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result sql = Effects of pH, temperature, and sucrose on benzyl alcohol-induced aggregation of recombinant human granulocyte colony stimulating factor.
Effects of pH, temperature, and sucrose on benzyl alcohol-induced aggregation of recombinant human granulocyte colony stimulating factor.
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char = t;code = 116
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = r;code = 114
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = r;code = 114
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = r;code = 114
char = h;code = 104
char = G;code = 71
char = C;code = 67
char = S;code = 83
char = F;code = 70
char = ;code = 32
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = b;code = 98
char = o;code = 111
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = p;code = 112
char = H;code = 72
char = ,;code = 44
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = o;code = 111
char = u;code = 117
char = t;code = 116
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = t;code = 116
char = e;code = 101
char = c;code = 99
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = l;code = 108
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = d;code = 100
char = a;code = 97
char = r;code = 114
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = r;code = 114
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = S;code = 83
char = t;code = 116
char = r;code = 114
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = b;code = 98
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = w;code = 119
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = g;code = 103
char = r;code = 114
char = e;code = 101
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = 3;code = 51
char = 7;code = 55
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = g;code = 103
char = r;code = 114
char = e;code = 101
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = C;code = 67
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = 2;code = 50
char = 5;code = 53
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = g;code = 103
char = r;code = 114
char = e;code = 101
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = C;code = 67
char = .;code = 46
char = ;code = 32
char = A;code = 65
char = t;code = 116
char = ;code = 32
char = b;code = 98
char = o;code = 111
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = p;code = 112
char = H;code = 72
char = ;code = 32
char = 7;code = 55
char = .;code = 46
char = 0;code = 48
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = 3;code = 51
char = .;code = 46
char = 5;code = 53
char = ,;code = 44
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = o;code = 111
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = -;code = 45
char = d;code = 100
char = e;code = 101
char = u;code = 117
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = u;code = 117
char = m;code = 109
char = ;code = 32
char = (;code = 40
char = H;code = 72
char = -;code = 45
char = D;code = 68
char = );code = 41
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = c;code = 99
char = h;code = 104
char = a;code = 97
char = n;code = 110
char = g;code = 103
char = e;code = 101
char = ;code = 32
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = r;code = 114
char = h;code = 104
char = G;code = 71
char = C;code = 67
char = S;code = 83
char = F;code = 70
char = ;code = 32
char = w;code = 119
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = c;code = 99
char = r;code = 114
char = e;code = 101
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = 0;code = 48
char = .;code = 46
char = 9;code = 57
char = %;code = 37
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = n;code = 110
char = z;code = 122
char = y;code = 121
char = l;code = 108
char = ;code = 32
char = a;code = 97
char = l;code = 108
char = c;code = 99
char = o;code = 111
char = h;code = 104
char = o;code = 111
char = l;code = 108
char = .;code = 46
char = ;code = 32
char = S;code = 83
char = u;code = 117
char = c;code = 99
char = r;code = 114
char = o;code = 111
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = 1;code = 49
char = .;code = 46
char = 0;code = 48
char = ;code = 32
char = M;code = 77
char = );code = 41
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = u;code = 117
char = n;code = 110
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = n;code = 110
char = z;code = 122
char = y;code = 121
char = l;code = 108
char = ;code = 32
char = a;code = 97
char = l;code = 108
char = c;code = 99
char = o;code = 111
char = h;code = 104
char = o;code = 111
char = l;code = 108
char = -;code = 45
char = i;code = 105
char = n;code = 110
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = b;code = 98
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = r;code = 114
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = r;code = 114
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = c;code = 99
char = r;code = 114
char = e;code = 101
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = H;code = 72
char = -;code = 45
char = D;code = 68
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = c;code = 99
char = h;code = 104
char = a;code = 97
char = n;code = 110
char = g;code = 103
char = e;code = 101
char = ;code = 32
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = h;code = 104
char = u;code = 117
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = n;code = 110
char = z;code = 122
char = y;code = 121
char = l;code = 108
char = ;code = 32
char = a;code = 97
char = l;code = 108
char = c;code = 99
char = o;code = 111
char = h;code = 104
char = o;code = 111
char = l;code = 108
char = ;code = 32
char = a;code = 97
char = c;code = 99
char = c;code = 99
char = e;code = 101
char = l;code = 108
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = g;code = 103
char = r;code = 114
char = e;code = 101
char = g;code = 103
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = r;code = 114
char = h;code = 104
char = G;code = 71
char = C;code = 67
char = S;code = 83
char = F;code = 70
char = ;code = 32
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = p;code = 112
char = H;code = 72
char = ;code = 32
char = 7;code = 55
char = .;code = 46
char = 0;code = 48
char = ,;code = 44
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = c;code = 99
char = a;code = 97
char = u;code = 117
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = t;code = 116
char = ;code = 32
char = f;code = 102
char = a;code = 97
char = v;code = 118
char = o;code = 111
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = f;code = 102
char = o;code = 111
char = l;code = 108
char = d;code = 100
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = g;code = 103
char = r;code = 114
char = e;code = 101
char = g;code = 103
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = -;code = 45
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = t;code = 116
char = e;code = 101
char = i;code = 105
char = n;code = 110
char = .;code = 46
char = ;code = 32
char = S;code = 83
char = u;code = 117
char = c;code = 99
char = r;code = 114
char = o;code = 111
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = u;code = 117
char = n;code = 110
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = s;code = 115
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = n;code = 110
char = z;code = 122
char = y;code = 121
char = l;code = 108
char = ;code = 32
char = a;code = 97
char = l;code = 108
char = c;code = 99
char = o;code = 111
char = h;code = 104
char = o;code = 111
char = l;code = 108
char = -;code = 45
char = i;code = 105
char = n;code = 110
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = r;code = 114
char = h;code = 104
char = G;code = 71
char = C;code = 67
char = S;code = 83
char = F;code = 70
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = g;code = 103
char = r;code = 114
char = e;code = 101
char = g;code = 103
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = h;code = 104
char = i;code = 105
char = f;code = 102
char = t;code = 116
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = e;code = 101
char = c;code = 99
char = u;code = 117
char = l;code = 108
char = a;code = 97
char = r;code = 114
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = p;code = 112
char = u;code = 117
char = l;code = 108
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = w;code = 119
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = p;code = 112
char = e;code = 101
char = c;code = 99
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char = e;code = 101
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = w;code = 119
char = a;code = 97
char = r;code = 114
char = d;code = 100
char = s;code = 115
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = r;code = 114
char = e;code = 101
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char = c;code = 99
char = o;code = 111
char = m;code = 109
char = p;code = 112
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = s;code = 115
char = .;code = 46
char = ;code = 32
char = W;code = 87
char = e;code = 101
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = s;code = 115
char = t;code = 116
char = u;code = 117
char = l;code = 108
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = b;code = 98
char = s;code = 115
char = e;code = 101
char = n;code = 110
char = c;code = 99
char = e;code = 101
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = g;code = 103
char = r;code = 114
char = e;code = 101
char = g;code = 103
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = p;code = 112
char = H;code = 72
char = ;code = 32
char = 3;code = 51
char = .;code = 46
char = 5;code = 53
char = ,;code = 44
char = ;code = 32
char = e;code = 101
char = v;code = 118
char = e;code = 101
char = n;code = 110
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = n;code = 110
char = z;code = 122
char = y;code = 121
char = l;code = 108
char = ;code = 32
char = a;code = 97
char = l;code = 108
char = c;code = 99
char = o;code = 111
char = h;code = 104
char = o;code = 111
char = l;code = 108
char = -;code = 45
char = i;code = 105
char = n;code = 110
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = r;code = 114
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = u;code = 117
char = r;code = 114
char = b;code = 98
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ,;code = 44
char = ;code = 32
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = d;code = 100
char = u;code = 117
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = f;code = 102
char = a;code = 97
char = v;code = 118
char = o;code = 111
char = r;code = 114
char = a;code = 97
char = b;code = 98
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = r;code = 114
char = g;code = 103
char = e;code = 101
char = t;code = 116
char = i;code = 105
char = c;code = 99
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = e;code = 101
char = c;code = 99
char = u;code = 117
char = l;code = 108
char = a;code = 97
char = r;code = 114
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = c;code = 99
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result sql = Antimicrobial preservatives (e.g., benzyl alcohol), which are required in multidose formulations, can induce protein aggregation. In this study, the mechanism of benzyl alcohol-induced aggregation of recombinant human granulocyte colony-stimulating factor (rhGCSF) was investigated by determining the effects of temperature, pH, and sucrose on this process. rhGCSF was incubated at 25 and 37 degrees C and at pH 7.0 (phosphate-buffered saline, PBS) and pH 3.5 (HCl). Benzyl alcohol (0.9% w/v) accelerated aggregation of rhGCSF at pH 7.0, an effect that was much greater at 37 degrees C than at 25 degrees C and partially counteracted by 1.0 M sucrose. At pH 3.5, benzyl alcohol did not induce aggregation of rhGCSF. Spectroscopic studies showed that 0.9% benzyl alcohol altered the tertiary structure of rhGCSF at both pH, without detectably altering secondary structure. Structural perturbation was greater at 37 degrees C than at 25 degrees C. At both pH 7.0 and 3.5, the hydrogen-deuterium (H-D) exchange rate for rhGCSF was increased by 0.9% benzyl alcohol. Sucrose (1.0 M) partially counteracted the benzyl alcohol-induced perturbation of tertiary structure and the increase in H-D exchange rate. Thus, benzyl alcohol accelerates aggregation of rhGCSF at pH 7.0, because it favors partially unfolded aggregation-prone conformations of the protein. Sucrose partially counteracts benzyl alcohol-induced rhGCSF aggregation by shifting the molecular population away from these species and towards more compact conformations. We postulate that the absence of aggregation at pH 3.5, even with benzyl alcohol-induced structural perturbation, is due to the unfavorable energetics of intermolecular interactions (i.e., colloidal stability) between rhGCSF molecules at this pH.

Publication: J Pharm Sci 2006 Jul ;95(7)::1480-97.
Publication Date: 2006 Jul
Study Author(s): Thirumangalathu, Renuka;Krishnan, Sampathkumar;Brems, David N;Randolph, Theodore W;Carpenter, John F;
Institution: Department of Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, University of Colorado, Denver, 80262, USA.
Shortcut link to this study:
Antimicrobial preservatives (e.g., benzyl alcohol), which are required in multidose formulations, can induce protein aggregation. In this study, the mechanism of benzyl alcohol-induced aggregation of recombinant human granulocyte colony-stimulating factor (rhGCSF) was investigated by determining the effects of temperature, pH, and sucrose on this process. rhGCSF was incubated at 25 and 37 degrees C and at pH 7.0 (phosphate-buffered saline, PBS) and pH 3.5 (HCl). Benzyl alcohol (0.9% w/v) accelerated aggregation of rhGCSF at pH 7.0, an effect that was much greater at 37 degrees C than at 25 degrees C and partially counteracted by 1.0 M sucrose. At pH 3.5, benzyl alcohol did not induce aggregation of rhGCSF. Spectroscopic studies showed that 0.9% benzyl alcohol altered the tertiary structure of rhGCSF at both pH, without detectably altering secondary structure. Structural perturbation was greater at 37 degrees C than at 25 degrees C. At both pH 7.0 and 3.5, the hydrogen-deuterium (H-D) exchange rate for rhGCSF was increased by 0.9% benzyl alcohol. Sucrose (1.0 M) partially counteracted the benzyl alcohol-induced perturbation of tertiary structure and the increase in H-D exchange rate. Thus, benzyl alcohol accelerates aggregation of rhGCSF at pH 7.0, because it favors partially unfolded aggregation-prone conformations of the protein. Sucrose partially counteracts benzyl alcohol-induced rhGCSF aggregation by shifting the molecular population away from these species and towards more compact conformations. We postulate that the absence of aggregation at pH 3.5, even with benzyl alcohol-induced structural perturbation, is due to the unfavorable energetics of intermolecular interactions (i.e., colloidal stability) between rhGCSF molecules at this pH.
PMID: 16729274

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