-type sequences. Sequences in bold typeface are those that were integrated into one molecule. Sequences written in italic are high-affinity-generating mutants derived from our unpublished work. Molecular models were generated using CCP4MG.](bsr2012-0113i001){#F1}

![Binding and dissociation kinetics obtained by BtR175-immobilized CM5 Biacore sensor chips using concentrated phage clone-derived mutant toxins\
Mutant toxins (12.5, 25, 50, 100 and 200 nM) were prepared using PBST (0.005% Tween 20) and injected over the CM5 Biacore sensor chip immobilized with BtR175 for 120 s. For dissociation measurements, the toxin flow was replaced by PBST buffer and recorded for at least 240 s. RU, response units.](bsr2012-0113i002){#F2}

###### Binding of 30 mutant and wild-type toxins for BtR175 obtained by fitting the curves indicated in [Figures 2](#F2){ref-type="fig"} and [5](#F5){ref-type="fig"} (wild-type) to a 1:1 Langmuir binding models

  Clone names      *K~a~* (×10^5^ M^−1^·s^−1^)   *k~d~* (× 10^−3^·s^−1^)   *K*~D~ (×10^−8^ M)
  ---------------- ----------------------------- ------------------------- --------------------
  Wild-type        0.96                          2.66                      2.77
  G^275^EGG^278^   1.15                          1.66                      1.45
  D^278^DIY^281^   1.12                          2.40                      2.14
  F^280^RWE^283^   0.85                          1.92                      2.24
  A^310^QMA^313^   1.12                          1.80                      1.6
  K^310^ASR^313^   2.51                          6.75                      2.69
  L^365^EAQ^368^   2.57                          4.28                      1.66
  H^365^AGG^368^   2.38                          4.39                      1.84
  F^365^EPK^368^   0.84                          3.1                       3.68
  F^367^SQA^370^   1.26                          4.14                      3.29
  A^367^PGP^370^   0.94                          3.82                      4.06
  S^367^TRS^370^   1.27                          2.26                      1.77
  T^367^LGP^370^   0.88                          4.00                      4.54
  L^367^PRP^370^   1.34                          1.78                      1.33
  L^367^PAP^370^   0.85                          2.55                      2.98
  P^367^RRP^370^   1.26                          1.53                      1.21
  S^367^QAA^367^   0.71                          2.6                       3.64
  H^367^NAG^370^   0.71                          1.82                      2.55
  A^367^PAP^370^   0.81                          2.7                       3.31
  S^367^PSA^370^   0.79                          2.93                      3.68
  S^371^APN^374^   1.25                          1.67                      1.33
  E^371^PRH^374^   2.90                          3.28                      1.13
  R^375^GPD^378^   1.85                          1.22                      0.65
  V^375^GCA^378^   1.06                          3.01                      2.84
  G^435^LVG^438^   4.14                          5.59                      1.35
  S^435^VVG^438^   1.90                          3.79                      1.99
  E^439^PPA^442^   1.21                          1.40                      1.16
  T^439^LRT^442^   1.51                          1.58                      1.05
  D^439^TGN^442^   1.20                          1.62                      1.35
  I^443^VPV^446^   1.58                          3.16                      1.99
  V^443^ELL^446^   1.33                          2.72                      2.04

Insecticidal activities of the Cry1Aa loop mutants
--------------------------------------------------

Insecticidal activities toward *B*. *mori* larvae were assessed using 23 Cry1Aa loop mutants. Although no activity was observed in seven mutants (H^365^AGG^368^, F^365^EPK^368^, A^367^PAP^370^, S^367^PSA^370^, S^371^APN^374^, R^375^GPD^378^ and T^439^LRT^442^), insecticidal activities were confirmed for the other 16 mutants ([Figure 3](#F3){ref-type="fig"}). At least one mutant indicated that insecticidal activity was associated with replacement of the eight sites covering all areas of loops 1, 2 and 3.

![Insecticidal activities of wild-type and mutant toxins\
Mutant toxins with a high binding affinity for BtR175 and wild-type (wt) toxin were added to the diet in the protoxin form, resulting in a final concentration of 1.3 μg/g diet. Twenty-five third-instar larvae were reared on each diet at 25°C. Dead larvae were confirmed after 48 (**A**) and 72 h (**B**).](bsr2012-0113i003){#F3}

Defects in the activation process of non-active loop-mutants in larval midgut juice
-----------------------------------------------------------------------------------

To determine whether non-active mutants contain defects in the activation process in the midgut fluid, two non-active mutants (R^375^GPD^378^ and T^439^LRT^442^), five active mutants (K^310^ASR^313^, H^367^NAG^370^, P^367^RRP^370^, V^375^GCA^378^ and V^443^ELL^446^) and wild-type toxins were incubated in midgut fluid and analysed by SDS/PAGE ([Figure 4](#F4){ref-type="fig"}). The non-active loop-mutant R^375^GPD^378^ underwent complete degradation, leaving no observable fluid-tolerant fragments ([Figure 4](#F4){ref-type="fig"}B). Another non-active loop-mutant (T^439^LRT^442^) contained only a faint band of activated toxin at 60 kDa ([Figure 4](#F4){ref-type="fig"}D). The remaining mutants, as well as the wild-type toxin, had larvicidal activities with stable, activated forms present at 60 kDa. These results indicate the non-activity of R^375^GPD^378^ and T^439^LRT^442^ owing to a defect in the activation process, resulting in the suggestion that only mutants with defects in the activation process might have lost insecticidal activity.

![Activation profile of wild-type and mutant toxins containing high binding affinities for BtR175 using the midgut fluid of *B*. *mori* larvae\
Wild-type and mutant toxins were prepared as inclusions using GST-protoxin fusion proteins. The inclusions were mixed with a 1:4 dilution of midgut fluid from fifth-instar *B*. *mori* larvae, incubated at 25°C for 0.5, 1, 2 and 4 h, and subjected to SDS/PAGE. Asterisks indicate 160 kDa GST--protoxin fusion proteins and arrowheads indicate 60 kDa midgut fluid-tolerant toxins. (**A**) wild-type and K^310^ASR^313^; (**B**) H^367^NAG^370^ and R^375^GPD^378^; (**C**) P^367^RRP^370^ and ^375^VGCA^378^; (**D**) T^439^LRT^442^ and V^443^ELL^446^. Lane 1: before digestion; Lane 2: 0.5 h after digestion; Lane 3: 1 h after digestion; Lane 4: 2 h after digestion; Lane 5: 4 h after digestion.](bsr2012-0113i004){#F4}

Assessment of BtR175 binding affinity of mutants containing double mutations in different loops
-----------------------------------------------------------------------------------------------

Phage-displayed Q^439^PRG^442^, H^439^MPR^442^ and R^443^LGR^446^ mutants were selected by bio-panning during a trial of directed evolution directing Cry1Aa affinity against BtR175 (Y. Fujii, S. Tanaka, M. Otsuki, Y. Hoshino, H. Endo and R. Sato, unpublished work). Simultaneous two-site replacement was performed in different loops to assess the effects of multiple mutation sites on BtR175-binding affinity. Specifically, we were interested in determining whether binding affinity would decrease when two mutations were combined. The loop 3 mutants Q^439^PRG^442^, H^439^MPR^442^ and R^443^LGR^446^ had higher affinities (13, 42 and 15 times, respectively) than that of wild-type ([Table 2](#T2){ref-type="table"}) (Y. Fujii, S. Tanaka, M. Otsuki, Y. Hoshino, H. Endo and R. Sato, unpublished work) and were integrated with the R^375^GPD^378^ loop 2 mutation (four times higher affinity) into one molecule. The binding affinities of R^375^GPD^378^/Q^439^PRG^442^, R^375^GPD^378^/H^439^MPR^442^ and R^375^GPD^378^/R^443^LGR^446^ double mutants were assessed by Biacore and resulted in *K*~D~ values of 0.34, 0.056 and 0.27×10^−18^ M, respectively ([Table 2](#T2){ref-type="table"}), indicating affinity values that were 8, 49 and 10 times higher than the wild-type toxin. In case of R^375^GPD^378^/H^439^MPR^442^ double mutant, an increase in binding affinity was observed compared with the original mutants ([Table 2](#T2){ref-type="table"}).

###### BtR175 binding by mutant toxins containing two mutated loops in one molecule

Values were obtained by fitting the curves indicated in [Figure 5](#F5){ref-type="fig"} to a 1:1 Langmuir binding model.

  Clone names                                        *k*~a~ [\*](#T2TFN1){ref-type="table-fn"} (×10^5^ M^−1^·s^−1^)   *k*~d~[\*](#T2TFN1){ref-type="table-fn"} (×10^−3^·s^−1^)   *K*~D~ (×10^−8^ M)
  -------------------------------------------------- ---------------------------------------------------------------- ---------------------------------------------------------- ----------------------------------------
  Wild-type                                          0.960                                                            2.66                                                       2.77
  Q^439^PRG^442^ [†](#T2TFN2){ref-type="table-fn"}   7.96[†](#T2TFN2){ref-type="table-fn"}                            1.62[†](#T2TFN2){ref-type="table-fn"}                      0.211[†](#T2TFN2){ref-type="table-fn"}
  H^439^MPR^442^ [†](#T2TFN2){ref-type="table-fn"}   15.9[†](#T2TFN2){ref-type="table-fn"}                            1.05[†](#T2TFN2){ref-type="table-fn"}                      0.066[†](#T2TFN2){ref-type="table-fn"}
  R^443^LGR^446^ [†](#T2TFN2){ref-type="table-fn"}   7.12[†](#T2TFN2){ref-type="table-fn"}                            1.32[†](#T2TFN2){ref-type="table-fn"}                      0.185[†](#T2TFN2){ref-type="table-fn"}
  R^375^GPD^378^/PQ^439^RG^442^                      5.13                                                             1.75                                                       0.340
  R^375^GPD^378^/H^439^MPR^442^                      21.6                                                             1.22                                                       0.056
  R^375^GPD^378^/R^443^LGR^446^                      6.46                                                             1.75                                                       0.270

\*To calculate parameters, the 1:1 Langmuir binding fit model was applied to the curves indicated in [Figure 5](#F5){ref-type="fig"}.

†Y. Fujii, S. Tanaka, M. Otsuki, Y. Hoshino, H. Endo and R. Sato, unpublished work.

DISCUSSION
==========

Replacement of any four sequential amino acids within any of the four loops did not reduce BtR175-binding affinity ([Table 1](#T1){ref-type="table"}, [Figure 2](#F2){ref-type="fig"}). In addition, insecticidal activities remained high in many of these mutants ([Figure 3](#F3){ref-type="fig"}). No relationship was observed between the identity of the four replaced amino acid sequences of the mutants and those of the wild-type toxin ([Figure 1](#F1){ref-type="fig"}). Pigott et al. \[[@B33]\] reported that replacement of loop 1, 2 and 3 regions with CDR3 fragments from human IgG heavy chain did not affect the binding affinity between Cry1Aa and *M*. *sexta* BtR (Bt-R1). These results suggest that the amino-acid sequences of the four loops of Cry1Aa are not critical to BtR175 binding.

Many proteins do require conserved amino acid sequences for ligand binding. For example, the RGD motif is necessary for the binding of several mammalian extracellular matrix proteins, such as fibronectin, to integrin \[[@B34]\]. However, our results suggest that Cry1Aa activity does not depend on conserved amino-acid sequences in loops, indicating an unusual binding character compared with other proteins.

One binding mechanism that does not require specific amino-acid loop sequences for BtR binding is multipoint attachment. Indeed, loop 2 and 3 of Cry1Aa and loops 2 and α8 of Cry1Ab are reported to have a binding affinity for Bt-R1 \[[@B21],[@B22]\], and loops 2 and 3 of Cry1Ab are reported to have a binding affinity for *Heliothis virescens* BtR \[[@B24]\]. Furthermore, all four loop regions are candidate Cry1A-binding regions for BtRs \[[@B25],[@B35]--[@B38]\]. Thus, it is possible that these loops bind to BtR by multipoint attachment because replacing a restricted region (four amino acids in one loop) with random amino acids would not be expected to significantly affect BtR-binding affinity, which we observed in the present study.

Even if the multipoint-attachment mechanism applies to Cry toxins, it is reasonable to assume that reduction of the binding site should reduce the binding affinity. However, none of the mutants had a BtR175-binding affinity two times lower than that of wild-type toxins. Indeed, Gomez et al. \[[@B21]\] reported that one of the BtR1-binding sites of Cry1Aa and Cry1Ab includes 367--373 of loop 2, although only two of the seven amino acids overlap between the toxins \[[@B21]\]. It was postulated that amino-acid sequences displaying inverted hydropathic profiles against ligand proteins are important for binding. If binding in the Cry loop regions were governed by this rule, hydropathic profiles would be conserved in all BtR-binding loops. To test this hypothesis, we compared the replaced sequences of the 30 mutants with the wild-type sequence. In only a few cases did the hydropathic profile of the replaced sequences coincide with the original sequences.

Short peptides and small linear molecules can adopt different conformations, resulting in different molecules binding to the same protein. For example, various peptide fragments can bind to human HLA-A2 Class I MHC molecules \[[@B39]\]. Further, many kinds of volatile compounds with disparate structures can bind to the same odorant binding protein \[[@B40]\]. These phenomena are possible due to conformation changes or the generation of new binding sites.

It has also been reported that loops are flexible in nature, which affects the activities of loop-harbouring molecules. Indeed, the flexible nature of a catalytic loop plays an important role in the activity of a nucleotide hydrolase from *Trypanosoma vivax* \[[@B41]\]. The flexibility of a loop in *E*. *coli* class II fructose-1,6-bisphosphate aldolase was found to be important to its catalytic activity \[[@B42]\]. Cry1Aa loops were reported to be highly mobile and their flexible nature was postulated to play an important role in receptor recognition \[[@B14]\]. It is possible that mutated loops may change conformation to present suitable hydropathic profiles to BtR175, preventing a reduction in BtR175-binding affinity. The sequences of the four loop regions of domain II of Cry1Aa, Ab and Ac generally contain low homology, with exceptionally low homology in loop 2. However, all of these toxins are reported to bind to *M*. *sexta* BtR1 \[[@B21]\]. Thus, our experimental results suggest that the high BrR175-binding affinities found for all 30 mutants containing four serially replaced amino acids depend on the flexible nature of the loops. This flexibility may facilitate multipoint attachment by exposing suitable hydropathic profiles for the receptors without the requirement for specially conserved amino acid sequences in the loops. It is conceivable that faster association rates seen in those mutants ([Tables 1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}) could be a consequence of larger conformational flexibility of loops involved in binding, allowing for a larger available fraction of loop conformations compatible with toxin--receptor complex formation.

The Q^439^PRG^442^, H^439^MPR^442^ and R^443^LGR^446^ loop 3 mutants had 13, 42 and 15 times higher affinity for BtR, respectively, than the wild-type toxin (Y. Fujii, S. Tanaka, M. Otsuki, Y. Hoshino, H. Endo and R. Sato, unpublished work). Each of these loop 3 mutants was independently integrated with a R^375^GPD^378^ loop 2 mutant having an affinity four times higher than that of the wild-type. Initially, it was expected that the integration of these separate sequences into the same mutant toxin would reduce the binding affinity of the mutant for BtR. However, all three mutants containing the two simultaneous replacements maintained higher binding affinity for BtR175 at levels comparable to that of the wild-type toxin ([Table 2](#T2){ref-type="table"}, [Figure 5](#F5){ref-type="fig"}). Moreover, the R^375^GPD^378^/H^439^MPR^442^ double mutant further increased the binding affinity for BtR175, which suggests that loops do not require a specific amino acid sequence for BtR binding. Moreover, these results indicate that the mutated version containing mutations at both loops 2 and 3 possessed new binding sites for BtR175, which probably resulted from a conformational change in the double mutant. Indeed, Pigott et al. \[[@B33]\] reported that replacing loops 1 and 2, or 2 and 3 simultaneously with CDR3 peptide mutant toxins maintained the binding affinity for Bt-R1 \[[@B33]\]. This is also consistent with the idea that loops do not require specific amino acid sequences for BtR-binding and that newly created loops can form new binding sites.

![Binding and dissociation kinetics obtained with BtR175-immobilized CM5 Biacore sensor chips using two-site mutants\
Toxins were prepared in five concentrations using PBST \[0.005% (v/v) Tween 20\] and injected over the CM5 Biacore sensor chip immobilized with BtR175 for 120 s. For dissociation constants, the toxin flow was replaced by PBST buffer and recorded for at least 240 s. (**A**) R^375^GPD^378^/Q^439^PRG^442^ mutant; (**B**) R^375^GPD^378^/H^439^MPR^442^ mutant; (**C**) R^375^GPD^378^/R^443^LGR^446^ mutant; (**D**) wild-type. RU, response units.](bsr2012-0113i005){#F5}

In the case of scFv (single-chain fragment variable), a mutation generating 133 times higher binding affinity and a mutation derived from a different loop region generating 59 times higher affinity to an antigen were integrated into one molecule, resulting in generation of a 1200 times higher affinity mutant in contrast with the wild-type version \[[@B43]\]. We found that integration of the R^375^GPD^378^ mutation (four times higher affinity) with the H^439^MPR^442^ mutation (42 times higher affinity) into a R^375^GPD^378^/H^439^MPR^442^ double mutant had a resulting binding affinity 49 times higher than that of wild type. These results suggest that the binding affinity of the Cry toxin to the receptor can be increased by integrating two different high-affinity-generating mutations simultaneously into one molecule. Affinity maturation of the Cry1A toxin can be enhanced by introducing several different loop-derived high-affinity-generating mutations into one molecule. In fact, in addition to mutations on loop 3, such as H^439^MPR^442^, we succeeded in finding several mutations on loop 2 containing affinities 20--50 times higher than that of the wild-type toxin. We anticipate the generation of mutants with more than 100 greater binding affinities for BtR than the wild-type toxin by integrating multiple mutations into one molecule.

AUTHOR CONTRIBUTION
===================

Ryoichi Sato conceived and designed the projects and wrote the paper. Yuki Fujii performed the experiments, analysed the data and wrote the paper. Manami Otsuki, Yasushi Hoshino, Chinatsu Morimoto, Takuya Kotani, Yuko Harashima, Haruka Endo, Shiho Tanaka and Yasutaka Yoshizawa performed the experiments and analysed the data.

FUNDING
=======

This work was supported by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan \[grant number 24310054\].


Sodium-free contractures in frog myocardium damaged by catecholamines.
1. Sodium-free contractures were studied in myocardial strips from R. pipiens with extracellular sodium (Na+o) replaced by choline chloride and extracellular calcium (Ca2+o) varied with EGTA-buffer. Normal myocardium was compared with that damaged by adrenaline (ADR) or isoproterenol (ISO). 2. Frog myocardium, damaged by in vivo injections of catecholamines, remained relaxed when exposed to Na+/Ca2+-free solutions. Only in 2 out of 18 experiments were small contractures observed after several hours. 3. Addition of KCN to the Na+/Ca2+-free solution caused small contractures after several hours in 7 out of 10 experiments. 4. The time to maximum Na+-free contractures was correlated to Ca2+o in a dose-dependent manner, but not influenced by catecholamine-induced myocardial damage. 5. Cell injury in the frog heart after in vivo injections of catecholamines does not affect the sarcolemmal Na+/Ca2+-exchange and is not associated with passive leakage of Ca2+ from the extracellular to the intracellular space.


In microelectronic circuit packages, circuits and units are prepared in packaging levels of increasing scale. Generally, the smallest scale packaging levels are semiconductor chips housing multiple microcircuits and/or other components. Such chips are usually made from ceramics, silicon, and the like. Intermediate package levels (i.e., “chip carriers”) comprising multi-layer substrates may have attached thereto a plurality of small-scale chips housing many microelectronic circuits. Likewise, these intermediate package levels themselves can be attached to larger scale circuit cards, motherboards, and the like. The intermediate package levels serve several purposes in the overall circuit assembly including structural support, transitional integration of the smaller scale microcircuits and circuits to larger scale boards, and the dissipation of heat from the circuit assembly. Substrates used in conventional intermediate package levels have included a variety of materials, for example, ceramic, fiberglass reinforced polyepoxides, and polyimides.
Dielectric materials used as coatings on the substrates must meet several requirements, including conformality, flame resistance, and compatible thermal expansion properties. Conventional dielectric materials include, for example, polyimides, polyepoxides, phenolics, and fluorocarbons. A common method of applying conformal coatings is by vapor deposition. Electrophoretic deposition has also been explored; however, polyimide resins such as bismaleimide resins, while desired for their superior dielectric and thermal stability properties, are intractable in most solvents, let alone aqueous dispersions, making electrophoretic deposition of such resins virtually impossible. Reactivity of the resins with amines increases the difficulty of formulation in both solvent based and aqueous dispersed coatings.
Accordingly, it would be desirable to provide a composition that provides the dielectric and thermal stability properties necessary for electronic circuit applications, while allowing for convenient electrophoretic deposition thereof.


Why Is The IOT Important To Your Business?

Two of the key focuses of my blog are Business and Entrepreneurship and Technology. As we move forward, understanding new technologies will be critical to conducting business. This will involve understanding new applications for the internet and how devices can become smarter and more connected. The following contributed post is thus entitled, Why Is The IoT Important To Your Business?

Along with entering the digital era, a lot of people are starting to feel like the world has entered an age of abbreviations. When you work in business, it’s especially hard to escape this reality, with practically every buzzword and phrase being shortened in an effort to make it easier to digest. Of course, though, this doesn’t always work. As a great example of this, there has been a lot of confusion around the field of IoT in recent years, with a lot of businesses having no idea what this side of technology actually is. To help you out with this, while also showing you the importance of IoT to your business, this post will be showing you what it means.

What is IoT?

When broken down into the words which its letters stand for, the Internet of Things sounds like something from a sci-fi universe aimed at children. In reality, though, this area of technology is becoming one of the biggest around, with areas like automation, manufacturing, and computer networking all heavily relying on it to keep working. This phrase simply refers to networks of devices which go further than computers being connected to one another. In the future, it’s not inconceivable that every piece of electronics in a building will be working together, but this will rely on developing in the IoT field to be able to advance.

What is IoT Used for?

Currently, this side of technology is in relatively early stages of existence. Up until recently, a lot of businesses have worked hard to keep their devices from being too well connected to each other, helping to prevent issues like cybercrime. As time has gone on, though, the value in having things like lights, machinery, and even basic office logistics being handled by connected devices is becoming more apparent. Companies like Cisco are working very hard to make this sort of technology work, with research into areas like intuitive networking being a priority for their vast teams.

Getting Started with IoT

As with many emerging tech trends, it can be hard to know where a field like the IoT will go over the next few years. If you’re running a business which could benefit from this sort of infrastructure, though, it will be worth taking the time to learn more here. As time goes on, being part of an evolution like this could save you a lot of time and money, especially as practices like this become standardised. Of course, though, if you’re unsure, it could be worth waiting a few years before you get stuck in, while keeping the idea of connecting your business together in the back of your mind for another day.

Hopefully, this post will give you everything you need to assess whether or not you could use a system like IoT to run your company. As time goes on, this is only going to become more prevalent in society, especially as more and more people start to use smartphones and other devices which can connect to the internet.

Author: anwaryusef

Anwar Y. Dunbar is a Regulatory Scientist. Being a naturally curious person, he is also a student of all things. He earned his Ph.D. in Pharmacology from the University of Michigan and his Bachelor’s Degree in General Biology from Johnson C. Smith University (JCSU). Prior to starting the Big Words Blog Site, Anwar published and contributed to numerous research articles in competitive scientific journals reporting on his research from graduate school and postdoctoral years. After falling in love with writing, he contributed to the now defunct Examiner.com, and the Edvocate where he regularly wrote about: Education-related stories/topics, Science, Technology, Engineering and Mathematics (STEM), Financial Literacy; as well as conducted interviews with notable individuals such as actor and author Hill Harper. Having many influences, one of his most notable heroes is author, intellectual and speaker, Malcolm Gladwell, author of books including Outliers and David and Goliath.
Anwar has his hands in many, many activities. In addition to writing, Anwar actively mentors youth, works to spread awareness of STEM careers, serves on the Board of Directors of the Friends of the David M. Brown Arlington Planetarium, serves as Treasurer for the JCSU Washington, DC Alumni Chapter, and is active in the Dave Ramsey Financial Peace Ministry at the Alfred Street Baptist Church. He also tutors in the subjects of biology, chemistry and physics.
Along with his multi-talented older brother Amahl Dunbar (designer of the Big Words logos, inventor and a plethora of other things), Anwar is a “Fanboy” and really enjoys Science-Fiction and Superhero movies including but not restricted to Captain America Civil War, Batman vs. Superman: Dawn of Justice, and Prometheus. He is a proud native of Buffalo, NY.
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Outbreak of diffuse lamellar keratitis caused by marking-pen toxicity.
To examine the evidence that a series of cases of diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK) was caused by a type of marker pen. Eye Institute, Auckland, New Zealand. During a 10-week period, 522 consecutive LASIK procedures were performed using a 60 Hz IntraLase femtosecond laser (IntraLase Corp.) to create the LASIK flap and a 217Z 100 Hz excimer laser (Bausch & Lomb) to perform the refractive ablation. As standard practice, a marking pen was used to enable accurate flap realignment. Three weeks after a sudden increase in the incidence of DLK was identified, one of the 5 surgeons performed 5 consecutive bilateral cases using the marking pen in the right eyes but not in the left eyes. Of the 522 LASIK cases (119 without marking pen, 403 with marking pen), DLK developed in 49 (9.4%). No eye treated without the marking pen developed DLK; of those in which the marking pen was used, 49 (12.2%) developed DLK (P<0.0001, Fischer exact test; odds ratio, 27). In the 5 consecutive bilateral cases in which the marking pen was used in the right eye but not the left eye, 4 right eyes and no left eye developed DLK (P=0.03). Forty-five of the 49 eyes with DLK quickly recovered. The other 4 developed central toxic keratopathy. There is strong statistical evidence that the marking pen was a factor in the occurrence of DLK.