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Ernestina Lecuona
Ernestina Lecuona y Casado (16 January 1882 – 3 September 1951) was a Cuban pianist, music educator and composer.
Life
Ernestina Lecuona y Casado was born in Matanzas into a musical family. Her brother was pianist and composer Ernesto Lecuona. Leo Brouwer, a classical guitarist, was her grandson, and gymnast-political scientist Rafael A. Lecuona, an anti-communist, was her nephew. She studied music at the Centro Asturiano de La Habana and with French teacher Lucía Calderón.
At the age of 15, Lecuona completed her first work Habanera Luisa, which was published widely in Cuba and Spain by Anselmo López in 1897. She gave early music lessons to her brother Ernesto, and in 1936 was invited to New York City by the Pan American Union, where she accompanied the Mexican tenor Tito Guizar. She made contact with singer Jessica Dragonette, who added some of Lecuona's works to her repertoire.
In 1937 she founded a women's orchestra in Cuba, which debuted at the Teatro Alkazar, and in 1938 performed in concerts at the National Theatre. In 1939 she toured Mexico, Chile and Argentina and in 1940-42 traveled to South America again. She traveled with her brother on tour, and sometimes played with him as a duo for four hands at radio stations and concert venues including Carnegie Hall in 1948. She died in Havana.
Works
Selected works include:
Bolero
Amarte es mi destino
Anhelo besarte
Mi sueño eres tú
Mi vida es soñar
No lo dudes
¿Por qué me dejaste?
Te has cansado de mi amor
ú serás en mis noches
Tus besos de pasión
Ya que te vas
Canción-bolero
Ahora que eres mío
Te has cansado de mi amor
Canción
References
External links
"Ahora que eres mia" by Ernestina Lecuona - performed by Juan Arvizu from YouTube
Category:1882 births
Category:1951 deaths
Category:19th-century classical composers
Category:20th-century classical composers
Category:Cuban classical composers
Category:Cuban music educators
Category:Female classical composers
Category:People from Matanzas
Category:Cuban pianists
Category:Cuban women pianists
Category:20th-century women musicians
Category:19th-century Cuban educators
Category:20th-century Cuban educators
Category:19th-century Cuban musicians
Category:20th-century Cuban musicians
Category:19th-century women musicians
Category:20th-century pianists
Category:Women music educators |
#### suite/funcs_1/triggers/triggers_03.inc
#======================================================================
#
# Trigger Tests
# (test case numbering refer to requirement document TP v1.1)
#======================================================================
# WL#4084: enable disabled parts. 2007-11-15, hhunger
# This test cannot be used for the embedded server because we check here
# privilgeges.
--source include/not_embedded.inc
USE test;
--source suite/funcs_1/include/tb3.inc
--disable_abort_on_error
###########################################
################ Section 3.5.3 ############
# Check for the global nature of Triggers #
###########################################
# General setup to be used in all testcases of 3.5.3
let $message= Testcase 3.5.3:;
--source include/show_msg.inc
--disable_warnings
drop database if exists priv_db;
--enable_warnings
create database priv_db;
use priv_db;
--replace_result $engine_type <engine_to_be_used>
eval create table t1 (f1 char(20)) engine= $engine_type;
create User test_noprivs@localhost;
set password for test_noprivs@localhost = password('PWD');
create User test_yesprivs@localhost;
set password for test_yesprivs@localhost = password('PWD');
#Section 3.5.3.1 / 3.5.3.2
# Test case: Ensure TRIGGER privilege is required to create a trigger
#Section 3.5.3.3 / 3.5.3.4
# Test case: Ensure that root always has the TRIGGER privilege.
# OMR - No need to test this since SUPER priv is an existing one and not related
# or added for triggers (TP 2005-06-06)
#Section 3.5.3.5 / 3.5.3.6
# Test case: Ensure that the TRIGGER privilege is required to drop a trigger.
let $message= Testcase 3.5.3.2/6:;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant ALL on *.* to test_noprivs@localhost;
revoke TRIGGER on *.* from test_noprivs@localhost;
show grants for test_noprivs@localhost;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
# Adding the minimal priv to be able to set to the db
grant SELECT on priv_db.t1 to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (no_privs,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
let $message= Testcase 3.5.3.2:;
--source include/show_msg.inc
connection no_privs;
select current_user;
use priv_db;
--error ER_TABLEACCESS_DENIED_ERROR
create trigger trg1_1 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.2_1-no';
connection default;
use priv_db;
insert into t1 (f1) values ('insert 3.5.3.2-no');
select f1 from t1 order by f1;
connection yes_privs;
select current_user;
use priv_db;
create trigger trg1_2 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.2_2-yes';
connection default;
select current_user;
use priv_db;
--error ER_COLUMNACCESS_DENIED_ERROR
insert into t1 (f1) values ('insert 3.5.3.2-yes');
select f1 from t1 order by f1;
grant UPDATE on priv_db.t1 to test_yesprivs@localhost;
insert into t1 (f1) values ('insert 3.5.3.2-yes');
select f1 from t1 order by f1;
let $message= Testcase 3.5.3.6:;
--source include/show_msg.inc
connection no_privs;
use priv_db;
--error ER_TABLEACCESS_DENIED_ERROR
drop trigger trg1_2;
connection default;
use priv_db;
insert into t1 (f1) values ('insert 3.5.3.6-yes');
select f1 from t1 order by f1;
connection yes_privs;
use priv_db;
drop trigger trg1_2;
connection default;
use priv_db;
insert into t1 (f1) values ('insert 3.5.3.6-no');
select f1 from t1 order by f1;
# Cleanup
--disable_warnings
connection default;
--error 0, ER_TRG_DOES_NOT_EXIST
drop trigger trg1_2;
disconnect no_privs;
disconnect yes_privs;
--enable_warnings
#Section 3.5.3.7
# Test case: Ensure that use of the construct "SET NEW. <column name> = <value>"
# fails at CREATE TRIGGER time, if the current user does not have the
# UPDATE privilege on the column specified
# --- 3.5.3.7a - Privs set on a global level
let $message=Testcase 3.5.3.7a:;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant ALL on *.* to test_noprivs@localhost;
revoke UPDATE on *.* from test_noprivs@localhost;
show grants for test_noprivs@localhost;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER, UPDATE on *.* to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (no_privs_424a,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_424a,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection no_privs_424a;
select current_user;
use priv_db;
show grants;
select f1 from t1 order by f1;
create trigger trg4a_1 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.7-1a';
connection default;
--error ER_COLUMNACCESS_DENIED_ERROR
insert into t1 (f1) values ('insert 3.5.3.7-1a');
select f1 from t1 order by f1;
drop trigger trg4a_1;
connection yes_privs_424a;
use priv_db;
select current_user;
show grants;
create trigger trg4a_2 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.7-2a';
connection default;
insert into t1 (f1) values ('insert 3.5.3.7-2b');
select f1 from t1 order by f1;
# Cleanup
--disable_warnings
drop trigger trg4a_2;
disconnect no_privs_424a;
disconnect yes_privs_424a;
--enable_warnings
# --- 3.5.3.7b - Privs set on a database level
let $message= Testcase 3.5.3.7b:;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant TRIGGER on *.* to test_noprivs;
grant ALL on priv_db.* to test_noprivs@localhost;
revoke UPDATE on priv_db.* from test_noprivs@localhost;
show grants for test_noprivs;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
grant UPDATE on priv_db.* to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (no_privs_424b,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_424b,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
connection no_privs_424b;
show grants;
use priv_db;
create trigger trg4b_1 before UPDATE on t1 for each row
set new.f1 = 'trig 3.5.3.7-1b';
connection default;
insert into t1 (f1) values ('insert 3.5.3.7-1b');
select f1 from t1 order by f1;
update t1 set f1 = 'update 3.5.3.7-1b' where f1 = 'insert 3.5.3.7-1b';
select f1 from t1 order by f1;
drop trigger trg4b_1;
connection yes_privs_424b;
show grants;
use priv_db;
create trigger trg4b_2 before UPDATE on t1 for each row
set new.f1 = 'trig 3.5.3.7-2b';
connection default;
insert into t1 (f1) values ('insert 3.5.3.7-2b');
select f1 from t1 order by f1;
update t1 set f1 = 'update 3.5.3.7-2b' where f1 = 'insert 3.5.3.7-2b';
select f1 from t1 order by f1;
# Cleanup
--disable_warnings
drop trigger trg4b_2;
disconnect no_privs_424b;
disconnect yes_privs_424b;
--enable_warnings
# --- 3.5.3.7c - Privs set on a table level
let $message= Testcase 3.5.3.7c;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant TRIGGER on *.* to test_noprivs@localhost;
grant ALL on priv_db.t1 to test_noprivs@localhost;
revoke UPDATE on priv_db.t1 from test_noprivs@localhost;
show grants for test_noprivs;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
grant UPDATE on priv_db.t1 to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (no_privs_424c,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_424c,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
connection no_privs_424c;
show grants;
use priv_db;
create trigger trg4c_1 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.7-1c';
connection default;
insert into t1 (f1) values ('insert 3.5.3.7-1c');
select f1 from t1 order by f1;
drop trigger trg4c_1;
connection yes_privs_424c;
show grants;
use priv_db;
create trigger trg4c_2 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.7-2c';
connection default;
insert into t1 (f1) values ('insert 3.5.3.7-2c');
select f1 from t1 order by f1;
# Cleanup
--disable_warnings
drop trigger trg4c_2;
disconnect no_privs_424c;
disconnect yes_privs_424c;
--enable_warnings
# --- 3.5.3.7d - Privs set on a column level
--disable_query_log
let $message= Testcase 3.5.3.7d:;
--enable_query_log
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant TRIGGER on *.* to test_noprivs@localhost;
# There is no ALL privs on the column level
grant SELECT (f1), INSERT (f1) on priv_db.t1 to test_noprivs@localhost;
show grants for test_noprivs;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
grant UPDATE (f1) on priv_db.t1 to test_yesprivs@localhost;
show grants for test_noprivs;
connect (no_privs_424d,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_424d,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
connection no_privs_424d;
show grants;
use priv_db;
create trigger trg4d_1 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.7-1d';
connection default;
insert into t1 (f1) values ('insert 3.5.3.7-1d');
select f1 from t1 order by f1;
drop trigger trg4d_1;
connection yes_privs_424d;
show grants;
use priv_db;
create trigger trg4d_2 before INSERT on t1 for each row
set new.f1 = 'trig 3.5.3.7-2d';
connection default;
insert into t1 (f1) values ('insert 3.5.3.7-2d');
select f1 from t1 order by f1;
# Cleanup
--disable_warnings
drop trigger trg4d_2;
disconnect no_privs_424d;
disconnect yes_privs_424d;
--enable_warnings
#Section 3.5.3.8
# Test case: Ensure that use of the construct "SET <target> = NEW. <Column name>" fails
# at CREATE TRIGGER time, if the current user does not have the SELECT privilege
# on the column specified.
# --- 3.5.3.8a - Privs set on a global level
let $message= Testcase 3.5.3.8a:;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant ALL on *.* to test_noprivs@localhost;
revoke SELECT on *.* from test_noprivs@localhost;
show grants for test_noprivs@localhost;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER, SELECT on *.* to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (no_privs_425a,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_425a,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
connection no_privs_425a;
select current_user;
use priv_db;
show grants;
create trigger trg5a_1 before INSERT on t1 for each row
set @test_var = new.f1;
connection default;
set @test_var = 'before trig 3.5.3.8-1a';
select @test_var;
insert into t1 (f1) values ('insert 3.5.3.8-1a');
select @test_var;
drop trigger trg5a_1;
connection yes_privs_425a;
use priv_db;
select current_user;
show grants;
create trigger trg5a_2 before INSERT on t1 for each row
set @test_var= new.f1;
connection default;
set @test_var= 'before trig 3.5.3.8-2a';
select @test_var;
insert into t1 (f1) values ('insert 3.5.3.8-2a');
select @test_var;
# Cleanup
--disable_warnings
drop trigger trg5a_2;
disconnect no_privs_425a;
disconnect yes_privs_425a;
--enable_warnings
# --- 3.5.3.8b - Privs set on a database level
let $message= Testcase: 3.5.3.8b;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant TRIGGER on *.* to test_noprivs@localhost;
grant ALL on priv_db.* to test_noprivs@localhost;
revoke SELECT on priv_db.* from test_noprivs@localhost;
show grants for test_noprivs@localhost;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
grant SELECT on priv_db.* to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (no_privs_425b,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_425b,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
connection no_privs_425b;
show grants;
use priv_db;
create trigger trg5b_1 before UPDATE on t1 for each row
set @test_var= new.f1;
connection default;
set @test_var= 'before trig 3.5.3.8-1b';
insert into t1 (f1) values ('insert 3.5.3.8-1b');
select @test_var;
update t1 set f1= 'update 3.5.3.8-1b' where f1 = 'insert 3.5.3.8-1b';
select @test_var;
drop trigger trg5b_1;
connection yes_privs_425b;
show grants;
use priv_db;
create trigger trg5b_2 before UPDATE on t1 for each row
set @test_var= new.f1;
connection default;
set @test_var= 'before trig 3.5.3.8-2b';
insert into t1 (f1) values ('insert 3.5.3.8-2b');
select @test_var;
update t1 set f1= 'update 3.5.3.8-2b' where f1 = 'insert 3.5.3.8-2b';
select @test_var;
# Cleanup
--disable_warnings
drop trigger trg5b_2;
disconnect no_privs_425b;
disconnect yes_privs_425b;
--enable_warnings
# --- 3.5.3.8c - Privs set on a table level
let $message= Testcase 3.5.3.8c:;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant TRIGGER on *.* to test_noprivs@localhost;
grant ALL on priv_db.t1 to test_noprivs@localhost;
revoke SELECT on priv_db.t1 from test_noprivs@localhost;
show grants for test_noprivs@localhost;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
grant SELECT on priv_db.t1 to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (no_privs_425c,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_425c,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
connection no_privs_425c;
show grants;
use priv_db;
create trigger trg5c_1 before INSERT on t1 for each row
set @test_var= new.f1;
connection default;
set @test_var= 'before trig 3.5.3.8-1c';
insert into t1 (f1) values ('insert 3.5.3.8-1c');
select @test_var;
drop trigger trg5c_1;
connection yes_privs_425c;
show grants;
use priv_db;
create trigger trg5c_2 before INSERT on t1 for each row
set @test_var= new.f1;
connection default;
set @test_var='before trig 3.5.3.8-2c';
insert into t1 (f1) values ('insert 3.5.3.8-2c');
select @test_var;
# Cleanup
--disable_warnings
drop trigger trg5c_2;
disconnect no_privs_425c;
disconnect yes_privs_425c;
--enable_warnings
# --- 3.5.3.8d - Privs set on a column level
let $message=Testcase: 3.5.3.8d:;
--source include/show_msg.inc
revoke ALL PRIVILEGES, GRANT OPTION FROM test_noprivs@localhost;
grant TRIGGER on *.* to test_noprivs@localhost;
# There is no ALL prov on the column level
grant UPDATE (f1), INSERT (f1) on priv_db.t1 to test_noprivs@localhost;
show grants for test_noprivs@localhost;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
grant SELECT (f1) on priv_db.t1 to test_yesprivs@localhost;
show grants for test_noprivs@localhost;
connect (no_privs_425d,localhost,test_noprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connect (yes_privs_425d,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection default;
connection no_privs_425d;
show grants;
use priv_db;
create trigger trg5d_1 before INSERT on t1 for each row
set @test_var= new.f1;
connection default;
set @test_var='before trig 3.5.3.8-1d';
insert into t1 (f1) values ('insert 3.5.3.8-1d');
select @test_var;
drop trigger trg5d_1;
connection yes_privs_425d;
show grants;
use priv_db;
create trigger trg5d_2 before INSERT on t1 for each row
set @test_var= new.f1;
connection default;
set @test_var='before trig 3.5.3.8-2d';
insert into t1 (f1) values ('insert 3.5.3.8-2d');
select @test_var;
# Cleanup 3.5.3.8
--disable_warnings
drop trigger trg5d_2;
--enable_warnings
# --- 3.5.3.x to test for trigger definer privs in the case of trigger
# actions (insert/update/delete/select) performed on other
# tables.
let $message=Testcase: 3.5.3.x:;
--source include/show_msg.inc
use priv_db;
--disable_warnings
drop table if exists t1;
drop table if exists t2;
--enable_warnings
--replace_result $engine_type <engine_to_be_used>
eval create table t1 (f1 int) engine= $engine_type;
--replace_result $engine_type <engine_to_be_used>
eval create table t2 (f2 int) engine= $engine_type;
revoke ALL PRIVILEGES, GRANT OPTION FROM test_yesprivs@localhost;
grant TRIGGER on *.* to test_yesprivs@localhost;
grant SELECT, UPDATE on priv_db.t1 to test_yesprivs@localhost;
grant SELECT on priv_db.t2 to test_yesprivs@localhost;
show grants for test_yesprivs@localhost;
connect (yes_353x,localhost,test_yesprivs,PWD,test,$MASTER_MYPORT,$MASTER_MYSOCK);
connection yes_353x;
select current_user;
use priv_db;
create trigger trg1 before insert on t1 for each row
insert into t2 values (new.f1);
connection default;
use priv_db;
insert into t1 (f1) values (4);
revoke SELECT on priv_db.t2 from test_yesprivs@localhost;
grant INSERT on priv_db.t2 to test_yesprivs@localhost;
insert into t1 (f1) values (4);
select f1 from t1 order by f1;
select f2 from t2 order by f2;
connection yes_353x;
use priv_db;
drop trigger trg1;
create trigger trg2 before insert on t1 for each row
update t2 set f2=new.f1-1;
connection default;
use priv_db;
insert into t1 (f1) values (2);
revoke INSERT on priv_db.t2 from test_yesprivs@localhost;
grant UPDATE on priv_db.t2 to test_yesprivs@localhost;
insert into t1 (f1) values (2);
select f1 from t1 order by f1;
select f2 from t2 order by f2;
connection yes_353x;
use priv_db;
drop trigger trg2;
create trigger trg3 before insert on t1 for each row
select f2 into @aaa from t2 where f2=new.f1;
connection default;
use priv_db;
insert into t1 (f1) values (1);
revoke UPDATE on priv_db.t2 from test_yesprivs@localhost;
grant SELECT on priv_db.t2 to test_yesprivs@localhost;
insert into t1 (f1) values (1);
select f1 from t1 order by f1;
select f2 from t2 order by f2;
select @aaa;
connection yes_353x;
use priv_db;
drop trigger trg3;
create trigger trg4 before insert on t1 for each row
delete from t2;
connection default;
use priv_db;
insert into t1 (f1) values (1);
revoke SELECT on priv_db.t2 from test_yesprivs@localhost;
grant DELETE on priv_db.t2 to test_yesprivs@localhost;
insert into t1 (f1) values (1);
select f1 from t1 order by f1;
select f2 from t2 order by f2;
# Cleanup 3.5.3
--disable_warnings
drop database if exists priv_db;
drop user test_yesprivs@localhost;
drop user test_noprivs@localhost;
drop user test_noprivs;
--enable_warnings
use test;
drop table tb3;
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g truck mounted spreaders. Our snow plowing service in Monmouth County is performed by experienced operators, many of whom have provided commercial snow plowing service to Monmouth County for over a decade. When considering a snow plowing service or commercial snow removal service be sure you are confident in the snow plowing service provider. We treat our customers the way they deserve to be treated and we are proud of the amount of clients we retain year after year.Snow plowing…
…and Rack/Grain bodies that can be combined with a large selection of snow and ice removal equipment including snow plows, spreaders and a wide range of other vehicle accessories Our many years of experience…
Members of the Smithfield Town Council, The Town of Smithfield and the Northern Rhode Island Chamber of Commerce gathered for the ceremonial groundbreaking of a 45,000 square foot facility and the new home of Dejan…
Traffic Safety & Equipment Co Our business has expanded to include divisions for traffic safety products, signs and graphics and snowplows and spreaders 44 years, 3 generations, and thousands of satisfied…
Members of the Smithfield Town Council, The Town of Smithfield and the Northern Rhode Island Chamber of Commerce gathered for the ceremonial groundbreaking of a 45,000 square foot facility and the new home of Dejan…
Traffic Safety & Equipment Co Our business has expanded to include divisions for traffic safety products, signs and graphics and snowplows and spreaders 44 years, 3 generations, and thousands of satisfied…
For over 70 years, Certified Products Inc. has offered our customer quality products and services. Through out the years, we have established a prosperous relationship with key government agencies and private businesses… |
Even as California leads the country by signing up uninsured people, Covered California has stumbled by failing adequately to extend coverage to Latinos, the largest uninsured population in the state.
Covered California executive director Peter Lee’s persistent efforts to “accentuate the positive” and “eliminate the negative,” as the song goes, should not mask that hard reality. The first major deadline for coverage was Dec. 31. California’s target for overall enrollment was 611,000; the number enrolled was 498,794.
Exchange officials expect a surge of enrollment by March 31, the end of the open enrollment period for this year. A surge probably will occur. But to reach the target of 1.3 million, more than 800,000 people will have to sign up. That means the state must do a better job of reaching Latinos, who account for 59 percent of the state’s uninsured population.
Critics have pointed to technical issues, such as the Spanish-language version of the website not working until late November, and lack of a Spanish-language paper application until late December.
These startup problems undoubtedly will be repaired for the 25 percent of Latinos who are Spanish-language dominant and generally older.
That alone won’t solve the problem: 58 percent of California Latinos are bilingual and 17 percent speak English-only, according to the health exchange’s 2012 marketing plan. The exchange has boosted spending on television and radio ads in English and Spanish.
But mending the website and doing more advertising is no substitute for addressing the real issue, which is to connect with target communities in person. Word of mouth from trusted sources matters more than ads, especially among people who never have had insurance and don’t use the Internet.
Further, in a state with a long tradition of immigration, a major issue is that many people come from “mixed-status” immigration families. Nearly half of California’s immigrants are U.S. citizens; 26 percent have other legal status, including green cards and visas.
In-person help is needed to work through these complexities.
The dearth of boots on the ground is reflected in 40-minute average wait times at call centers. It also is reflected in the low numbers of enrollment counselors. Covered California originally had plans to train 20,000 enrollment counselors by Dec. 31; the actual number was 3,000.
As a group, Latinos are younger and healthier than the general population. The state needs Latino enrollment to make the new insurance pool work as intended, with a mix of old and young, healthy and sick people. |
Snowman Christmas Countdown Figurine - Personalized
Snowman Christmas Countdown Figurine - Personalized
Item# BLPC378
Price: $44.00
Sale Price: $64.00
You Save: $-20.00 (-45%)
Name or Message:
Gift Message
We’re sorry, this item is sold out! Thank you.
Here's the perfect remedy for the never-ending question, "How many days till Christmas?" Our dapper Snowman Christmas Countdown Figurine - Personalized holds a sign with number blocks that kids can turn every day as the holiday approaches.
*We are sorry but gift wrapping or gift card is not available for this item.
Made of resin.
Measures 15" H x 6" W.
We personalize the base with any 1 line message up to 15 characters.
Designer: Gifts by Pepper Carrie
Shipping Time: This item is custom made especially for you upon order. Please allow 4 to 7 days to ship out. |
1. Field of the Invention
The present invention relates to surface acoustic wave (SAW) touch sensitive technology, and more particularly, to apparatus and method for determining SAW touch sensitive area size.
2. Description of the Prior Art
Touch screen is an important human-machine interface of modern electronic product. It is widely adopted in various consumer electronics, such as smartphone, tablet computer, notebook computer, and etc. Touch screen can comprise but not limit to the following types: resistive, matrix resistive, capacitive, projected capacitive, electromagnetic sensing, infra-red sensing, and in-cell. The present invention relates to surface acoustic wave (SAW) touch sensitive technology, especially related to apparatus and method for determining SAW touch area size.
Usually, a touch screen comprises a display module and a sensor module stacked on the display module. User may use one part of human body such as finger or stylus to touch or approximate the sensor module. A processing device of the touch screen receives sensing information from the sensor module, accordingly. The working principle of SAW touch sensitive technology relies on the propagation of acoustic wave on the surface of object. In case another object touches the surface, the acoustic wave propagation is disrupted. The disruptive event is detected and used to determine the coordinates of touch.
The object providing surface mentioned above is usually a glass substrate such that user can see the underlying display module though the transparent glass substrate. The SAW is propagated on the surface of the glass substrate. Since the propagation speed on the surface of the glass substrate is constant, it is possible to calculate the coordinates where the object touches the surface according to the signal change measured and the constant wave propagation speed. In other words, the processing device coupled to the sensor module has to know parameters of SAW touch panel in advanced. Thus, the coordinate values can be derived accordingly.
Usually, one system vendor has more than one brand customer. Each brand customer may order more than one type of product. Each product may feature different sizes. For example, one brand may sell several All-In-One computers covering every aspect of market. They may comprise 10 to 12 inch light weight computers, 17 to 19 inch mainstream computers, and 20 inch high level computers. No matter their sizes, these products featuring different sizes of touch screen usually use a common processing device in considerations of design, purchase, manufacturing, inventory, and maintenance. The manufacture vendor of this common processing device is usually different to the system vendor and the brands. As mentioned above, the processing device coupled to different sized SAW sensor modules must be configured in order to connect these sensor modules.
From the view point of commerce, if the manufacture vendor of the processing device can provide a product which is capable of automatically detecting the size of SAW sensor module, the configuration step can be omitted accordingly. Thus, the manufacture sequence, cost, and time are also reduced as a result. Naturally, it enhances the competence of the processing device which is capable of automatic detecting the size of SAW sensor module. There is impossible to delay system integration due to configuration errors. Hence, there is a need of processing device and method for automatically detecting the size of SAW sensor module in the market in order to reduce the tool, cost, and time for parameter configuration.
From the above it is clear that prior art still has shortcomings. In order to solve these problems, efforts have long been made in vain, while ordinary products and methods offering no appropriate structures and methods. Thus, there is a need in the industry for a novel technique that solves these problems. |
Q:
Vivado simulation stuck at 0 fs
I am trying to simulate a D flip flop using Vivado 2018.2.2.
But upon running the simulation a window pops up stating Current time: 0 fs. The program doesn't freeze, it just doesn't progress. Here is the code:
LIBRARY IEEE;
USE IEEE.std_logic_1164.ALL;
ENTITY Dff IS
port (d, clk, rst: in std_logic;
q : out std_logic);
END ENTITY Dff;
ARCHITECTURE behav OF Dff IS
BEGIN
main : PROCESS
BEGIN
IF rst='1' THEN
q <= '0';
ELSIF rising_edge(clk) THEN
q <= d;
END IF;
END PROCESS main;
END ARCHITECTURE behav;
And the test bench:
LIBRARY IEEE;
USE IEEE.std_logic_1164.all;
ENTITY Dff_tb IS
END Dff_tb;
ARCHITECTURE behav OF Dff_tb IS
CONSTANT T : time := 10 ns;
CONSTANT N : INTEGER := 3;
COMPONENT Dff
PORT(
d : IN std_logic;
clk : IN std_logic;
rst : IN std_logic;
q : OUT std_logic
);
END COMPONENT;
SIGNAL d : std_logic := '0';
SIGNAL clk : std_logic := '0';
SIGNAL rst : std_logic := '0';
SIGNAL q : std_logic;
SIGNAL sim_data : std_logic_vector (N downto 0) := "0011";
BEGIN
Dff_0 : Dff PORT MAP (d => d, clk => clk, rst=>rst, q => q);
clk_pr : PROCESS
BEGIN
clk <= '0';
WAIT FOR T/2;
clk <= '1';
WAIT FOR T/2;
END PROCESS clk_pr;
main_pr : PROCESS
VARIABLE i : INTEGER := 0;
BEGIN
rst <= '1';
wait for T*2;
rst <= '0';
d <= '0';
wait for T*2;
rst <= '0';
d <= '1';
wait;
END PROCESS main_pr;
END ARCHITECTURE behav;
I am new to VHDL so It is probably something obvious.
Any help is appreciated.
EDIT:
Following a comment, I edited my test bench code like this:
LIBRARY IEEE;
USE IEEE.std_logic_1164.all;
ENTITY Dff_tb IS
END Dff_tb;
ARCHITECTURE behav OF Dff_tb IS
CONSTANT T : time := 10 ns;
CONSTANT N : INTEGER := 3;
COMPONENT Dff
PORT(
d : IN std_logic;
clk : IN std_logic;
rst : IN std_logic;
q : OUT std_logic
);
END COMPONENT;
SIGNAL d : std_logic := '0';
SIGNAL clk : std_logic := '0';
SIGNAL rst : std_logic := '0';
SIGNAL q : std_logic;
SIGNAL sim_data : std_logic_vector (N downto 0) := "0011";
SHARED VARIABLE sim_end : boolean := false;
BEGIN
Dff_0 : Dff PORT MAP (d => d, clk => clk, rst=>rst, q => q);
clk_pr : PROCESS
BEGIN
IF sim_end = false THEN
clk <= '0';
WAIT FOR T/2;
clk <= '1';
WAIT FOR T/2;
ELSE
WAIT;
END IF;
END PROCESS clk_pr;
main_pr : PROCESS
VARIABLE i : INTEGER := 0;
BEGIN
rst <= '1';
wait for T*2;
rst <= '0';
d <= '0';
wait for T*2;
rst <= '0';
d <= '1';
sim_end := true;
wait;
END PROCESS main_pr;
END ARCHITECTURE behav;
But the problem stil persists.
A:
EDIT: Besides the problem mentioned below, I found the problem in the Dff entity itself (I was focused on the testbench, sorry for that): The main process hasn't any wait statement of any sort. Either add a sensitivity list to main process:
main : PROCESS (clk, rst)
Or add a wait statement inside main process:
wait on clk, rst;
That should fix it.
Your simulation doesn't terminate since the clk_pr process will run forever. Here's a simple solution:
--- clock generator
clk <= not clk after 50 ns when finished /= '1' else '0';
--- rest of your test bench
main_pr : PROCESS
--- test code
--- stop clock
finished <= '1';
wait;
END PROCESS;
Of course you can replace 50 ns with the constant you use, remember to add a finished signal to the test bench initialized to '0'.
|
Elementary school children's knowledge and intended behavior toward hearing conservation.
The purposes of the study were to investigate children's knowledge about hearing conservation, the types of protective behaviors they would adopt in noise, the agreement between children's knowledge and intended behaviors in hearing protection, and reasons why they would not take any protective action in noise. A questionnaire was administered to 479 fourth and fifth graders in their school classrooms. Results indicated that children scored low (62.0%) on this hearing conservation questionnaire. They scored the highest in strategies of hearing protection (69.9%), followed by their knowledge in general hearing health (62.6%) and noise hazards (49.6%). Only 55% of children knew that hearing protective devices could protect them against noise. Approximately 28% of children did not intend to adopt any protective behavior in noise and the major reason for this was lack of knowledge. Children's knowledge and their noise protective behavior were correlated ( P < .05). However, possessing knowledge did not guarantee that children would adopt such behaviors when they were exposed to loud sounds. It is important to increase children's knowledge about hearing protection and hazardous noise as well as to encourage actual protective actions. |
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Daw Aung San Suu Kyi's Congressional Gold Medal
Wed, 09/19/2012 - 20:32 — ub
News
US Rep. Joe Crowley (D-Queens, the Bronx), a leader in the House on foreign affairs and Burma, delivered remarks as a featured speaker at today’s Congressional Gold Medal ceremony honoring recently released Nobel Peace Prize recipient and leader of Burma’s democracy movement Aung San Suu Kyi. Crowley spearheaded legislation awarding Daw Suu Kyi with the Congressional Gold Medal – the highest civilian award bestowed by the U.S. Congress.
Below are Rep. Crowley’s remarks as prepared for delivery. Click here to watch his speech.
“Thank you Speaker Boehner and Father Conroy. Thank you to all of my House colleagues, in particular Leader Pelosi, Whip Hoyer, and Representative Manzullo, as well as the distinguished Senators with us, including Leader McConnell, Senator Feinstein and Senator McCain.
“Mrs. Bush and Madam Secretary – our thanks and appreciation to both of you for not only taking the time to be here today, but for your many contributions to this effort and your commitment to advancing the cause of freedom and democracy in Burma.
“I would be remiss if I didn’t also mention someone who is not here with us today, Congressman Tom Lantos. Tom, his wife Annette, and his staff were mentors to me and all worked so hard on Burma for so many years. I wish he was here today to share this moment in history with us, because I think he’d agree:
“Today is an amazing day! It’s an incredible day!
“Who would have thought that when this bill was introduced in the House in 2008, when Daw Aung San Suu Kyi was still under house arrest, that in a few short years she would be standing here with us, on U.S. soil receiving this honor – as a Member of the Burmese Parliament?
“Back then, we thought about granting the Medal in abstentia – which may have made her the only person in history to receive the Congressional Gold Medal while in detention. Who would have imagined this change was possible? Who would have thought? Well, let me tell you who: Daw Aung San Suu Kyi.
“She might be too humble to admit it, but I know she always thought this day, this moment, would be possible. Not because she is someone who worries about awards and honors – because I can tell you she does not.
“She believed it because she and the Burmese people always believed in change. They hoped, they fought, they knew change must come to their country.
“She knew the Burmese people yearned for human rights and, most importantly, deserved democratic governance. She stoked the flames in a peaceful way for lasting change, even amongst those already in a position of power. Her efforts have helped lead us here today.
“There has been a lot of advancements made in Burma over the last year or two. And, we must recognize and give thanks to those who have had the courage to help lead and support the changes, including those in the government.
“But, we also must honor and remember those who have made great sacrifices – imprisonment, lives lost – to get where we are. Far too many have paid too high a price in the effort to bring about freedom and democratic governance in Burma.
“It is with those people in mind; those who have sacrificed so much, that we acknowledge the work is not done. We must ensure that the momentum unfolds into sustained progress, into permanent freedoms and into a solidified democracy. Because as much as I’d like to believe that the change in Burma is irreversible, as much as I’d like to revel in blind optimism and believe the battle for freedom has been won, it has not.
“The tides of progress can reverse just as easily as they flow if we do not remain vigilant and demand further progress.
“But, let there be no doubt – today is a moment of joy; a moment to honor a genuine hero. Someone who has endured solitude. Someone who has been forced to watch others struggle and suffer. Someone who has put country before self. Someone who has inspired thousands of others to stand up for human rights and for justice. Someone who has given voice to a movement. Someone who has led with unwavering commitment.
“That person is Daw Aung San Suu Kyi, and we are so, so proud to stand here and honor you today.” |
Sheridan reports that the police have not ruled out mechanical failure, but the meticulous planning required to sneak a massive plane with 238 passengers into radar darkness points to human action.
Senior U.S. officials have said that the westward turn that diverted the missing Malaysia airplane from its original path toward Beijing was carried out by a computer that was most likely programmed by someone in the plane's cockpit.
Captain Zaharie Ahmad Shah operated a flight simulator at his home, and files containing records of simulations carried out on the program were deleted on February 3.
Investigators told The Times that evidence from the deleted data includes routes programmed into the machine that took a plane far out into the Indian Ocean and simulated landing a short runway on an island.
Shah, 53, was reportedly unique among others onboard in that he had no recorded social or work commitments after the date of the March 8 flight.
He joined Malaysia Airlines in 1981 and had more than 18,000 hours of flying experience.
“The police investigation is still ongoing. To date no conclusions can be made as to the contributor to the incident and it would be sub judice to say so," a spokesperson for the Malaysian police told the Times. "Nevertheless, the police are still looking into all possible angles.” |
package org.broadinstitute.hellbender.utils.codecs.sampileup;
import htsjdk.samtools.SAMUtils;
import htsjdk.samtools.util.StringUtil;
import htsjdk.tribble.Feature;
import org.apache.commons.lang.ArrayUtils;
import org.broadinstitute.hellbender.utils.Utils;
import java.util.List;
import java.util.stream.Collectors;
import java.util.stream.Stream;
/**
* A tribble feature representing a SAM pileup.
*
* Allows intake of simple mpileups. Simple pileup features will contain only basic information, no reconstructed reads.
*
* @author Danil Gomez-Sanchez (magiDGS)
*/
public class SAMPileupFeature implements Feature {
// genomic location
private final String contig;
private final int position;
// reference base
private final byte refBase;
// list of pileup elements
private final List<SAMPileupElement> pileupElements;
SAMPileupFeature(final String contig, final int position, final byte refBase, final List<SAMPileupElement> pileupElements) {
Utils.nonNull(pileupElements);
this.contig = contig;
this.position = position;
this.refBase = refBase;
this.pileupElements = pileupElements;
}
@Override
@Deprecated
public String getChr() {
return getContig();
}
@Override
public String getContig() {
return contig;
}
@Override
public int getStart() {
return position;
}
@Override
public int getEnd() {
return position;
}
/**
* Returns pile of obseved qualities over the genomic location
*
* Note: this call costs O(n) and allocates fresh array each time
*/
public String getQualsString() {
return SAMUtils.phredToFastq(getBaseQuals());
}
/**
* Returns pile of observed bases over the genomic location.
*
* Note: this call costs O(n) and allocates fresh array each time
*/
public String getBasesString() {
return StringUtil.bytesToString(getBases());
}
/**
* Returns the reference basse
*/
public byte getRef() {
return refBase;
}
/**
* Return the number of observed bases over the genomic location
*/
public int size() {
return pileupElements.size();
}
/**
* Format in a samtools-like string.
* Each line represents a genomic position, consisting of chromosome name, coordinate,
* reference base, read bases and read qualities
*/
public String getPileupString() {
// In the pileup format,
return String.format("%s %s %c %s %s",
getContig(), getStart(), // chromosome name and coordinate
getRef(), // reference base
getBasesString(),
getQualsString());
}
/**
* Gets the bases in byte array form
*
* Note: this call costs O(n) and allocates fresh array each time
*/
public byte[] getBases() {
final List<Byte> bases = getBasesStream().collect(Collectors.toList());
return ArrayUtils.toPrimitive(bases.toArray(new Byte[bases.size()]));
}
/**
* Gets the PHRED base qualities.
*
* Note: this call costs O(n) and allocates fresh array each time
*/
public byte[] getBaseQuals() {
final List<Byte> quals = getQualsStream().collect(Collectors.toList());
return ArrayUtils.toPrimitive(quals.toArray(new Byte[quals.size()]));
}
/**
* Get the bases as a stream
*/
public Stream<Byte> getBasesStream() {
return pileupElements.stream().map(SAMPileupElement::getBase);
}
/**
* Get the qualities as a stream
*/
public Stream<Byte> getQualsStream() {
return pileupElements.stream().map(SAMPileupElement::getBaseQuality);
}
}
|
{
"id":"35291671",
"url":"http:\/\/collection.cooperhewitt.org\/types\/35291671\/",
"name":"Candy dish\nCandy dish",
"count_objects":"1",
"supersedes":"0",
"superseded_by":"0"
} |
"""Remove deprecated and unused ApiTokens
Revision ID: 6cd62c61bc1a
Revises: d37e30db3df1
Create Date: 2019-10-02 12:05:01.207728
"""
# revision identifiers, used by Alembic.
revision = "6cd62c61bc1a"
down_revision = "d37e30db3df1"
from alembic import op # noqa: E402
import sqlalchemy as sa # noqa: E402
def upgrade():
op.drop_table("apitoken")
def downgrade():
op.create_table(
"apitoken",
sa.Column("id", sa.INTEGER(), autoincrement=True, nullable=False),
sa.Column("user_id", sa.INTEGER(), autoincrement=False, nullable=False),
sa.Column("token", sa.VARCHAR(length=255), autoincrement=False, nullable=False),
sa.Column("secret", sa.VARCHAR(length=255), autoincrement=False, nullable=False),
sa.ForeignKeyConstraint(["user_id"], ["user.id"], name="apitoken_user_id_fkey"),
sa.PrimaryKeyConstraint("id", name="apitoken_pkey"),
sa.UniqueConstraint("secret", name="apitoken_secret_key"),
sa.UniqueConstraint("token", name="apitoken_token_key"),
)
|
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Stockholm Royal Seaport area transformed in lighting event
On the 26 November in the evening, in cooperation with the City of Stockholm, ÅF Lighting conducted a lighting event in the Norra Djurgårdstaden area. With the help of 130 volunteers, carrying LED torches and filters, the usually dark historic industrial area was lit up and transformed for the evening.
The lighting event in Stockholm Royal Seaport came about as a result of the City of Stockholm winning a free lighting design proposal in a contest organised by ÅF Lighting. Devised to raise public awareness about the importance of light and lighting design, the “Win a Lighting Makeover” contest was part of the official programme for the International Year of Light 2015, organised by the Lighting-Related Organizations under the patronage of UNESCO.
In January 2015, ÅF Lighting called for nominations for public spaces to be enhanced by lighting design. In their winning proposal, the City of Stockholm nominated two historic gas holders in the Royal Seaport area. Here, extensive urban development is taking place, with plans for some 12 000 new homes, 35 000 new work spaces, infrastructure, amenities, and facilities for arts and culture. Situated adjacent to the Frihamnen docks, the area was previously mainly used for gas production and other industry. Its relative proximity to Stockholm City centre, in combination with its location close to water, woods and parkland, means that the development is attracting great interest.
Having discussed various options and lighting scenarios, the parties decided to organise a lighting event where members of the general public would help create temporary installations, in cooperation with professional lighting designers and a photographer. In this way, the group would experience the transformative power of lighting design first hand. The lighting scenarios would be documented with long expose photography and shared with the general public. Using powerful hand held LED torches, filters and a so-called pixel stick, four dramatic and playful lighting scenarios were created.
“I’m delighted with the results. This evening, we created art together”, says Kai Piippo, Head of Design at ÅF Lighting. “Using light, we managed to bring out the soul and the architectural character of these fascinating sites.”
“People love seeing these spectacular buildings up close”, says Staffan Lorentz, Head of Development of Stockholm Royal Seaport. “When we invited the public to register online for the event, it was fully booked almost immediately.” |
package us.codecraft.jobhunter.pipeline;
import org.springframework.stereotype.Component;
import us.codecraft.jobhunter.dao.JobInfoDAO;
import us.codecraft.jobhunter.model.LieTouJobInfo;
import us.codecraft.webmagic.Task;
import us.codecraft.webmagic.pipeline.PageModelPipeline;
import javax.annotation.Resource;
/**
* @author code4crafer@gmail.com
* Date: 13-6-23
* Time: 下午8:56
*/
@Component("JobInfoDaoPipeline")
public class JobInfoDaoPipeline implements PageModelPipeline<LieTouJobInfo> {
@Resource
private JobInfoDAO jobInfoDAO;
@Override
public void process(LieTouJobInfo lieTouJobInfo, Task task) {
jobInfoDAO.add(lieTouJobInfo);
}
}
|
Taiwan Beer
Taiwan Beer (, or ) is a large-market beer brewed by the Taiwan Tobacco and Liquor Corporation (TTL). The brand, an icon of Taiwanese culture, began as a monopoly product but has remained the best-selling beer in the territory in the era of free trade.
History
The company today known as TTL had its origins in a government agency established by Taiwan's Japanese rulers in 1901. The Monopoly Bureau of the Taiwan Governor's Office was responsible for all liquor and tobacco products sold in Taiwan as well as opium, salt, and camphor.
In 1922 the Monopoly Bureau began brewing Takasago Beer through the Takasago Malted Beer Company. Light and dark varieties were produced. The price of Takasago Beer varied widely over the course of its manufacture, depending on the availability of imported Japanese beers in Taiwan and on the contingencies of the economy. As World War II reached its conclusion in the 1940s, matches, petroleum, and standard weights and measures also came under the Monopoly Bureau's authority.
After the end of World War II the incoming Chinese Nationalists preserved the monopoly system for alcohol and tobacco. Production of beer was assigned in 1945 to the Taiwan Provincial Monopoly Bureau. The name Taiwan Beer was adopted in 1946. The following year, production of the beer was assigned to the Taiwan Tobacco and Wine Monopoly Bureau.
In the 1960s locally produced was added to the fermentation process, resulting in the distinctive local flavour for which the beer is known today.
Taiwan entered its modern period of pluralistic democracy in the 1990s. Free trade and open markets became priorities as Taiwan prepared for admission to the World Trade Organization (WTO) in 2002. Laws went into force that year that opened Taiwan's market to competing products. On 2002-07-01 the Monopoly Bureau passed into history. Its successor, the Taiwan Tobacco and Liquor Corporation, is a publicly owned company that competes in the marketplace. TTL introduced a new brew, Taiwan Beer Gold Medal, by the end of its first year. Since then the product line has expanded to include Taiwan Beer Draft (a lager for restaurant sales), two malt beers under the Mine label (Amber and Dark), and four fruit-flavoured beers.
Taiwan Beer remains the island's best-selling beer brand and is one of the most recognized brands in Taiwan's business world.
Beer
Taiwan Beer is an amber lager beer with a distinct taste produced by the addition of locally produced ponlai rice ("Formosa rice" 蓬萊米) during the fermentation process. Like all large-market beers, the original Taiwan Beer brew is filtered and pasteurized. It is served cold and best complements Taiwanese and Japanese cuisine, especially seafood dishes such as sushi and sashimi. Taiwan Beer has won international awards, including the International Monde Selection in 1997 and the Brewing Industry International Awards in 2002. Taiwan Beer is mass-produced at the Taiwan Beer Factory (烏日啤酒廠行政大樓前廣場, Wūrì Píjiǔ Chǎng Xíngzhèng Dàlóu qián guǎngchǎng) in Wuri District, Taichung City. It is also brewed on site at the Taiwan Beer Bar in Taipei.
Three lager-style brews bear the label Taiwan Beer. The Original brew is sold in brown bottles with a cream-coloured label and in white cans bearing a blue and green design. The Gold Medal brew, introduced in April 2003, is sold in green bottles and cans that reproduce the white, red and green label seen on the bottle. Original Taiwan beer is 4.5% and Gold is 5% by volume and are regularly seen in Taiwan's convenience and grocery stores. The newest lager, Taiwan Beer 18 days, bears a signature solid green bottle with no paper label; was sold in cans at a later date. Draft appears most often in bars and restaurants, where it is available on tap or from refrigerators. The brew, designed to be sold fresh, is less often seen in stores due to its expiration just 18 days after production. In 2013 more stores began to stock the brew as ads and labels trumpeted its early expiration date.
Taiwan Beer began selling a malt brew in 2008 bearing the Mine label. Brisk sales of Mine, an amber, led to the introduction of Mine Dark two years later. Mine malts are 5% alcohol by volume. Both malts are regularly stocked in Taiwan convenience stores. Bottles wear paper labels whose designs are repeated on cans. Mine sales have reportedly fallen since the launch of the new summer fruit beer brews.
In 2012 Taiwan Beer introduced two new brews with tropical fruit flavours added: mango and pineapple. With 2.8% alcohol content and sweeter flavour, these proved popular with summer drinkers. Two new flavors, grape and orange, were introduced to the line in 2013. The fruit-enhanced brews, like all Taiwan Beer brews, are made available in bottles or cans.
With the increasing popularity of wheat beer such as Kronenbourg Blanc and Hoegaarden in Taiwan, Taiwan Beer launched a wheat beer of their own, Taiwan Weissbier Draft, in late 2013. It is sold in 600ml bottles and 330ml cans in most convenience stores.
Culture
The iconic status of the Taiwan Beer brand in Taiwanese society is reinforced by TTL marketing strategies. Ads feature celebrity endorsements by popular Taiwanese figures such as A-Mei. A basketball team named Taiwan Beer, popularly nicknamed 'The Brew Crew,' is sponsored by the company. The Taiwan Beer Bar and Beer Garden is a popular restaurant/brewery in Taipei. Restaurants and nightspots are also proliferating at the Taiwan Beer Factory in Wuri District, Taichung City. The Factory, near the Wujih station of the Taiwan High Speed Rail, is the site of an annual Taiwan Beer Festival (台灣啤酒節, Táiwān Píjiǔjié) held every summer.
Competition
Taiwan Beer leads its namesake market. Its main large-market competitor is Long Chuan, owned by the Taiwan Tsing Beer Corporation and brewed in Kaohsiung City. Long Chuan launched a range of fruit beers in 2012.
Microbrews, handcrafted beers and other limited-distribution beers represent a separate category. Leading Taiwan artisan beers include Three Giants Brewing Company (巨人啤酒) with a range of Lagers, Pale Ales, IPA's, Wheat Beer and Dark Lager, Redpoint Brewing, which produced the island's first IPA called 台PA,Formosa Bird Beer and Lychee Beer (both by North Taiwan Brewing) along with the house brews served in two locally owned restaurant chains, Jolly Brewery and Restaurant (operated by Great Reliance Food & Beverage) and Le ble d'or.
References
Bibliography
Lin, Jackie. "Beer fight is about politics: TTL." Taipei Times, 2004-07-02.
"2008 Taiwan Beer Festival." Taipei Times, 2008-08-01.
External links
Taiwan Tobacco and Liquor Corporation (TTL) - Official Site
Taiwan Beer - Ads and Features (YouTube)
Review: Taiwan Beer Bar. Taiwan Fun magazine, 2005-08.
Category:Beer in Taiwan
Category:Taiwanese brands |
[Detection of lymphocytotoxic HLA-A, B, C antibodies in the blood of married female blood donors. I. Serological study].
Bleedings from 1.843 blood donors married women have been checked, according to NIH standard technique, against panels of 17-20 samples of lymphocytes from random healthy HLA-A, -B, -C typed individuals of the Turin population. Lymphocytotoxic activity has been found in 209 sera, but only 90 of them showed strong reaction(s) (at least 60-80% of killed lymphocytes) and 20 of these were HLA typing (or easily transformable in HLA typing) sera. Very few sera killed only 20-50% of lymphocytes in some samples and this cytotoxic activity remained even if dilutions as far as 1:16 and, sometimes, 1:32 were made. Bleedings from blood donors married women give a small yield (1%) of ;typing sera, but these sera are easily available and, practically, without problems of quantity. |
That photographic media, in one way or the other, record, depict and represent truth, realities and the past, is a staple. In theory this relation has been called into question, in particular with the advent of digital image manipulation, and the doubts have been extended since to analogue photography, too. Yet despite these doubts the notion of photographs being somewhat true permeates most, if not all, practices with these media: in science and humanities, photographic images replace and represent the object of research; in an ID, the portrait connects a face and a body with a name and other personal data; photographs in family albums and books allow to look back into the past. And though it may have been the reason for recent doubts in photography’s veracity, digital photography thrives on this promise as well: we share meals with our social networks the moment they are served, video telephony lets us talk not only to a voice but a face, and GPS metadata tells us where on Earth we took a certain picture. Moreover, photography is hybridized when the camera in our smartphones becomes a scanner for QR-codes, drones are equipped with face-recognition software, and augmented reality systems transform the material world into a space and surface for digital data.
Under these conditions, photography has ceased to be a specific medium generating still images. It has become a dispositif in the sense of being a network of applications, institutions, materialisations and theoretical settings such as its privileged relation in representing truth – which, looking back, it has always been. The discussions concerning the re-evaluation of photography, however, usually give most attention to individual pictures as products and as depictions. What we would like to focus upon with our next conference are the modes of the technical, optical, chemical and social conditions of pre-, post-, mass and over-production, of the distribution, consumption, circulation and archiving of what is so commonly known as photographs. We welcome in particular submissions concerned with new theoretical and empirical approaches and perspectives on these fields. And we would be delighted to receive papers dealing with rarely researched topics such as photographic optics, photochemistry and the applications of soft- and hardware for generating photorealistic images. We plan to arrange the talks in four panels:
Ça eu été? What photography has been and will become.
What is needed. The material bases of photography
How to use them. Production, dissemination, application and perception of photographic images
What else is new? Photographic practices at the fringes of photography.
Please submit your application, including a short summary of your paper (250-400 words) in English using the following link: https://easychair.org/conferences/?conf=app4 no later than 20 December 2017. Note that you should register at the Easychair website in order to submit your application. There is no participation fee.
We shall consider the possibility of online participation for a limited number of participants. The working languages of the conference are Russian and English. Conference materials are planned for publication in 2018-2019. For programs of After Post-Photography since 2015 and past publications, please see http://www.after-post.photography
We would appreciate it if you would circulate the call to your own networks and other mailing lists. |
NBC will keep Jay Leno five nights a week, but in prime time, competing not with David Letterman, but with shows like “CSI: Miami.”
The network will announce Tuesday that Mr. Leno’s new show will appear at 10 o’clock each weeknight in a format similar to “The Tonight Show,” which he has hosted since 1992.
Five years ago NBC announced that it would hand the job of host of that franchise show to Conan O’Brien in May 2009. Since then the network has maneuvered to try to keep Mr. Leno, who continues to be the late-night ratings leader, fearing that he could leave and start a new late-night show on a competitor’s network. “The Tonight Show” is seen at 11:35 weeknights.
Mr. Leno, 58, was known to have suitors, including ABC, the Fox network and the Sony television studio. But he was apparently persuaded to stay at NBC after aggressive personal wooing by Jeff Zucker, the chief executive of NBC Universal, a unit of General Electric. |
Camo Buck Necklace
Custom coated in your choice of many different patterns, see images for choices. Custom patterns available, email us for more info at camoring@nwhydroprint.com
Your choice of 13 different camo patterns! (see images for patterns)
Camo Buck pendants measure approx. 1" oval.
Necklace is either Stainless steel snake chain or ball chain with clasp. Choose Black, silver or pink in ball chains.
Great gift ideas for birthdays, bridesmaid gifts, graduation, etc.
Discounted for quantity purchase.
Shipping is FREE via USPS First Class Mail. Please allow 4-5 days for delivery. If ordered with a camo ring or other custom coated item, they will ship together with your whole order, so check timelines. |
Who is lisa rogers dating
If you don’t want to read the rest of this article, there is one surefire way to know if your soldier is fake: If a soldier you’re “dating” online asks you for money for ANY reason, it’s a scam. What he really means is she’s going to be his next victim. He is in a special operations unit and therefore cannot share any information with you.
If there were soldiers being denied leave after being overseas for years at a time, it would be ALL over the news. Now, we certainly do have troops in other countries.And this is not a post office box and it’s not in Nigeria!!It also has nothing to do with a Western Union office.If all an Army spouse had to do was email her soldier’s commander to get him home from deployment, don’t you think ALL Army spouses would be doing this? The Army does not allow leave requests from Army family members. There are ATMs on any main post and the PX accepts debit and credit cards. Last time I checked we aren’t on a lot of peacekeeping missions in Nigeria and Syria.In fact, even in the case of the death of an immediate family member, the information has to be verified by the American Red Cross before the soldier’s command is contacted for possible leave by officials at the Red Cross – they don’t just take your word for it. He wants you to pay for a phone line, cell phone or calling card so you can talk to each other. My husband deployed multiple times and we never paid for a single phone call. Not to mention, soldiers are making enough to buy a phone card if they really had to. He’s about to retire and then he can marry you and live happily ever after. LOL A General will have well over 20 years of service and less than 1% of officers will make it to the rank of General. If he says he’s somewhere that there isn’t an ATM or another way to get money, there is also nothing for him to spend money on. And if we were, a real soldier wouldn’t tell you that. I originally had other questions you could ask regarding his training, etc, but I’m removing those because many people believed whatever ridiculous information he came up with when they asked. |
Three-factor model posits that a three-way interaction between weight concern, perfectionism, and self-efficacy predict changes in disordered eating but has proved difficult to replicate. The aim of this study was to investigate a revised model, examining a three-way interaction between concerns, perfectionism, and self-compassion in predicting changes in disordered eating. Women (N=55) with a mean age of 23.89 years completed questionnaires on two occasions, 4 to 6 months apart. The three-way interaction was significant, as was the contribution of self-compassion, the concern score from the Eating Disorder Examination, and all of the 2-way interaction terms. The whole model accounted for 43% of the variance of the change in disordered eating. Women experienced decreases in disordered eating if they had high level of concerns but low levels of perfectionism and high levels of self-compassion. This suggests that interventions that tackle both perfectionism and self-compassion may be useful in the prevention and treatment of eating disorders. Subsequent examination of a short self-compassion intervention confirmed that, compared to a distraction and control condition, it increased weight satisfaction. Additionally, in comparison to the control condition, it decreased belief in perfectionistic thinking.
This abstract was presented in the **Disordered Eating -- Characteristics & Treatment** stream of the 2013 ANZAED Conference.
|
/*
* Copyright (c) MuleSoft, Inc. All rights reserved. http://www.mulesoft.com
* The software in this package is published under the terms of the CPAL v1.0
* license, a copy of which has been included with this distribution in the
* LICENSE.txt file.
*/
package org.mule.runtime.module.extension.soap.internal.runtime.operation;
import static org.mule.runtime.api.metadata.DataType.INPUT_STREAM;
import static org.mule.runtime.api.metadata.DataType.XML_STRING;
import static org.mule.runtime.core.api.rx.Exceptions.wrapFatal;
import static org.mule.runtime.module.extension.soap.internal.loader.SoapInvokeOperationDeclarer.ATTACHMENTS_PARAM;
import static org.mule.runtime.module.extension.soap.internal.loader.SoapInvokeOperationDeclarer.BODY_PARAM;
import static org.mule.runtime.module.extension.soap.internal.loader.SoapInvokeOperationDeclarer.HEADERS_PARAM;
import static org.mule.runtime.module.extension.soap.internal.loader.SoapInvokeOperationDeclarer.MESSAGE_GROUP;
import static org.mule.runtime.module.extension.soap.internal.loader.SoapInvokeOperationDeclarer.OPERATION_PARAM;
import static org.mule.runtime.module.extension.soap.internal.loader.SoapInvokeOperationDeclarer.SERVICE_PARAM;
import static org.mule.runtime.module.extension.soap.internal.loader.SoapInvokeOperationDeclarer.TRANSPORT_HEADERS_PARAM;
import org.mule.runtime.api.el.BindingContext;
import org.mule.runtime.api.el.CompiledExpression;
import org.mule.runtime.api.el.ExpressionLanguageSession;
import org.mule.runtime.api.el.MuleExpressionLanguage;
import org.mule.runtime.api.lifecycle.Initialisable;
import org.mule.runtime.api.meta.model.operation.OperationModel;
import org.mule.runtime.api.metadata.DataType;
import org.mule.runtime.api.metadata.TypedValue;
import org.mule.runtime.api.transformation.TransformationService;
import org.mule.runtime.core.api.transformer.MessageTransformerException;
import org.mule.runtime.core.api.transformer.TransformerException;
import org.mule.runtime.core.api.util.IOUtils;
import org.mule.runtime.extension.api.runtime.operation.CompletableComponentExecutor;
import org.mule.runtime.extension.api.runtime.operation.ExecutionContext;
import org.mule.runtime.extension.api.soap.SoapAttachment;
import org.mule.runtime.module.extension.internal.runtime.client.strategy.ExtensionsClientProcessorsStrategyFactory;
import org.mule.runtime.module.extension.internal.runtime.resolver.ConnectionArgumentResolver;
import org.mule.runtime.module.extension.internal.runtime.resolver.ExtensionsClientArgumentResolver;
import org.mule.runtime.module.extension.internal.runtime.resolver.StreamingHelperArgumentResolver;
import org.mule.runtime.module.extension.soap.internal.runtime.connection.ForwardingSoapClient;
import org.mule.runtime.soap.api.client.SoapClient;
import org.mule.runtime.soap.api.exception.error.SoapExceptionEnricher;
import org.mule.runtime.soap.api.message.SoapRequest;
import org.mule.runtime.soap.api.message.SoapRequestBuilder;
import org.mule.runtime.soap.api.message.SoapResponse;
import java.io.InputStream;
import java.util.HashMap;
import java.util.Map;
import java.util.Optional;
import javax.inject.Inject;
/**
* {@link CompletableComponentExecutor} implementation that executes SOAP operations using a provided {@link SoapClient}.
*
* @since 4.0
*/
public final class SoapOperationExecutor implements CompletableComponentExecutor<OperationModel>, Initialisable {
@Inject
private MuleExpressionLanguage expressionExecutor;
@Inject
private TransformationService transformationService;
@Inject
private ExtensionsClientProcessorsStrategyFactory extensionsClientProcessorsStrategyFactory;
private final ConnectionArgumentResolver connectionResolver = new ConnectionArgumentResolver();
private final StreamingHelperArgumentResolver streamingHelperArgumentResolver = new StreamingHelperArgumentResolver();
private final SoapExceptionEnricher soapExceptionEnricher = new SoapExceptionEnricher();
private ExtensionsClientArgumentResolver extensionsClientArgumentResolver;
private CompiledExpression headersExpression;
/**
* {@inheritDoc}
*/
@Override
public void execute(ExecutionContext<OperationModel> context, ExecutorCallback callback) {
try {
String serviceId = context.getParameter(SERVICE_PARAM);
ForwardingSoapClient connection = (ForwardingSoapClient) connectionResolver.resolve(context);
Map<String, String> customHeaders = connection.getCustomHeaders(serviceId, getOperation(context));
SoapRequest request = getRequest(context, customHeaders);
SoapClient soapClient = connection.getSoapClient(serviceId);
SoapResponse response = connection.getExtensionsClientDispatcher(() -> extensionsClientArgumentResolver
.resolve(context))
.map(d -> soapClient.consume(request, d))
.orElseGet(() -> soapClient.consume(request));
callback.complete((response.getAsResult(streamingHelperArgumentResolver.resolve(context))));
} catch (MessageTransformerException | TransformerException e) {
callback.error(e);
} catch (Exception e) {
callback.error(soapExceptionEnricher.enrich(e));
} catch (Throwable t) {
callback.error(wrapFatal(t));
}
}
public void initialise() {
this.extensionsClientArgumentResolver = new ExtensionsClientArgumentResolver(extensionsClientProcessorsStrategyFactory);
headersExpression = expressionExecutor.compile(
"%dw 2.0 \n"
+ "output application/java \n"
+ "---\n"
+ "payload.headers mapObject (value, key) -> {\n"
+ " '$key' : write((key): value, \"application/xml\")\n"
+ "}",
BindingContext.builder()
.addBinding("payload", new TypedValue<>("", XML_STRING)).build());
}
/**
* Builds a Soap Request with the execution context to be sent using the {@link SoapClient}.
*/
private SoapRequest getRequest(ExecutionContext<OperationModel> context, Map<String, String> fixedHeaders)
throws MessageTransformerException, TransformerException {
SoapRequestBuilder builder = SoapRequest.builder().operation(getOperation(context));
builder.soapHeaders(fixedHeaders);
Optional<Object> optionalMessageGroup = getParam(context, MESSAGE_GROUP);
if (optionalMessageGroup.isPresent()) {
Map<String, Object> message = (Map<String, Object>) optionalMessageGroup.get();
InputStream body = (InputStream) message.get(BODY_PARAM);
if (body != null) {
builder.content(body);
}
InputStream headers = (InputStream) message.get(HEADERS_PARAM);
if (headers != null) {
builder.soapHeaders((Map<String, String>) evaluateHeaders(headers));
}
Map<String, TypedValue<?>> attachments = (Map<String, TypedValue<?>>) message.get(ATTACHMENTS_PARAM);
if (attachments != null) {
toSoapAttachments(attachments).forEach(builder::attachment);
}
}
getParam(context, TRANSPORT_HEADERS_PARAM)
.ifPresent(th -> builder.transportHeaders((Map<String, String>) th));
return builder.build();
}
private String getOperation(ExecutionContext<OperationModel> context) {
return (String) getParam(context, OPERATION_PARAM)
.orElseThrow(
() -> new IllegalStateException("Execution Context does not have the required operation parameter"));
}
private <T> Optional<T> getParam(ExecutionContext<OperationModel> context, String param) {
return context.hasParameter(param) ? Optional.ofNullable(context.getParameter(param)) : Optional.empty();
}
private Object evaluateHeaders(InputStream headers) {
String hs = IOUtils.toString(headers);
BindingContext context = BindingContext.builder().addBinding("payload", new TypedValue<>(hs, XML_STRING)).build();
try (ExpressionLanguageSession session = expressionExecutor.openSession(context)) {
return session.evaluate(headersExpression).getValue();
}
}
private Map<String, SoapAttachment> toSoapAttachments(Map<String, TypedValue<?>> attachments)
throws MessageTransformerException, TransformerException {
Map<String, SoapAttachment> soapAttachmentMap = new HashMap<>();
for (Map.Entry<String, TypedValue<?>> attachment : attachments.entrySet()) {
SoapAttachment soapAttachment =
new SoapAttachment(toInputStream(attachment.getValue()), attachment.getValue().getDataType().getMediaType());
soapAttachmentMap.put(attachment.getKey(), soapAttachment);
}
return soapAttachmentMap;
}
private InputStream toInputStream(TypedValue typedValue) throws MessageTransformerException, TransformerException {
Object value = typedValue.getValue();
if (value instanceof InputStream) {
return (InputStream) value;
}
return (InputStream) transformationService.transform(value, DataType.fromObject(value), INPUT_STREAM);
}
}
|
"use strict";
const conversions = require("webidl-conversions");
const utils = require("./utils.js");
const convertAttr = require("./Attr.js").convert;
const isNode = require("./Node.js").is;
const impl = utils.implSymbol;
const Node = require("./Node.js");
const ChildNode = require("./ChildNode.js");
const NonDocumentTypeChildNode = require("./NonDocumentTypeChildNode.js");
const ParentNode = require("./ParentNode.js");
function Element() {
throw new TypeError("Illegal constructor");
}
Object.setPrototypeOf(Element.prototype, Node.interface.prototype);
Object.setPrototypeOf(Element, Node.interface);
Object.defineProperty(Element, "prototype", {
value: Element.prototype,
writable: false,
enumerable: false,
configurable: false
});
Element.prototype.hasAttributes = function hasAttributes() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl].hasAttributes();
};
Element.prototype.getAttributeNames = function getAttributeNames() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.tryWrapperForImpl(this[impl].getAttributeNames());
};
Element.prototype.getAttribute = function getAttribute(qualifiedName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'getAttribute' on 'Element': 1 argument required, but only " + arguments.length + " present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getAttribute' on 'Element': parameter 1"
});
args.push(curArg);
}
return this[impl].getAttribute(...args);
};
Element.prototype.getAttributeNS = function getAttributeNS(namespace, localName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 2) {
throw new TypeError(
"Failed to execute 'getAttributeNS' on 'Element': 2 arguments required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
if (curArg === null || curArg === undefined) {
curArg = null;
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getAttributeNS' on 'Element': parameter 1"
});
}
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getAttributeNS' on 'Element': parameter 2"
});
args.push(curArg);
}
return this[impl].getAttributeNS(...args);
};
Element.prototype.setAttribute = function setAttribute(qualifiedName, value) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 2) {
throw new TypeError(
"Failed to execute 'setAttribute' on 'Element': 2 arguments required, but only " + arguments.length + " present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'setAttribute' on 'Element': parameter 1"
});
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'setAttribute' on 'Element': parameter 2"
});
args.push(curArg);
}
return this[impl].setAttribute(...args);
};
Element.prototype.setAttributeNS = function setAttributeNS(namespace, qualifiedName, value) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 3) {
throw new TypeError(
"Failed to execute 'setAttributeNS' on 'Element': 3 arguments required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
if (curArg === null || curArg === undefined) {
curArg = null;
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'setAttributeNS' on 'Element': parameter 1"
});
}
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'setAttributeNS' on 'Element': parameter 2"
});
args.push(curArg);
}
{
let curArg = arguments[2];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'setAttributeNS' on 'Element': parameter 3"
});
args.push(curArg);
}
return this[impl].setAttributeNS(...args);
};
Element.prototype.removeAttribute = function removeAttribute(qualifiedName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'removeAttribute' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'removeAttribute' on 'Element': parameter 1"
});
args.push(curArg);
}
return this[impl].removeAttribute(...args);
};
Element.prototype.removeAttributeNS = function removeAttributeNS(namespace, localName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 2) {
throw new TypeError(
"Failed to execute 'removeAttributeNS' on 'Element': 2 arguments required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
if (curArg === null || curArg === undefined) {
curArg = null;
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'removeAttributeNS' on 'Element': parameter 1"
});
}
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'removeAttributeNS' on 'Element': parameter 2"
});
args.push(curArg);
}
return this[impl].removeAttributeNS(...args);
};
Element.prototype.hasAttribute = function hasAttribute(qualifiedName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'hasAttribute' on 'Element': 1 argument required, but only " + arguments.length + " present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'hasAttribute' on 'Element': parameter 1"
});
args.push(curArg);
}
return this[impl].hasAttribute(...args);
};
Element.prototype.hasAttributeNS = function hasAttributeNS(namespace, localName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 2) {
throw new TypeError(
"Failed to execute 'hasAttributeNS' on 'Element': 2 arguments required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
if (curArg === null || curArg === undefined) {
curArg = null;
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'hasAttributeNS' on 'Element': parameter 1"
});
}
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'hasAttributeNS' on 'Element': parameter 2"
});
args.push(curArg);
}
return this[impl].hasAttributeNS(...args);
};
Element.prototype.getAttributeNode = function getAttributeNode(qualifiedName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'getAttributeNode' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getAttributeNode' on 'Element': parameter 1"
});
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].getAttributeNode(...args));
};
Element.prototype.getAttributeNodeNS = function getAttributeNodeNS(namespace, localName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 2) {
throw new TypeError(
"Failed to execute 'getAttributeNodeNS' on 'Element': 2 arguments required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
if (curArg === null || curArg === undefined) {
curArg = null;
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getAttributeNodeNS' on 'Element': parameter 1"
});
}
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getAttributeNodeNS' on 'Element': parameter 2"
});
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].getAttributeNodeNS(...args));
};
Element.prototype.setAttributeNode = function setAttributeNode(attr) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'setAttributeNode' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = convertAttr(curArg, { context: "Failed to execute 'setAttributeNode' on 'Element': parameter 1" });
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].setAttributeNode(...args));
};
Element.prototype.setAttributeNodeNS = function setAttributeNodeNS(attr) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'setAttributeNodeNS' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = convertAttr(curArg, { context: "Failed to execute 'setAttributeNodeNS' on 'Element': parameter 1" });
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].setAttributeNodeNS(...args));
};
Element.prototype.removeAttributeNode = function removeAttributeNode(attr) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'removeAttributeNode' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = convertAttr(curArg, { context: "Failed to execute 'removeAttributeNode' on 'Element': parameter 1" });
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].removeAttributeNode(...args));
};
Element.prototype.closest = function closest(selectors) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'closest' on 'Element': 1 argument required, but only " + arguments.length + " present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, { context: "Failed to execute 'closest' on 'Element': parameter 1" });
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].closest(...args));
};
Element.prototype.matches = function matches(selectors) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'matches' on 'Element': 1 argument required, but only " + arguments.length + " present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, { context: "Failed to execute 'matches' on 'Element': parameter 1" });
args.push(curArg);
}
return this[impl].matches(...args);
};
Element.prototype.webkitMatchesSelector = function webkitMatchesSelector(selectors) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'webkitMatchesSelector' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'webkitMatchesSelector' on 'Element': parameter 1"
});
args.push(curArg);
}
return this[impl].webkitMatchesSelector(...args);
};
Element.prototype.getElementsByTagName = function getElementsByTagName(qualifiedName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'getElementsByTagName' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getElementsByTagName' on 'Element': parameter 1"
});
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].getElementsByTagName(...args));
};
Element.prototype.getElementsByTagNameNS = function getElementsByTagNameNS(namespace, localName) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 2) {
throw new TypeError(
"Failed to execute 'getElementsByTagNameNS' on 'Element': 2 arguments required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
if (curArg === null || curArg === undefined) {
curArg = null;
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getElementsByTagNameNS' on 'Element': parameter 1"
});
}
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getElementsByTagNameNS' on 'Element': parameter 2"
});
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].getElementsByTagNameNS(...args));
};
Element.prototype.getElementsByClassName = function getElementsByClassName(classNames) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'getElementsByClassName' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'getElementsByClassName' on 'Element': parameter 1"
});
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].getElementsByClassName(...args));
};
Element.prototype.insertAdjacentHTML = function insertAdjacentHTML(position, text) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 2) {
throw new TypeError(
"Failed to execute 'insertAdjacentHTML' on 'Element': 2 arguments required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'insertAdjacentHTML' on 'Element': parameter 1"
});
args.push(curArg);
}
{
let curArg = arguments[1];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'insertAdjacentHTML' on 'Element': parameter 2"
});
args.push(curArg);
}
return this[impl].insertAdjacentHTML(...args);
};
Element.prototype.getClientRects = function getClientRects() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.tryWrapperForImpl(this[impl].getClientRects());
};
Element.prototype.getBoundingClientRect = function getBoundingClientRect() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.tryWrapperForImpl(this[impl].getBoundingClientRect());
};
Element.prototype.before = function before() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
const args = [];
for (let i = 0; i < arguments.length; i++) {
let curArg = arguments[i];
if (isNode(curArg)) {
curArg = utils.implForWrapper(curArg);
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'before' on 'Element': parameter " + (i + 1)
});
}
args.push(curArg);
}
return this[impl].before(...args);
};
Element.prototype.after = function after() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
const args = [];
for (let i = 0; i < arguments.length; i++) {
let curArg = arguments[i];
if (isNode(curArg)) {
curArg = utils.implForWrapper(curArg);
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'after' on 'Element': parameter " + (i + 1)
});
}
args.push(curArg);
}
return this[impl].after(...args);
};
Element.prototype.replaceWith = function replaceWith() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
const args = [];
for (let i = 0; i < arguments.length; i++) {
let curArg = arguments[i];
if (isNode(curArg)) {
curArg = utils.implForWrapper(curArg);
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'replaceWith' on 'Element': parameter " + (i + 1)
});
}
args.push(curArg);
}
return this[impl].replaceWith(...args);
};
Element.prototype.remove = function remove() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl].remove();
};
Element.prototype.prepend = function prepend() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
const args = [];
for (let i = 0; i < arguments.length; i++) {
let curArg = arguments[i];
if (isNode(curArg)) {
curArg = utils.implForWrapper(curArg);
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'prepend' on 'Element': parameter " + (i + 1)
});
}
args.push(curArg);
}
return this[impl].prepend(...args);
};
Element.prototype.append = function append() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
const args = [];
for (let i = 0; i < arguments.length; i++) {
let curArg = arguments[i];
if (isNode(curArg)) {
curArg = utils.implForWrapper(curArg);
} else {
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'append' on 'Element': parameter " + (i + 1)
});
}
args.push(curArg);
}
return this[impl].append(...args);
};
Element.prototype.querySelector = function querySelector(selectors) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'querySelector' on 'Element': 1 argument required, but only " + arguments.length + " present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'querySelector' on 'Element': parameter 1"
});
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].querySelector(...args));
};
Element.prototype.querySelectorAll = function querySelectorAll(selectors) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
if (arguments.length < 1) {
throw new TypeError(
"Failed to execute 'querySelectorAll' on 'Element': 1 argument required, but only " +
arguments.length +
" present."
);
}
const args = [];
{
let curArg = arguments[0];
curArg = conversions["DOMString"](curArg, {
context: "Failed to execute 'querySelectorAll' on 'Element': parameter 1"
});
args.push(curArg);
}
return utils.tryWrapperForImpl(this[impl].querySelectorAll(...args));
};
Object.defineProperty(Element.prototype, "namespaceURI", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["namespaceURI"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "prefix", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["prefix"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "localName", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["localName"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "tagName", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["tagName"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "id", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
const value = this.getAttribute("id");
return value === null ? "" : value;
},
set(V) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
V = conversions["DOMString"](V, { context: "Failed to set the 'id' property on 'Element': The provided value" });
this.setAttribute("id", V);
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "className", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
const value = this.getAttribute("class");
return value === null ? "" : value;
},
set(V) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
V = conversions["DOMString"](V, {
context: "Failed to set the 'className' property on 'Element': The provided value"
});
this.setAttribute("class", V);
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "classList", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.getSameObject(this, "classList", () => {
return utils.tryWrapperForImpl(this[impl]["classList"]);
});
},
set(V) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
this.classList.value = V;
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "attributes", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.getSameObject(this, "attributes", () => {
return utils.tryWrapperForImpl(this[impl]["attributes"]);
});
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "innerHTML", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["innerHTML"];
},
set(V) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
V = conversions["DOMString"](V, {
context: "Failed to set the 'innerHTML' property on 'Element': The provided value",
treatNullAsEmptyString: true
});
this[impl]["innerHTML"] = V;
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "outerHTML", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["outerHTML"];
},
set(V) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
V = conversions["DOMString"](V, {
context: "Failed to set the 'outerHTML' property on 'Element': The provided value",
treatNullAsEmptyString: true
});
this[impl]["outerHTML"] = V;
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "scrollTop", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["scrollTop"];
},
set(V) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
V = conversions["unrestricted double"](V, {
context: "Failed to set the 'scrollTop' property on 'Element': The provided value"
});
this[impl]["scrollTop"] = V;
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "scrollLeft", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["scrollLeft"];
},
set(V) {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
V = conversions["unrestricted double"](V, {
context: "Failed to set the 'scrollLeft' property on 'Element': The provided value"
});
this[impl]["scrollLeft"] = V;
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "scrollWidth", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["scrollWidth"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "scrollHeight", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["scrollHeight"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "clientTop", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["clientTop"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "clientLeft", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["clientLeft"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "clientWidth", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["clientWidth"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "clientHeight", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["clientHeight"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "previousElementSibling", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.tryWrapperForImpl(this[impl]["previousElementSibling"]);
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "nextElementSibling", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.tryWrapperForImpl(this[impl]["nextElementSibling"]);
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "children", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.getSameObject(this, "children", () => {
return utils.tryWrapperForImpl(this[impl]["children"]);
});
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "firstElementChild", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.tryWrapperForImpl(this[impl]["firstElementChild"]);
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "lastElementChild", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return utils.tryWrapperForImpl(this[impl]["lastElementChild"]);
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, "childElementCount", {
get() {
if (!this || !module.exports.is(this)) {
throw new TypeError("Illegal invocation");
}
return this[impl]["childElementCount"];
},
enumerable: true,
configurable: true
});
Object.defineProperty(Element.prototype, Symbol.toStringTag, {
value: "Element",
writable: false,
enumerable: false,
configurable: true
});
const iface = {
// When an interface-module that implements this interface as a mixin is loaded, it will append its own `.is()`
// method into this array. It allows objects that directly implements *those* interfaces to be recognized as
// implementing this mixin interface.
_mixedIntoPredicates: [],
is(obj) {
if (obj) {
if (utils.hasOwn(obj, impl) && obj[impl] instanceof Impl.implementation) {
return true;
}
for (const isMixedInto of module.exports._mixedIntoPredicates) {
if (isMixedInto(obj)) {
return true;
}
}
}
return false;
},
isImpl(obj) {
if (obj) {
if (obj instanceof Impl.implementation) {
return true;
}
const wrapper = utils.wrapperForImpl(obj);
for (const isMixedInto of module.exports._mixedIntoPredicates) {
if (isMixedInto(wrapper)) {
return true;
}
}
}
return false;
},
convert(obj, { context = "The provided value" } = {}) {
if (module.exports.is(obj)) {
return utils.implForWrapper(obj);
}
throw new TypeError(`${context} is not of type 'Element'.`);
},
create(constructorArgs, privateData) {
let obj = Object.create(Element.prototype);
obj = this.setup(obj, constructorArgs, privateData);
return obj;
},
createImpl(constructorArgs, privateData) {
let obj = Object.create(Element.prototype);
obj = this.setup(obj, constructorArgs, privateData);
return utils.implForWrapper(obj);
},
_internalSetup(obj) {
Node._internalSetup(obj);
},
setup(obj, constructorArgs, privateData) {
if (!privateData) privateData = {};
privateData.wrapper = obj;
this._internalSetup(obj);
Object.defineProperty(obj, impl, {
value: new Impl.implementation(constructorArgs, privateData),
writable: false,
enumerable: false,
configurable: true
});
obj[impl][utils.wrapperSymbol] = obj;
if (Impl.init) {
Impl.init(obj[impl], privateData);
}
return obj;
},
interface: Element,
expose: {
Window: { Element }
}
}; // iface
module.exports = iface;
ChildNode._mixedIntoPredicates.push(module.exports.is);
NonDocumentTypeChildNode._mixedIntoPredicates.push(module.exports.is);
ParentNode._mixedIntoPredicates.push(module.exports.is);
const Impl = require("../nodes/Element-impl.js");
|
// Copyright 2020 Open Source Robotics Foundation, Inc.
//
// Licensed under the Apache License, Version 2.0 (the "License");
// you may not use this file except in compliance with the License.
// You may obtain a copy of the License at
//
// http://www.apache.org/licenses/LICENSE-2.0
//
// Unless required by applicable law or agreed to in writing, software
// distributed under the License is distributed on an "AS IS" BASIS,
// WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
// See the License for the specific language governing permissions and
// limitations under the License.
#include <gtest/gtest.h>
#include <chrono>
#include <map>
#include <memory>
#include <string>
#include <utility>
#include <vector>
#include "rclcpp/node.hpp"
#include "test_msgs/msg/empty.hpp"
#include "test_msgs/msg/empty.h"
#include "rclcpp/exceptions.hpp"
#include "rclcpp/executors.hpp"
#include "rclcpp/executor.hpp"
#include "rclcpp/rclcpp.hpp"
using namespace std::chrono_literals;
template<typename T>
class TestAddCallbackGroupsToExecutor : public ::testing::Test
{
public:
static void SetUpTestCase()
{
rclcpp::init(0, nullptr);
}
static void TearDownTestCase()
{
rclcpp::shutdown();
}
};
using ExecutorTypes =
::testing::Types<
rclcpp::executors::SingleThreadedExecutor,
rclcpp::executors::MultiThreadedExecutor,
rclcpp::executors::StaticSingleThreadedExecutor>;
class ExecutorTypeNames
{
public:
template<typename T>
static std::string GetName(int idx)
{
(void)idx;
if (std::is_same<T, rclcpp::executors::SingleThreadedExecutor>()) {
return "SingleThreadedExecutor";
}
if (std::is_same<T, rclcpp::executors::MultiThreadedExecutor>()) {
return "MultiThreadedExecutor";
}
if (std::is_same<T, rclcpp::executors::StaticSingleThreadedExecutor>()) {
return "StaticSingleThreadedExecutor";
}
return "";
}
};
TYPED_TEST_CASE(TestAddCallbackGroupsToExecutor, ExecutorTypes, ExecutorTypeNames);
/*
* Test adding callback groups.
*/
TYPED_TEST(TestAddCallbackGroupsToExecutor, add_callback_groups) {
using ExecutorType = TypeParam;
auto node = std::make_shared<rclcpp::Node>("my_node", "/ns");
auto timer_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive);
rclcpp::TimerBase::SharedPtr timer_ = node->create_wall_timer(
2s, timer_callback, cb_grp);
ExecutorType executor;
executor.add_callback_group(cb_grp, node->get_node_base_interface());
ASSERT_EQ(executor.get_all_callback_groups().size(), 1u);
ASSERT_EQ(executor.get_manually_added_callback_groups().size(), 1u);
ASSERT_EQ(executor.get_automatically_added_callback_groups_from_nodes().size(), 0u);
const rclcpp::QoS qos(10);
auto options = rclcpp::SubscriptionOptions();
auto callback = [](const test_msgs::msg::Empty::SharedPtr) {};
rclcpp::CallbackGroup::SharedPtr cb_grp2 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive);
options.callback_group = cb_grp2;
auto subscription =
node->create_subscription<test_msgs::msg::Empty>("topic_name", qos, callback, options);
executor.add_callback_group(cb_grp2, node->get_node_base_interface());
ASSERT_EQ(executor.get_all_callback_groups().size(), 2u);
ASSERT_EQ(executor.get_manually_added_callback_groups().size(), 2u);
ASSERT_EQ(executor.get_automatically_added_callback_groups_from_nodes().size(), 0u);
executor.add_node(node);
ASSERT_EQ(executor.get_manually_added_callback_groups().size(), 2u);
ASSERT_EQ(executor.get_automatically_added_callback_groups_from_nodes().size(), 1u);
executor.remove_node(node);
ASSERT_EQ(executor.get_manually_added_callback_groups().size(), 2u);
ASSERT_EQ(executor.get_automatically_added_callback_groups_from_nodes().size(), 0u);
executor.remove_callback_group(cb_grp);
ASSERT_EQ(executor.get_manually_added_callback_groups().size(), 1u);
ASSERT_EQ(executor.get_automatically_added_callback_groups_from_nodes().size(), 0u);
executor.remove_callback_group(cb_grp2);
ASSERT_EQ(executor.get_manually_added_callback_groups().size(), 0u);
ASSERT_EQ(executor.get_automatically_added_callback_groups_from_nodes().size(), 0u);
}
/*
* Test removing callback groups.
*/
TYPED_TEST(TestAddCallbackGroupsToExecutor, remove_callback_groups) {
using ExecutorType = TypeParam;
auto node = std::make_shared<rclcpp::Node>("my_node", "/ns");
auto timer_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive);
rclcpp::TimerBase::SharedPtr timer_ = node->create_wall_timer(
2s, timer_callback, cb_grp);
ExecutorType executor;
executor.add_callback_group(cb_grp, node->get_node_base_interface());
const rclcpp::QoS qos(10);
auto options = rclcpp::SubscriptionOptions();
auto callback = [](const test_msgs::msg::Empty::SharedPtr) {};
rclcpp::CallbackGroup::SharedPtr cb_grp2 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive);
options.callback_group = cb_grp2;
auto subscription =
node->create_subscription<test_msgs::msg::Empty>("topic_name", qos, callback, options);
executor.add_callback_group(cb_grp2, node->get_node_base_interface());
executor.remove_callback_group(cb_grp);
ASSERT_EQ(executor.get_all_callback_groups().size(), 1u);
executor.remove_callback_group(cb_grp2);
ASSERT_EQ(executor.get_all_callback_groups().size(), 0u);
}
/*
* Test adding duplicate callback groups to executor.
*/
TYPED_TEST(TestAddCallbackGroupsToExecutor, add_duplicate_callback_groups)
{
rclcpp::executors::MultiThreadedExecutor executor;
auto node = std::make_shared<rclcpp::Node>("my_node", "/ns");
auto timer_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive);
rclcpp::TimerBase::SharedPtr timer_ = node->create_wall_timer(
2s, timer_callback, cb_grp);
executor.add_callback_group(cb_grp, node->get_node_base_interface());
EXPECT_THROW(
executor.add_callback_group(cb_grp, node->get_node_base_interface()),
std::exception);
}
/*
* Test adding callback group after node is added to executor.
*/
TYPED_TEST(TestAddCallbackGroupsToExecutor, add_callback_groups_after_add_node_to_executor)
{
rclcpp::executors::MultiThreadedExecutor executor;
auto node = std::make_shared<rclcpp::Node>("my_node", "/ns");
executor.add_node(node->get_node_base_interface());
ASSERT_EQ(executor.get_all_callback_groups().size(), 1u);
std::atomic_int timer_count {0};
auto timer_callback = [&executor, &timer_count]() {
if (timer_count > 0) {
ASSERT_EQ(executor.get_all_callback_groups().size(), 3u);
executor.cancel();
}
timer_count++;
};
rclcpp::CallbackGroup::SharedPtr cb_grp = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive);
rclcpp::TimerBase::SharedPtr timer_ = node->create_wall_timer(
2s, timer_callback, cb_grp);
rclcpp::CallbackGroup::SharedPtr cb_grp2 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, false);
auto timer2_callback = []() {};
rclcpp::TimerBase::SharedPtr timer2_ = node->create_wall_timer(
2s, timer2_callback, cb_grp2);
rclcpp::CallbackGroup::SharedPtr cb_grp3 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, true);
auto timer3_callback = []() {};
rclcpp::TimerBase::SharedPtr timer3_ = node->create_wall_timer(
2s, timer3_callback, cb_grp3);
executor.spin();
}
/*
* Test adding unallowable callback group.
*/
TYPED_TEST(TestAddCallbackGroupsToExecutor, add_unallowable_callback_groups)
{
auto node = std::make_shared<rclcpp::Node>("my_node", "/ns");
auto timer_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, false);
rclcpp::TimerBase::SharedPtr timer_ = node->create_wall_timer(
2s, timer_callback, cb_grp);
rclcpp::executors::MultiThreadedExecutor executor;
executor.add_callback_group(cb_grp, node->get_node_base_interface());
ASSERT_EQ(executor.get_all_callback_groups().size(), 1u);
const rclcpp::QoS qos(10);
auto options = rclcpp::SubscriptionOptions();
auto callback = [](const test_msgs::msg::Empty::SharedPtr) {};
rclcpp::CallbackGroup::SharedPtr cb_grp2 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, false);
options.callback_group = cb_grp2;
auto subscription =
node->create_subscription<test_msgs::msg::Empty>("topic_name", qos, callback, options);
executor.add_callback_group(cb_grp2, node->get_node_base_interface());
ASSERT_EQ(executor.get_all_callback_groups().size(), 2u);
auto timer2_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp3 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, false);
rclcpp::TimerBase::SharedPtr timer2_ = node->create_wall_timer(
2s, timer2_callback, cb_grp3);
executor.add_node(node->get_node_base_interface());
ASSERT_EQ(executor.get_all_callback_groups().size(), 3u);
}
/*
* Test callback groups from one node to many executors.
*/
TYPED_TEST(TestAddCallbackGroupsToExecutor, one_node_many_callback_groups_many_executors)
{
auto node = std::make_shared<rclcpp::Node>("my_node", "/ns");
auto timer_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, false);
rclcpp::TimerBase::SharedPtr timer_ = node->create_wall_timer(
2s, timer_callback, cb_grp);
rclcpp::executors::MultiThreadedExecutor timer_executor;
rclcpp::executors::MultiThreadedExecutor sub_executor;
timer_executor.add_callback_group(cb_grp, node->get_node_base_interface());
const rclcpp::QoS qos(10);
auto options = rclcpp::SubscriptionOptions();
auto callback = [](const test_msgs::msg::Empty::SharedPtr) {};
rclcpp::CallbackGroup::SharedPtr cb_grp2 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, false);
options.callback_group = cb_grp2;
auto subscription =
node->create_subscription<test_msgs::msg::Empty>("topic_name", qos, callback, options);
sub_executor.add_callback_group(cb_grp2, node->get_node_base_interface());
ASSERT_EQ(sub_executor.get_all_callback_groups().size(), 1u);
ASSERT_EQ(timer_executor.get_all_callback_groups().size(), 1u);
auto timer2_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp3 = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive, false);
rclcpp::TimerBase::SharedPtr timer2 = node->create_wall_timer(
2s, timer2_callback, cb_grp3);
sub_executor.add_node(node);
ASSERT_EQ(sub_executor.get_all_callback_groups().size(), 2u);
timer_executor.add_callback_group(cb_grp3, node->get_node_base_interface());
ASSERT_EQ(timer_executor.get_all_callback_groups().size(), 2u);
}
/*
* Test removing callback group from executor that its not associated with.
*/
TYPED_TEST(TestAddCallbackGroupsToExecutor, remove_callback_group)
{
rclcpp::executors::MultiThreadedExecutor executor;
auto node = std::make_shared<rclcpp::Node>("my_node", "/ns");
auto timer_callback = []() {};
rclcpp::CallbackGroup::SharedPtr cb_grp = node->create_callback_group(
rclcpp::CallbackGroupType::MutuallyExclusive);
rclcpp::TimerBase::SharedPtr timer_ = node->create_wall_timer(
2s, timer_callback, cb_grp);
EXPECT_THROW(
executor.remove_callback_group(cb_grp),
std::exception);
executor.add_callback_group(cb_grp, node->get_node_base_interface());
EXPECT_NO_THROW(executor.remove_callback_group(cb_grp));
EXPECT_THROW(
executor.remove_callback_group(cb_grp),
std::exception);
}
|
Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium.
Vascular endothelial growth factor (VEGF) is a secreted protein that is a specific growth factor for endothelial cells. We have recently demonstrated that nitric oxide (NO) donors and vasoactive peptides promoting NO-mediated vasorelaxation induce angiogenesis in vivo as well as endothelial cell growth and motility in vitro; in contrast, inhibitors of NO synthase suppress angiogenesis. In this study we investigated the role of NO in mediating the mitogenic effect of VEGF on cultured microvascular endothelium isolated from coronary postcapillary venules. VEGF induced a dose-dependent increase in cell proliferation and DNA synthesis. The role of NO was determined by monitoring proliferation or guanosine 3',5'-cyclic monophosphate (cGMP) levels in the presence and absence of NO synthase blockers. The proliferative effect evoked by VEGF was reduced by pretreatment of the cells with NO synthase inhibitors. Exposure of the cells to VEGF induced a significant increment in cGMP levels. This effect was potentiated by superoxide dismutase addition and was abolished by NO synthase inhibitors. VEGF stimulates proliferation of postcapillary endothelial cells through the production of NO and cGMP accumulation. |
Introduction {#s1}
============
The *T-box* gene family encodes transcription factors that play critical roles during embryonic development, organogenesis, and tissue homeostasis. Mutations in *TBX* genes in humans cause multiple developmental dysmorphic syndromes and disease predispositions ([@bib64]; [@bib82]). Heterozygous mutation of *TBX3* causes Ulnar-mammary syndrome (UMS), initially described as a constellation of congenital limb defects, apocrine and mammary gland hypoplasia, and genital abnormalities ([@bib69]). Recently, heart and conduction system defects have also been described in mice and humans with abnormal *Tbx3* (mice) and *TBX3* (humans) function ([@bib3]; [@bib21]; [@bib49]; [@bib58]; [@bib59]). Germline deletion of *Tbx3* in mice results in embryonic lethality with heart, limb, and mammary defects ([@bib15]; [@bib21]; [@bib22]). Tbx3 also regulates pluripotency and cell fate in early development ([@bib11]; [@bib25]; [@bib37]; [@bib66]; [@bib97]).
TBX3 transcriptional repression controls expression of cell proliferation and senescence factors ([@bib7]; [@bib41]); abnormal *TBX3* expression occurs in multiple cancers ([@bib52]; [@bib55]; [@bib71]). TBX3 also regulates splicing and RNA metabolism ([@bib42]). Although these studies highlight the important pleiotropic molecular functions of TBX3, little is known about the core pathways it regulates in developing structures that require its function, such as the developing limb.
UMS limb phenotypes are variable ranging in severity from hypoplasia of digit 5 to complete absence of forearm and hand (OMIM \#181450). Mouse *Tbx3^tm1Pa/tm1Pa^* ([@bib15]) and *Tbx3^Δfl/Δfl^*([@bib22]) mutant forelimbs lack posterior digits and the ulna. Hindlimbs of *Tbx3^tm1Pa/tm1Pa^* and *Tbx3^Δfl/Δfl^*null mutants have only a single digit, but *Tbx3^Δfl/Δfl^*mutants also have pelvic defects ([@bib22]). Embryonic lethality of both types of mutants has prevented elucidation of Tbx3's limb-specific roles.
The Hedgehog pathway is a key regulator of limb development. Shh signaling in posterior mesenchyme promotes digit development and prevents processing of full length Gli3 (Gli3FL) to its repressor form, Gli3R, which constrains digit number ([@bib50]). The balance of Gli transcriptional activation and repression is critical for proper digit number and patterning ([@bib9]; [@bib31]; [@bib50]; [@bib87]; [@bib91]; [@bib94]; [@bib107]). In mammals, the limited, partial proteolytic processing of Gli3FL to Gli3R requires functional primary cilia, the ciliary protein Kif7 ([@bib24]; [@bib51]), as well as balanced activity of Sufu and the ubiquitin ligase adaptors βTrCP and Spop ([@bib10]; [@bib92]; [@bib95]; [@bib98])
In this study, conditional ablation of *Tbx3* reveals discrete roles for Tbx3 during limb initiation and compartment-specific functions during later limb development to regulate digit number. We discovered a novel molecular function of Tbx3 in the primary cilia where it interacts directly with Kif7 and is in a complex with Gli3. Loss of Tbx3 decreases Kif7-Sufu interactions, resulting in excess Gli3 proteolysis and decreased levels of both Gli3FL and Gli3R. The resulting preaxial polydactyly phenocopies limb defects seen in *Gli3* null heterozygotes and in mutants with abnormal structure or function of the primary cilia ([@bib12]; [@bib17]; [@bib24]; [@bib28]; [@bib48]; [@bib51]; [@bib68]; [@bib73]). Our findings reveal a novel mechanism where Tbx3 in the anterior mesenchyme is required for proper function of the Kif7/Sufu complex that regulates Gli3 stability and processing.
Results {#s2}
=======
Loss of Tbx3 in the limb bud mesenchyme results in preaxial polydactyly and postaxial oligodactyly {#s2-1}
--------------------------------------------------------------------------------------------------
*Tbx3* is expressed in discrete anterior and posterior mesenchymal domains in the limb buds from embryonic day (E) 9.5 ([Figure 1A,C,E](#fig1){ref-type="fig"}, [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}). To assess the role of these domains during limb development, we generated conditional mutants using our *Tbx3^flox^* allele ([@bib21]; [@bib22]) and the *Prx1Cre* transgene ([@bib53]) (genotype *Tbx3^flox/flox^;Prx1Cre*, henceforth referred to in the text as *Tbx3;PrxCre* mutants). This driver initiates Cre activity at \~14-somite stage (ss) in the forelimb-forming region of the lateral plate mesoderm (LPM) ([@bib27]). Its activity in the hindlimb is irregular, so our analysis focuses on the forelimb. In situ hybridization and immunohistochemistry confirm complete ablation of *Tbx3* mRNA and protein in *Tbx3;PrxCre* mutant forelimb mesenchyme by E9.5 ([Figure 1B--F](#fig1){ref-type="fig"}, [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}). Expression in the apical ectodermal ridge (AER) is preserved ([Figure 1B,D,F](#fig1){ref-type="fig"}). We previously reported the specificity of the custom anti-Tbx3 antibody used here and loss of limb mesenchymal protein production in *Tbx3;PrxCre* mutant forelimbs ([@bib21]; [@bib22]).10.7554/eLife.07897.003Figure 1.Tbx3 regulates anterior and posterior digit development.(**A**) *Tbx3* expression assayed by mRNA in situ hybridization in E9.5 forelimb bud (black line from a-p shows anterior-posterior axis). Red arrow points to *Tbx3* expression in apical ectodermal ridge (AER). Red ellipse encloses posterior mesenchymal expression domain. (**B**) *Tbx3* transcripts are absent in the limb bud mesenchyme of E9.5 *Tbx3^fl/fl^;PrxCre* mutants. *Tbx3* expression persists in the AER (red arrow) and adjacent posterior-lateral body wall (black arrowhead). (**C**, **D**) As in A and B except limb buds are E10.5. Red ellipses enclose anterior and posterior mesenchymal expression domains which are Tbx3 negative in the mutants. Red arrows highlight expression in AER. (**E**, **F**) Tbx3 immunohistochemistry on sectioned E10.5 limb. Tbx3 protein is lost in mesenchyme of *Tbx3^fl/fl^;PrxCre* mutants (F, red ellipses) but AER staining persists as expected (white arrowhead). Please see also [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}. (**G--J**) Skeleton preparations reveal preaxial polysyndactyly (duplicated/fused digit 1,red bracket, **H**, **H', J**) and postaxial oligodactyly (absent digit 5, red arrows in H' and J) in *Tbx3^fl/fl^;PrxCre* mutants at E15.5 (**H**, **H'**) and E19.5 (**J**). Note delayed ossification of the humerus (H, black arrowhead), loss of deltoid tuberosity (**J**, black arrowhead) and short, bowed ulna (**J**, black arrow) in mutant. s, scapula; h, humerus; oc, ossification center; dt, deltoid tuberosity r, radius; u, ulna; digits numbered 1--5 (**K**--**N**) *Sox9* mRNA expression shows evolving skeletal defects are already evident in *Tbx3^fl/fl^;PrxCre* mutants at E10.5- E11.5. Digit condensations are numbered. Bracket in L shows broadening of digit 1 forming region; red arrows highlight indentation in digit 5 forming region (**L**, **N**) and absence of Sox9 digit 5 condensation (**N**).**DOI:** [http://dx.doi.org/10.7554/eLife.07897.003](10.7554/eLife.07897.003)10.7554/eLife.07897.004Figure 1---figure supplement 1.Ablation of Tbx3 with PrxCre eliminates anterior and posterior mesenchymal protein production.From Frank et al., PLoSOne 2013, with permission. Confocal micrographs of sectioned E10.0 forelimb buds after fluorescent immunohistochemical detection of Tbx3 using custom antibody to its C-terminus. (**C1--C4**) Tbx3+/+ limb bud. C1) Merged color view of DAPI and FITC channels at 10X magnification. (**C2**--**C4**) 60X magnification of white boxed region in **C1**. (**C2**) DAPI channel showing DNA immunoreactivity. (**C3**) FITC channel showing Tbx3 immunoreactivity. (**C4**) Merged view. (**D1**--**C4**) *Tbx3^fl/fl^;PrxCre* limb bud. (**D1**) Merged color view of DAPI and FITC channels at low magnification. (**D2**--**C4**) 60X magnification of white boxed region in D1. (**D2**) DAPI channel showing DNA immunoreactivity. (**D3**) FITC channel showing lack of Tbx3 immunoreactivity in nucleus and cytoplasm. (**D4**) Merged view.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.004](10.7554/eLife.07897.004)10.7554/eLife.07897.005Figure 1---figure supplement 2.Increased severity of limb phenotypes in *Tbx3* null mutants (*Tbx3^Δfl/Δfl^*) compared to *Tbx3;PrxCre* is independent of Tbx3 in the AER.(**A**--**D**) Skeleton preparations comparing control (A: Tbx3*^Δfl/+^*, C:Tbx3*^fl/fl^), Tbx3^Δfl/Δfl^*(B, null), Tbx3*^fl/fl^* and *Tbx3^fl/fl^;PrxCre* (D, conditional mutant) forelimbs. Note single digit, absent ulna, and shortened humerus in *Tbx3^Δfl/Δfl^*mutant (**B**) compared to preaxial polysyndactyly and absent digit 5 in *Tbx3;PrxCre* mutants (**D**). s, scapula; h, humerus; r, radius; u, ulna; digits are numbered; red arrowhead highlights loss of digit 5. (**E**, **F**) X-gal stained E10.0 (**E**) and E11.5 (**F**) *Rosa^LacZ/+^;Fgf8^mcm/+^*embryos after the administration of tamoxifen at E8.5; black arrow indicates staining indicative of previous Cre activity in the AER. (**G**--**J**) mRNA in situ for *Tbx3* expression shows the absence of signal in the AER of *Tbx3^fl/fl^;Fgf8^mcm/mcm^*E9.5 and E10.5 mutants (**H**, **J**, respectively) compared to controls (**G**, **I**). White arrows point to AER in **G**--**J**; note persistent mesenchymal *Tbx3* expression as expected. (**K**--**N**) Skeleton preparations comparing E15.5 control (**K**,**M**), and *Tbx3 ^fl/fl^;Fgf8^mcm/mcm^* (**L**) and *Tbx3^fl/fl^;RarCre* (**N**) mutants. Forelimbs of *Tbx3 ^fl/fl^;Fgf8^mcm/mcm^* mutants are normal (**L**), while defects in *Tbx3^fl/fl^;RarCre* (**N**) phenocopy those of *Tbx3;PrxCre* mutants (compare panel N to D and also to [Figure 1](#fig1){ref-type="fig"}, panel **H**).**DOI:** [http://dx.doi.org/10.7554/eLife.07897.005](10.7554/eLife.07897.005)
Unlike mid-gestational lethality seen in constitutive *Tbx3* mutants ([@bib15]; [@bib22]), *Tbx3;PrxCre* mutants survive to adulthood with forelimb defects ([Video 1](#media1){ref-type="other"}): 100% have bilateral preaxial polysyndactyly of digit 1 (called PPD1 in humans \[[@bib56]\]), and 70% lack digit 5 ([Figure 1G--J](#fig1){ref-type="fig"}). Loss of digit 5 was bilateral in 6/18 and in the remaining, only affected the left forelimb ([Table 1](#tbl1){ref-type="table"}). This is not due to asymmetric activity of PrxCre because it is also observed in *Tbx3^Δfl/Δfl^*mutants (*^Δfl^* = recombined floxed conditional allele) where no Cre activity is involved ([@bib22]). Delayed ossification of the humerus ([Figure 1H](#fig1){ref-type="fig"}) and loss of the deltoid tuberosity ([Figure 1J](#fig1){ref-type="fig"}) were also observed. Abnormal limb bud morphology is evident by E10.5 ([Figure 1L](#fig1){ref-type="fig"}) and evolving skeletal defects at E11.5 by the altered pattern of *Sox9* expression ([Figure 1N](#fig1){ref-type="fig"}).10.7554/eLife.07897.006Table 1.Increased severity of left limb defects in Tbx3;PrxCre mutants.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.006](10.7554/eLife.07897.006)Skeletal phenotypes: E13.5-adultLoss of digit 5BilateralLeft onlyRight only*Tbx3 ^fl/+\ or\ fl/fl^*000*Tbx3^fl/fl^;PrxCre*6120Molecular phenotypes: gene expressionExpression pattern or levelLeft = RightLeft \>RightRight \> Left*Tbx3 ^fl/+\ or\ f/fl^*controlcontrolcontrol*Tbx3^fl/fl^;PrxCre*19301Video 1.Adult Tbx3;PrxCre mutant mouse is healthy and mobile despite forelimb deformities.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.007](10.7554/eLife.07897.007)10.7554/eLife.07897.007
*Tbx3;PrxCre* mutant limb defects are less severe than constitutive nulls {#s2-2}
-------------------------------------------------------------------------
Germline *Tbx3* null mutants (genotype *Tbx3^Δfl/Δfl^*) have more severe forelimb defects than *Tbx3;PrxCre* conditional mutants: of the few *Tbx3^Δfl/Δfl^* mutants that survive to E13.5, 100% have agenesis of the ulna and digits 3--5 ([Figure 1---figure supplement 2B](#fig1s2){ref-type="fig"}). Their hindlimbs have a single digit and no fibula ([@bib22]), phenocopying *Shh* and *Hand2* null mutants ([@bib23]). Variable timing of *Tbx3* loss of function by *PrxCre* may account for the disparate forelimb phenotypes of *Tbx3^Δfl/Δfl^*and *Tbx3;PrxCre* mutants, however, our skeletal data and phenotypes of *Tbx3^Δfl/fl^;PrxCre* mutants indicate that such variability manifests as incomplete penetrance of the ulnar and digit 5 defects ([Figure 1 H, H\',J](#fig1){ref-type="fig"}, and Colesanto et al., in preparation).
The AER is a critical signaling center, and *Tbx3* expression is preserved in the AER of *Tbx3;PrxCre* mutants ([Figure 1B,D,F](#fig1){ref-type="fig"}; [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}, [@bib22]). We tested whether AER Tbx3 has a required function using two Cre drivers: *RarbCre* (active in AER and mesenchyme from E9.0 \[[@bib63]\]), and a novel *Fgf8^mcm^* allele, which produces tamoxifen-inducible Cre in *Fgf8* expression domains (Moon et al., in preparation). Tamoxifen induction at E8.5 induces robust Cre activity in the AER in RosaLacZ/+;*Fgf8^mcm/+^* embryos ([Figure 1---figure supplement 2E,F](#fig1s2){ref-type="fig"}) and ablates *Tbx3* from forelimb AER by at least E9.5 ([Figure 1---figure supplement 2G--J](#fig1s2){ref-type="fig"}). *Tbx3^fl/fl^;Fgf8^mcm/mcm^*mutants have normal limbs ([Figure 1---figure supplement 2L](#fig1s2){ref-type="fig"}) and phenotypes of *Tbx3^fl/fl^;PrxCre* and *Tbx3^fl/fl^;RarbCre* are indistinguishable ([Figure 1---figure supplement 2D](#fig1s2){ref-type="fig"} versus N). The results with both Cre drivers indicate that the severe phenotypes of *Tbx3^Δfl/Δfl^*mutants are not due to a required function of *Tbx3* in the AER.
Tbx3 is required for normal limb bud initiation and *Tbx5* expression in the LPM {#s2-3}
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We next tested whether discrepant forelimb phenotypes in *Tbx3^Δfl/Δfl^* and *Tbx3;PrxCre* mutants reflect a role for *Tbx3* in an earlier expression domain than affected by *RarbCre* or *PrxCre*. Limb initiation in the LPM requires *Tbx5* expression in the prospective forelimb territory as early as the 8ss ([@bib61]), upstream of *Hand2* ([@bib1]). *Tbx3* is expressed in the LPM from E7.5 ([Figure 2A--C](#fig2){ref-type="fig"}'). Lineage tracing with a novel *Tbx3^mcm^*allele (Thomas et al., in preparation) revealed that *Tbx3*-expressing progenitors in the LPM at E8-8.5 give rise to most E10 forelimb mesenchyme in *Tbx3^mcm/+^;Rosa^LacZ/+^* embryos ([Figure 2D](#fig2){ref-type="fig"}). Consistent with a role for Tbx3 in limb initiation, *Tbx3^Δfl/Δfl^*mutants have decreased LPM expression of *Tbx5* ([Figure 2E,F](#fig2){ref-type="fig"}), visible defects in forelimb initiation and early limb bud morphology ([Figure 2G,H](#fig2){ref-type="fig"}), and disrupted expression of *Hand2* ([Figure 2I,J](#fig2){ref-type="fig"}). In contrast, early stage *Tbx5* expression and limb bud initiation are unaffected in *Tbx3;PrxCre* mutants ([Figure 2---figure supplement 1B,B\'](#fig2s1){ref-type="fig"})10.7554/eLife.07897.008Figure 2.Tbx3 is required for normal limb bud initiation.(**A**, **B**) *Tbx3* expression at E7.5 (**A**) and E8.5 (**B**). Anterior on left (**A**), posterior on right (P). NM (black arrow) indicates nascent mesoderm exiting primitive streak in panel **A**. (**C**, **C'**) *Tbx3* expression in the LPM (black arrow) of sectioned E8.5 embryo. Plane of section indicated by line in B. Panel D is magnification of red-boxed area in **C**. (**D**) X-gal stained E10.0 *Tbx3^MCM/+^; Rosa ^LacZ/+^* embryo after Cre induction at E8.5. FL, forelimb; HL, hindlimb. (**E**, **F**) 21 somite stage (ss) embryos assayed for *Tbx5* mRNA. White arrows denote forelimb bud. Left sided view. (**G**, **H**) Dorsal view of budding forelimbs of 24 ss embryo forelimbs (neural tube stained for *Shh* expression). Note abnormal shape and size of *Tbx3^Δfl/Δfl^*mutant forelimb buds indicative of disrupted initiation; white brackets are of equal size in both panels. (**I**, **J**) 22 ss embryos assayed for *Hand2* expression. Black arrows denote emerging forelimb bud.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.008](10.7554/eLife.07897.008)10.7554/eLife.07897.009Figure 2---figure supplement 1.Early *Tbx5* expression is normal in *Tbx3^fl/fl^;PrxCre* mutants.(**A--B'**) In situ hybridization for *Tbx5* mRNA on E9.5 control (**A**, **A'**) embryo versus *Tbx3;PrxCre* mutant (**B**, **B'**). Left sided views in A and B and dorsal views of dissected torsos with forelimbs in **A'**, **B'**. *Tbx5* expression and limb initiation are normal after conditional loss of Tbx3 in the limb bud mesenchyme. (**C**--**D'**) In situ hybridization for *Hand2* mRNA on E9.5 control (**A**, **A'**) embryo versus *Tbx3;PrxCre* mutant (**B**, **B'**). Left- sided views in C and D and dorsal views of dissected limbs in **C'**, **D'**. *Hand2* expression is affected by conditional loss of Tbx3 in limb bud mesenchyme, but not as severely as in *Tbx3^Δfl/Δfl^* mutants shown in [Figure 2J](#fig2){ref-type="fig"}. Boxed area in D encloses forelimb forming region shown from dorsal view in **D'**.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.009](10.7554/eLife.07897.009)
TBX3 positively regulates posterior digit development via the Hand2/Shh pathway in posterior mesenchyme {#s2-4}
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Our data reveal required functions for Tbx3 in limb initiation (demonstrated by *Tbx3^Δfl/Δfl^*mutants) and in later limb bud morphogenesis (demonstrated by *Tbx3;PrxCre* mutants). Most *Tbx3;PrxCre* mutants lack digit 5, whose specification and formation depend on \'early phase\' Shh signaling beginning at E9.5 ([@bib26]; [@bib80]; [@bib104]; [@bib105]). Hand2 protein is required to activate *Shh* expression in the limb bud ([@bib8]; [@bib23]). We found that *Hand2* transcripts are reduced in E9.5 and E10.5 *Tbx3;PrxCre* mutant forelimb buds ([Figure 2---figure supplement 1D,D\'](#fig2s1){ref-type="fig"}; [Figure 3A, A\',F](#fig3){ref-type="fig"}) as is *Shh* expression ([Figure 3B,B\',F](#fig3){ref-type="fig"}; [Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}). Expression of two targets and effectors of Shh signaling, *Ptch1* and *Grem1*, is also markedly reduced ([Figure 3C\',E\'](#fig3){ref-type="fig"}; [Figure 3---figure supplement 2](#fig3s2){ref-type="fig"}). *Tbx3* expression in posterior limb mesoderm begins earlier than in the anterior compartment ([Figure 1A,C](#fig1){ref-type="fig"}) and is required for normal *Hand2* in posterior mesoderm ([Figure 3A](#fig3){ref-type="fig"}, [Figure 2J](#fig2){ref-type="fig"} and [Figure 2---figure supplement 1D](#fig2s1){ref-type="fig"}') ([@bib75]). Thus, intact *Tbx5* expression in *Tbx3;PrxCre* E9.5 forelimbs ([Figure 2---figure supplement 1B](#fig2s1){ref-type="fig"}') indicates that post-initiation, Tbx3 functions downstream of Tbx5 and upstream of *Hand2*.10.7554/eLife.07897.010Figure 3.Loss of mesenchymal Tbx3 disrupts Shh signaling in the posterior limb bud and decreases Gli3 protein stability.(**A**--**E'**) In situ hybridization of control and mutant forelimb buds with probes and at embryonic stages as labeled. (**F**) qPCR of E10.75 (36-39ss) limb buds for transcripts listed confirms findings by detected by in situ. (**G**--**I**') In situ hybridization for *Zic3, Epha3* and *Hoxd13* transcripts in forelimb buds of *Tbx3 ^fl/+^*controls (**K**--**M**) and *Tbx3;PrxCre* mutants (**G'**--**I'**) at ages noted on panels. J) qPCR assay of *Zic3, Epha3, Hoxd13* transcript levels confirms findings detected by in situ. (**K**--**L'**) Representative images of E10.5 forelimb buds stained for DAPI (blue), pHH3 (green), TUNEL (red). K is *Tbx3* ^fl/+^ control and K' is digital zoom of posterior mesenchymal boxed area in K. Panel L is *Tbx3;PrxCre* mutant and L' is digital zoom of boxed area in L. This experiment is representative of data obtained from five biologic replicates. (**M**) Quantification of proliferating cells in anterior and posterior mesenchymal regions encompassing digit 1 and digit 5 progenitors from 20 control and 15 mutant sections. \*p=0.02. Control anterior limb (CA), *Tbx3;PrxCre* mutant anterior limb (MA), control posterior (CP), and mutant posterior (MP). (**N**--**O'**) Representative images of E11.5 whole mount forelimb buds stained for DAPI (blue) and pHH3 (green). N is *Tbx3* ^fl/+^ control and N' is digital zoom of boxed area. Panel O is *Tbx3;PrxCre* mutant and O' is digital zoom of boxed area in O. Note decreased pHH3+ cells in mutants, particularly cells in prophase and anaphase, which have the faint and speckled patterns compared to the bright staining of highly condensed S-phase chromatin. (**P**) Quantification of proliferating cells in anterior and posterior mesenchymal regions encompassing digit 1 and digit 5 progenitors from 50 control and 44 mutant sections at E11.5. \*p\<0.1. Control anterior limb (CA), *Tbx3;PrxCre* mutant anterior limb (MA), control posterior (CP), and mutant posterior (MP).**DOI:** [http://dx.doi.org/10.7554/eLife.07897.010](10.7554/eLife.07897.010)10.7554/eLife.07897.011Figure 3---figure supplement 1.Decreased Shh expression in E10.5 forelimb buds of Tbx3;PrxCre mutants.(**A**, **B**) Whole mount in situ hybridization for *Shh* transcripts on E10.5 embryos; left -sided views. (**C**, **D**) Dissected limbs from whole mount in situ hybridization for *Shh* transcripts on E11.0 embryos; dorsal views of left limb buds are shown.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.011](10.7554/eLife.07897.011)10.7554/eLife.07897.012Figure 3---figure supplement 2.No evidence of ectopic hedgehog pathway activity in Tbx3;PrxCre mutant forelimbs.(**A**, **B**) In situ hybridization for *Ptch1* at E11.5 in control (**A**) versus *Tbx3;PrxCre* mutant (**B**) limb buds. *Ptch1* expression is decreased in posterior mesenchyme, consistent with results in [Figure 3](#fig3){ref-type="fig"}. There is no ectopic *Ptch1* signal in anterior mesenchyme. Note decreased size of *Ptch1* negative zone in posterior mesenchyme (white ellipses), consistent with loss of digit 5 progenitors which are unresponsive to Shh signaling at this stage ([@bib79]) [@bib2]). a, anterior; p, posterior (**C**, **D**). In situ hybridization for *Grem1* at E11.5 reveals decreased expression throughout limb, consistent with qPCR and in situ results shown in [Figure 3](#fig3){ref-type="fig"} at E10.75.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.012](10.7554/eLife.07897.012)10.7554/eLife.07897.013Figure 3---figure supplement 3.Microdissection of E11 forelimb buds into anterior and posterior compartments for gene and protein expression analyses.(**A**) Intact left limb bud after in situ hybridization for *Tbx3* mRNA. Anterior (ant) at top, posterior (post) at bottom. (**B**, **C**) Microdissected anterior and posterior Tbx3+ compartments and list of example genes whose expression is confined to, or enriched in, each compartment.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.013](10.7554/eLife.07897.013)10.7554/eLife.07897.014Figure 3---figure supplement 4.qPCR of additional key transcripts in anterior and posterior forelimb compartments at E10.5--10.75 (36--39 somite stages).**DOI:** [http://dx.doi.org/10.7554/eLife.07897.014](10.7554/eLife.07897.014)10.7554/eLife.07897.015Figure 3---figure supplement 5.*Fgf8* expression and downstream in *Tbx3;PrxCre* mutant forelimb buds.(**A--F**) In situ hybridization for the transcripts listed on panels at ages specified. (**A**, **B**) View of AER stained for *Fgf8*. (**C**--**F**) Dorsal view of left forelimb buds stained for *Erm (Etv5)* and *Pea3 (Etv4)* transcripts which are regulated by FGF signaling in the limb bud.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.015](10.7554/eLife.07897.015)
To obtain a more comprehensive view of the transcriptional consequences of loss of Tbx3 in limb bud mesenchyme, we assayed gene expression of E10.25 limb buds (32--34 somite stage) by microarray. *Shh* and other hedgehog pathway members and target genes ([@bib45]; [@bib72]; [@bib90]; [@bib57])([@bib46]) were present among the significantly dysregulated transcripts ([Supplementary file 1](#SD12-data){ref-type="supplementary-material"}) consistent with the previous report of decreased *Shh* expression in *Tbx3^tm1Pa/tm1Pa^* mutant limb buds ([@bib15]). In addition to decreased levels of Shh- activated transcripts (*Gli1, Hand2, Osr1, Dkk1, Tbx2, Cntfr, Pkdcc*), we noted increased *Rprm, Zic3, Hand1,* and *Gli3* transcripts and confirmed these with qPCR and in situ hybridization at E10.5--11 ([Figure 3](#fig3){ref-type="fig"}, [Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}). The increase in expression of these putative targets of the Gli3 repressor ([@bib45]; [@bib72]; [@bib90]; [@bib57]) was intriguing as it suggested the possibility of alterations in both Gli3 activator and repressor function in *Tbx3;PrxCre* mutant forelimbs.
The transcriptional profiles of anterior and posterior limb mesenchyme are quite different: alterations in gene expression in either compartment in response to Tbx3 could mask some changes in the other if assayed simultaneously. Thus, we proceeded to microdissect anterior and posterior limb segments and assayed them independently using RNA-sequencing on 38--42 somite stage (\~E11; this later stage was needed in order to obtain sufficient RNA from accurately microdissected limb segments) wild type and *Tbx3;PrxCre* mutant forelimb buds ([Figure 3---figure supplement 3](#fig3s3){ref-type="fig"}). The resulting RNA-Seq data confirmed accurate dissection with the expected distribution of known compartment-specific transcripts ([Figure 3---figure supplement 3](#fig3s3){ref-type="fig"} and [Supplementary file 2](#SD13-data){ref-type="supplementary-material"}). *Shh, Fgf4* and anterior *Hoxd* family transcripts were over-represented in the posterior compartment, and *Alx* and *Pax* family members in the anterior.
Largely consistent with the previous microarray findings, we found evidence of aberrant cell differentiation/fate of posterior mesenchyme with downregulation of Shh-activated targets (*Osr1, Dkk1, Tbx2, Cntfr, Ptch2*, [Supplementary file 3](#SD14-data){ref-type="supplementary-material"}) that validated by qPCR ([Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}). It is known that *Gli3* expression increases with decreased Shh activity ([@bib91]), as we see here ([Figure 3D', F](#fig3){ref-type="fig"}). Although decreased levels of *Hand2, Shh, Ptch1 and Grem1* are clearly evident by in situ and qPCR at this stage ([Figure 3](#fig3){ref-type="fig"} and [figure upplements](#fig3s1){ref-type="fig"}), they were not detected on the RNA-Seq analysis for unclear reasons.
Shh signaling is required for proliferation to ensure sufficient cell numbers to form the normal complement of digits, and loss of Shh results in an increase in the number of cells in G1 arrest ([@bib105]). Assay of cell proliferation in E10.5 and E11.5 limb buds using anti-phosphohistone H3 immunohistochemistry revealed that at E10.5 there was a statistically significant decrease in the fraction of proliferating cells in the posterior mesenchyme ([Figure 3K--M](#fig3){ref-type="fig"}). At E11.5, proliferation was significantly decreased in both the anterior and posterior mesenchyme, indicating a global reduction in the number of mitotic cells in mutants ([Figure 3N--P](#fig3){ref-type="fig"}). This suggests that 5th digit agenesis is attributable, at least in part, to decreased cell number, as opposed to decreased proliferation specifically in digit 5 progenitors. Assay for apoptosis using TUNEL showed normal levels of anterior AER cell death at E10.5, as we have previously reported in this region ([@bib63]) ([Figure 3K,L](#fig3){ref-type="fig"}).
Proliferation of limb mesenchyme depends on activity of FGF8 and FGF4 from the AER ([@bib5]; [@bib63]; [@bib84]) and integrity of this structure requires Shh activity in posterior mesenchyme ([@bib13]). Despite decreased *Shh* expression in *Tbx3;PrxCre* mutants, *Fgf4* transcripts were increased in posterior mesenchyme while decreased in anterior (detected by RNA-Seq, [Supplementary files 3](#SD14-data){ref-type="supplementary-material"},[4](#SD15-data){ref-type="supplementary-material"} and qPCR, [Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}), the latter consistent with an expanded digit 1 region. qPCR detected increased *Fgf8* expression in the posterior AER ([Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}). Despite these changes in transcript levels, there was no evidence of altered downstream FGF signaling as expression of *Etv4, Etv5, Dusp6* and *Sprys* was unchanged ([Figure 3---figure supplement 5](#fig3s5){ref-type="fig"}, note these transcripts are not listed in [Supplementary files 3 or 4](#SD14-data){ref-type="supplementary-material"} because they did not meet criteria for differential expression). We conclude that despite the decrement in Shh pathway activity in posterior mesenchyme of *Tbx3;PrxCre* mutants, the level is sufficient to maintain ectodermal FGF signaling, consistent with preserved limb outgrowth.
Tbx3 stabilizes Gli3 protein in anterior limb mesenchyme {#s2-5}
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To understand the cause of the anterior PPD phenotype in *Tbx3;PrxCre* mutants, we pursued molecular mechanisms known to cause this defect in humans and mice: ectopic Hedgehog pathway activity ([@bib31]; [@bib32]; [@bib45]); decreased Gli3R activity ([@bib30]; [@bib65]; [@bib94]); and abnormal composition or function of the primary cilia ([@bib24]).
RNA-Seq analysis of control versus mutant anterior compartments ([Supplementary file 4](#SD15-data){ref-type="supplementary-material"}) showed no evidence of ectopic hedgehog activity in *Tbx3;PrxCre* mutants, and this was confirmed by in situ hybridization and qPCR for *Shh* and *Ptch1* ([Figure 3B--C\'](#fig3){ref-type="fig"}, [Figure 3---figure supplements 1](#fig3s1){ref-type="fig"} and [2](#fig3s2){ref-type="fig"}). Although *Gli3* transcripts were increased in mutant limb buds ([Figure 3D\',F](#fig3){ref-type="fig"}; [Supplementary file 1](#SD12-data){ref-type="supplementary-material"}), targets of Gli3R transcriptional repression such as *Zic3, Epha3, Hoxd13* ([@bib57]; [@bib90]) were overexpressed when assayed by microarray, RNA-Seq, qPCR and in situ hybridization ([Figure 3 G-J](#fig3){ref-type="fig"}, [Supplementary files 1](#SD12-data){ref-type="supplementary-material"},[3](#SD14-data){ref-type="supplementary-material"},[4](#SD15-data){ref-type="supplementary-material"}).
The discrepancy between *Gli3* RNA levels and increased expression of some repressor targets prompted examination of Gli3 protein levels. Gli3R constitutes the vast majority of Gli3 protein species in the anterior limb bud ([@bib91]) and [Figure 4A](#fig4){ref-type="fig"}). Gli3R protein was markedly decreased (7.4 fold on this representative immunoblot) with multiple bands of lower molecular weight than Gli3R present specifically in *Tbx3;PrxCre* mutant anterior mesenchyme (mutant anterior compartment: MA, [Figure 4A](#fig4){ref-type="fig"}, red box). Gli3FL was virtually undetectable in mutant anterior mesenchyme ([Figure 4A](#fig4){ref-type="fig"}'). This finding is not due to poor sample quality because it was reproducible (N=3), no degradation was present in simultaneously prepared posterior compartment lysates (mutant posterior, MP), and the β−tubulin control was intact. These findings indicate that Tbx3 is required for stability of Gli3FL and Gli3R proteins in the anterior limb mesenchyme, and are consistent with the PPD phenotype observed here and in other models of Gli3R deficiency ([@bib30]; [@bib65]; [@bib94]).10.7554/eLife.07897.016Figure 4.Loss of Tbx3 results in Gli3 protein instability and aberrant localization of Kif7 in limb bud cilia.(**A**) Representative immunoblot (N=3) blot of E10.75 forelimb bud lysates prepared from microdissected *Tbx3^fl/+^*control anterior limb (CA), *Tbx3;PrxCre* mutant anterior limb (MA), control posterior (CP), and mutant posterior (MP) probed for Gli3 and βtubulin loading control. Note decreased level of Gli3FL and Gli3R, and multiple bands of lower molecular weight than Gli3R in MA sample. Densitometry of Gli3R bands in red box in N revealed that in this representative experiment, the level of Gli3R was 7.4 fold decreased in mutant anterior relative to control anterior. (**A'**) Longer exposure of top of blot shown in panel A to examine Gli3FL band. The control (CA) Gli3FL band is 31 fold more intense than mutant (MA, virtually undetectable). (**B**) Immunoblot of lysates from E10.5 forelimb buds immunoprecipitated (IB) with antibodies listed at top and immunoblotted (IB) for Tbx3. Lane 5 shows that immunoprecipitation with anti-Kif7 antibody co-IPs Tbx3. (**C**) As in panel B, but assayed for Kif7. (**D**) Co-IP assay of Myc-tagged Tbx3 and Flag-tagged GFP overexpressed in HEK293 cells. IP was performed with antibodies listed at top and immunoblotted for Tbx3. Myc-tagged Tbx3 co-IPs with Flag-tagged Kif7. Input lane was 5 s exposure (5" exp) while other lanes were 15 s (15" exp). (**E**) As in **D**, but in this case, blot probed for Kif7; confirms interaction of tagged, overexpressed proteins. (**F**, **G**) Representative images of anterior mesenchyme in sectioned forelimbs of control (**F**, *Tbx3^fl/fl^*) and mutant (G, *Tbx3^fl/fl^;PrxCre*) E10.5 embryos stained for the ciliary marker Arl13b (red), Kif7 (green) and DAPI (DNA, blue). White arrowheads highlight cilia with multiple punctae or streak of ciliary Kif7 immunoreactivity (yellow) indicating translocation of Kif7 within the cilia. Please also see [Figure 5---source data 2](#SD3-data){ref-type="supplementary-material"} for z-stacks of additional Kif7 stained limb section. (**H**) Quantification of Kif7 staining pattern from multiple limb sections and three embryos of each genotype scored blinded to genotype. 10% fewer cilia have evidence of Kif7 translocation (multiple punctae or streak of Kif7 immunoreactivity) in *Tbx3^fl/fl^;PrxCre* mutants. N=1785 and 1792 cilia scored in controls and mutants, respectively. \* p\<0.001 There was no difference in the number of Kif7- cilia. Insets show digital zoom of cilia with representative pattern used for scoring. (**I**) Immunoblot assaying for Kif7 and b tubulin loading control in control anterior (CA), mutant anterior (MA), control posterior (CP) and mutant posterior (MP) e10.5 forelimb bud lysates. This is representative of four such experiments. (**J**) Western blot assaying for Sufu protein and b tubulin loading control in eE10.5 forelimb buds. Like *Kif7* mutants,*Tbx3;PrxCre* mutants have increased Sufu protein. Sufu is increased 1.8 fold when normalized to loading control in this representative immunoblot; N=4. K) Western blot assaying for Spop protein and actin loading control in E10.5 forelimb buds. Spop is increased 1.5 fold when normalized to loading control in this representative immunoblot; N=3. Both Sufu and Spop protein levels are increased in mutant forelimbs although their transcript levels are unchanged ([Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}).**DOI:** [http://dx.doi.org/10.7554/eLife.07897.016](10.7554/eLife.07897.016)10.7554/eLife.07897.017Figure 4---source data 1.CZI file containing z-stack of E10.5 sectioned limb shown in [Figure 4---figure supplement 1](#fig4s1){ref-type="fig"}.Kif7 is green, Arl13b red, DNA blue. Gli3 signal can be viewed if desired in the violet channel (channel 2). The entire z stack can be viewed using the free download of Zen software: <http://www.zeiss.com/microscopy/en_de/downloads/zen.html>.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.017](10.7554/eLife.07897.017)10.7554/eLife.07897.018Figure 4---figure supplement 1.Kif7 is also present in cytoplasm and nucleus.100 X confocal maximum image projection of E10.5 sectioned limb stained for Kif7 (green), Arl13b (red) and DNA (blue). Please also see [Figure 5---source data 2](#SD3-data){ref-type="supplementary-material"} for z-stack.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.018](10.7554/eLife.07897.018)10.7554/eLife.07897.019Figure 4---figure supplement 2.Anterior mesenchymal limb cilia are bigger in Tbx3;PrxCre mutants compared to controls.(**A**) Distribution of cilia volumes in control and mutant limb buds. (**B**) Average cilia volumes and 95% confidence intervals. (**C**) Average surface area to volume ratios over the range of cilia volumes measured. (**D**) Calculated of surface area and volume of both WT and mutant cilia fit the equation: Surface=8.01 X Volume^0.69^**DOI:** [http://dx.doi.org/10.7554/eLife.07897.019](10.7554/eLife.07897.019)
TBX3 interacts with Kif7 and is required for normal Kif7 trafficking {#s2-6}
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In an independent experiment to identify Tbx3 interacting partners, we performed Tbx3 co-immunoprecipitation (co-IP) on E10.5 mouse embryo lysates, followed by mass spectrometry ([@bib42]). Surprisingly, Kif7 was among the Tbx3 interacting proteins identified. Kif7 is a ciliary protein that modulates activity of the Hedgehog pathway ([@bib34]). It is required for proper formation of a \'cilium tip\' compartment that regulates Gli function ([@bib29]), and for the regulated, partial proteolytic processing of GliFL to Gli3R ([@bib10]; [@bib12]; [@bib17]; [@bib43]; [@bib77]). As in *Tbx3;PrxCre* and *Gli3^+/-^* mutants ([@bib30]), human and mouse *Kif7* mutants have PPD ([@bib12]; [@bib17]; [@bib73]).
Immunoprecipitation of protein lysates from E10.5 forelimb buds showed that Tbx3 co-immunoprecipitates (co-IPs) with Kif7, confirming interaction of Kif7 and Tbx3 in the developing limb ([Figure 4B](#fig4){ref-type="fig"}; specificity and efficiency of Kif7 IP in this experiment is shown in [Figure 4C](#fig4){ref-type="fig"}). We next tested whether these proteins directly interact by overexpressing Flag-tagged Kif7 and Myc-tagged Tbx3 in HEK293 cells and immunoprecipitating for either Flag or Myc, followed by immunoblotting for Tbx3 ([Figure 4D](#fig4){ref-type="fig"}) or Kif7 ([Figure 4E](#fig4){ref-type="fig"}). Both experiments confirmed direct interaction of the tagged proteins.
The interaction of Tbx3 with Kif7, and the shared PPD phenotypes of *Kif7* and *Tbx3;PrxCre* mutants, suggest that Tbx3 may be required for normal Kif7 function in the anterior limb bud, and that loss of Tbx3 may disrupt Gli3 stability and processing in part via a Kif7-dependent mechanism.
We examined Kif7 localization in E11 control and mutant forelimb buds, co-staining for the cilia marker Arl13b. Confocal fields spanning the anterior mesenchyme of controls and mutants were imaged and [Figure 4F and G](#fig4){ref-type="fig"} are representative 40X fields (a higher magnification image and confocal z-stack, which also show Kif7 in the cytoplasm and nucleus are shown in [Figure 4---figure supplement 1](#fig4s1){ref-type="fig"} and [Figure 4---source data 1](#SD1-data){ref-type="supplementary-material"}). Blinded to genotype, fields were scored for ciliary Kif7 immunoreactivity as a single puncta, multiple punctae/streak, or none (N=1785 and 1792 cilia scored in controls and mutants, respectively). In 16% of control cilia, Kif7 was detected in two punctae (presumed base and tip) or as a streak along the cilia, but this was only the case in 6% of mutant cilia ([Figure 4H](#fig4){ref-type="fig"}, p\<0.001). There was no significant difference in the number of Kif7+ cilia rather, there were more single puncta cilia in mutants than controls ([Figure 4H](#fig4){ref-type="fig"}, 84% vs 71%, p\<0.001). We did not detect any difference in the amount of Kif7 protein in control versus mutant limb bud compartments by western blot ([Figure 4I](#fig4){ref-type="fig"}, representative of four4 separate experiments). Levels of *Kif7* mRNA in the anterior limb mesenchyme were unaffected by loss of Tbx3 ([Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}). There was a decrease in the transcripts posterior compartment but as shown, the protein level was unchanged.
One feature of *Kif7^-/-^*mutants is excess Sufu due to increased protein stability ([@bib33]). Transcript levels of *Sufu* were unchanged in mutant forelimbs ([Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}, no difference was detected by microarray or RNA-Seq). However, increased amounts of Sufu (and Spop) protein were present in mutant limb buds ([Figure 4J,K](#fig4){ref-type="fig"}; increased 1.5 fold in both cases).
Humans and mice with *Kif7* mutations have abnormally long cilia because Kif7 reduces the rate of microtubule growth in the ciliary axoneme ([@bib29]; [@bib73]). With 3D images obtained from 100X confocal z-stacks of Arl13b stained limb sections, we used the 3D object counter from ImageJ to calculate the volume and surface area to volume ratio to derive the length of cilia. Consistent with aberrant, but not absent, Kif7 function, we found that while the range of cilia volumes detected were the same between mutants and controls, the distribution was not: mutants have an increased fraction of larger cilia and an average volume 18% greater ([Figure 4---figure supplement 2A, B](#fig4s2){ref-type="fig"}; 475 and 575 cilia assayed in controls and mutants, respectively). Mutants and controls had superimposable surface area/volume ratios ([Figure 4---figure supplement 2C](#fig4s2){ref-type="fig"}), indicating that the shape of cilia was not different thus, the derived length was 6% greater in mutants.
Together, these findings indicate that loss of Tbx3 results in aberrant ciliary localization of Kif7 and are consistent with abnormal Kif7 function.
Tbx3 is present in primary cilia and translocates in response to Hedgehog pathway activity {#s2-7}
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Kif7 and other proteins required for Gli3 processing and function are present in, or translocate to, primary cilia in response to Hedgehog pathway activity ([@bib24]; [@bib77]). Dual immunostaining for Tbx3 and Arl13b on whole mount optically sectioned E10.5 forelimbs shows that Tbx3 is present in control limb anterior mesenchymal cilia ([Figure 5 A-E](#fig5){ref-type="fig"}, [Figure 5---source data 1](#SD2-data){ref-type="supplementary-material"}). Specificity of Tbx3 staining was confirmed by loss of signal in mesenchymal cilia of *Tbx3;PrxCre* mutant limbs ([Figure 5F-J](#fig5){ref-type="fig"}, [Figure 5---source data 2](#SD3-data){ref-type="supplementary-material"}). The digital image overlap calculator in Zen software showed that 18/50 anterior mesenchymal cilia were Tbx3+ (36%, [Figure 5---figure supplement 1A,B](#fig5s1){ref-type="fig"}). Of note, no epithelial cilia were Tbx3+ in control limb epithelium, providing an internal negative control for the signal in mesenchymal cilia. This same calculation in *Tbx3;PrxCre* mutant anterior mesenchyme showed only 2/54 (\<4%) of mesenchymal cilia had background Tbx3 signal ([Figure 5---figure supplement 1, C, D](#fig5s1){ref-type="fig"}). These findings were reproduced with a commercially available anti-Tbx3 antibody (Abcam ab99302) which also showed Tbx3 ciliary staining on control limb mesenchymal cilia (24/87, 28%) that was virtually absent in *Tbx3* mutant limbs (2/52, \<4%; [Figure 5---figure supplement 2](#fig5s2){ref-type="fig"}).10.7554/eLife.07897.020Figure 5.Tbx3 localizes to the primary cilia in limb mesenchyme.(**A--D**, **F**--**I**) Confocal 100X single Z-plane immunofluorescence images from optically sectioned E10.5 control (top panels **A**--**D**) and *Tbx3;PrxCre* (**F**--**I**) anterior limb buds after immunostaining with: Hoechst (DNA, blue), C-terminal anti-Tbx3 antibody (green, [@bib22]), anti-Arl13b (red, cilia). Arrowheads demarcate Tbx3 colocalization with cilia marker. Panels E and J are further digital zooms of white boxed cells in D and I. The entire z-stacks containing these planes are in [Figure 5---source data 1](#SD2-data){ref-type="supplementary-material"},[2](#SD3-data){ref-type="supplementary-material"}.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.020](10.7554/eLife.07897.020)10.7554/eLife.07897.021Figure 5---source data 1.Czi file of z-stack through the region of control anterior limb shown in [Figure 5A--E](#fig5){ref-type="fig"}.The entire z stack can be viewed using the free download of Zen software <http://www.zeiss.com/microscopy/en_de/downloads/zen.html>.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.021](10.7554/eLife.07897.021)10.7554/eLife.07897.022Figure 5---source data 2.Czi file of z-stack through region of mutant anterior limb shown in [Figure 5F--J](#fig5){ref-type="fig"}.Please view as described above.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.022](10.7554/eLife.07897.022)10.7554/eLife.07897.023Figure 5---figure supplement 1.Digital image overlap of Tbx3 and Arl13b in limb bud anterior mesenchyme.(**A**) Maximum image projection of Arl13b channel from control limb z-stack shown in [Figure 5---source data 1](#SD2-data){ref-type="supplementary-material"}. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (**B**) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in control limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 50 mesenchymal cilia, 18 (36%) are Tbx3 positive. Please see Experimental Procedures for use of Zen and Image J software to calculate pixel overlap in separate channels. (**C**) Maximum image projection of Arl13b channel from control limb z-stack shown in [Figure 5---source data 2](#SD3-data){ref-type="supplementary-material"}. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (**D**) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in *Tbx3;PrxCre* limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 54 mesenchymal cilia, 2 (4%) are Tbx3 positive consistent with low level of background antibody staining in mutant ([Figure 5](#fig5){ref-type="fig"}, panels **G**, **I**).**DOI:** [http://dx.doi.org/10.7554/eLife.07897.023](10.7554/eLife.07897.023)10.7554/eLife.07897.024Figure 5---figure supplement 2.Tbx3 immunoreactivity in limb cilia is also detected by a commercial anti-Tbx3 antibody against the N-terminus of Tbx3.(**A**) Maximum image projection of Arl13b channel from control forelimb z-stack. (**B**) Calculated digital image overlap (see Methods section) of Arl13b (cilia) and Tbx3 positive pixels in control limb bud shown above using Abcam (Abcam ab99302) anti-Tbx3 antibody to the N-terminus of mouse Tbx3. 27/97 (28%) of mesenchymal cilia are Tbx3+. (**C**) Maximum image projection of Arl13b channel from *Tbx3;PrxCre* mutant forelimb z-stack. (**D**) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in *Tbx3;PrxCre* forelimb bud shown above. 2/52 mesenchymal cilia are Tbx3+. (**E**) Scatter plot obtained using ImageJ comparing Tbx3 and Arl13b intensities from control and mutant anterior forelimb buds stained with Abcam anti-Tbx3 antibody shown in **A--D**.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.024](10.7554/eLife.07897.024)
Murine embryonic fibroblasts (MEFs) are a robust system for studying ciliary proteins ([@bib10]; [@bib16]; [@bib47]; [@bib67]; [@bib76]), so we used them to further explore Tbx3 localization and trafficking. In untreated wild type MEFs, our custom C-terminal antibody detected Tbx3 in a subset of cilia (30%, N=56; [Figure 6A and B, a1-a5, b1-b5](#fig6){ref-type="fig"}; [Figure 6---source data 1](#SD4-data){ref-type="supplementary-material"},[2](#SD5-data){ref-type="supplementary-material"}). No Tbx3+ cilia were detected in *Tbx3* null MEFS ([Figure 6C--F](#fig6){ref-type="fig"}; [Figure 6---source data 3](#SD6-data){ref-type="supplementary-material"}). Treatment of wild type MEFs with the smoothened agonist SAG increased the number of Tbx3+ cilia from 30% to 75% ([Figure 6G-J](#fig6){ref-type="fig"} p\<0.005, [Figure 6---source data 4](#SD7-data){ref-type="supplementary-material"}, [Figure 6---figure supplement 1A](#fig6s1){ref-type="fig"}). Lysates from control and SAG-treated MEFs showed no detectable difference in Tbx3 protein levels indicating the increased Tbx3 signal in cilia was due to trafficking rather than increased protein levels ([Figure 6---figure supplement 1B](#fig6s1){ref-type="fig"}). The presence of Tbx3 in cilia and response to Hedgehog pathway activity were also detected with a commercially available anti-Tbx3 antibody with both SAG and Shh stimulation ([Figure 6---figure supplement 1C,D](#fig6s1){ref-type="fig"} and [Figure 6---source data 5](#SD8-data){ref-type="supplementary-material"},[6](#SD9-data){ref-type="supplementary-material"}). In total, these data show that Tbx3 is present at baseline in cilia, and is trafficked to cilia in response to hedgehog signaling.10.7554/eLife.07897.025Figure 6.Tbx3 is present in some cilia at baseline in Murine Embryonic Fibroblasts and trafficks to cilia in response to hedgehog pathway activation.(**A**, **B**) Confocal, 100X single z-plane immunofluorescence images from two different fields of wild type MEFS after immunostaining for: DAPI (DNA, blue), Tbx3 (green, c-terminal anti-Tbx3 antibody; [@bib22]), Arl13b (red, cilia). White boxed regions outline single cells that are shown at higher magnification in panels **a1--a5** and **b1**--**b5**. Please see [Figure 6---source data 1](#SD4-data){ref-type="supplementary-material"},[2](#SD5-data){ref-type="supplementary-material"} for z-stacks. **a1**--**a4**, **b1**--**b4**) Single cells from white boxed areas in panels A and B. Individual cilia are shown in a5 and b5. White arrowheads highlight Tbx3+ cilia. (**C**--**F**) Tbx3 null MEFs show loss of Tbx3 immunoreactivity in cilia and other cellular locations. Please see [Figure 6---source data 3](#SD6-data){ref-type="supplementary-material"} for z-stack. (**G**--**J**) As in **A** and **B**, but MEFs were treated with smoothened agonist (SAG). White arrowheads highlight Tbx3+ cilia. Please see [Figure 6---source data 4](#SD7-data){ref-type="supplementary-material"} for z-stack.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.025](10.7554/eLife.07897.025)10.7554/eLife.07897.026Figure 6---source data 1.Czi file showing z-stack of wild type MEFs imaged in [Figure 6](#fig6){ref-type="fig"} panel A.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.026](10.7554/eLife.07897.026)10.7554/eLife.07897.027Figure 6---source data 2.Czi file showing z-stack of wild type MEFs imaged in [Figure 6](#fig6){ref-type="fig"} panel B.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.027](10.7554/eLife.07897.027)10.7554/eLife.07897.028Figure 6---source data 3.Czi file showing z-stack of Tbx3 null MEFs imaged in [Figure 6](#fig6){ref-type="fig"} panel C--F.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.028](10.7554/eLife.07897.028)10.7554/eLife.07897.029Figure 6---source data 4.Czi file showing z-stack of SAG treated MEFs imaged in [Figure 6](#fig6){ref-type="fig"} panel G--J.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.029](10.7554/eLife.07897.029)10.7554/eLife.07897.030Figure 6---source data 5.Czi file showing z-stack of SAG treated MEFs imaged in [Figure 6---figure supplement 1](#fig6s1){ref-type="fig"} panel C.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.030](10.7554/eLife.07897.030)10.7554/eLife.07897.031Figure 6---source data 6.Czi file showing z-stack of SHH treated MEFs imaged in [Figure 6---figure supplement 1](#fig6s1){ref-type="fig"} panel D.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.031](10.7554/eLife.07897.031)10.7554/eLife.07897.032Figure 6---figure supplement 1.Tbx3 immunoreactivity in cilia increases in response to Hedgehog pathway stimulation without an overall increase in Tbx3 protein levels.(**A**) Quantitation of Tbx3+ cilia in wild type MEFS -/+ SAG shows marked increase in Tbx3 immunoreactive cilia in response to SAG. B) Western blot assaying Tbx3 and btubulin (loading control) protein levels in MEFs +/- SAG; the increase in number of Tbx3+ cilia occurs without an increase in amount of total Tbx3 protein. (**C**, **D**) Immunofluorescence images of SAG-treated (**C**) or SHH (**D**) MEFs assayed with a Santa Cruz commercial anti-Tbx3 antibody (A20) raised against an internal Tbx3 epitope (green) confirm colocalization with cilia/Arl13b (red). These merged images include DAPI in blue; white arrowheads highlight ciliary Tbx3. Please see [Figure 6---source data 5](#SD8-data){ref-type="supplementary-material"},[6](#SD9-data){ref-type="supplementary-material"} for z-stacks.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.032](10.7554/eLife.07897.032)
Tbx3 co-localizes with Gli3 in the primary cilia {#s2-8}
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The presence of Tbx3 in cilia and the known association of Gli3 and Kif7 in primary cilia ([@bib17]) led us to test for interaction between endogenous Tbx3 and Gli3. Co-immunoprecipitation of E10.5 forelimb lysates showed that Tbx3 co-IPs with endogenous Gli3 ([Figure 7A](#fig7){ref-type="fig"}, and with Kif7 as shown previously), and that both Gli3FL and Gli3R are complexed with Tbx3 in the limb bud ([Figure 7B](#fig7){ref-type="fig"}, lane 1). These interactions are also detected in whole embryos ([Figure 7C](#fig7){ref-type="fig"}; specificity/efficiency of Gli3 and Kif7 IPs are shown in [Figure 7C' and C"](#fig7){ref-type="fig"}, respectively. Additional experiments showing this interaction are in [Figure 7---figure supplement 1](#fig7s1){ref-type="fig"}). Overexpression of Flag-tagged Gli3 and Myc-tagged Tbx3 in HEK293 cells followed by co-immunoprecipitation did not indicate direct interaction in this cellular context ([Figure 7---figure supplement 2](#fig7s2){ref-type="fig"}), suggesting that their association occurs within a larger complex that includes Kif7 ([Figure 4](#fig4){ref-type="fig"}).10.7554/eLife.07897.033Figure 7.Tbx3 interacts with Gli3 in the limb bud and trafficks with Gli3 in primary cilia.(**A**, **B**) Immunoprecipitation (IP) of E10.5 forelimb bud protein lysates with antibodies listed at the top of panel and immunoblotted (IB) to detect Tbx3 (**A**) or Gli3 (**B**). Black arrowhead indicates IgG. Gli3FL and Gli3R (red arrowheads in B) both co-immunoprecipitate with Tbx3. (**C**) Immunoprecipitation (IP) of E10.5 whole embryo protein lysates with antibodies listed at the top of panel and immunoblotted to detect Tbx3. Tbx3 co-IPs with Gli3. Specificity and efficiency of anti-Gi3 and anti-Kif7 antibodies in whole embryo lysates tested are shown in panels **C'** and **C"**. Additional experiments demonstrating Tbx3/Gli3 interactions are in [Figure 7---figure supplement 1](#fig7s1){ref-type="fig"}. Em, empty lane (**D--I'**) Confocal 100X single Z-plane immunofluorescence images of vehicle (DMSO) treated MEFS after immunostaining for: DAPI (DNA, blue), Tbx3 (green, [@bib22]), Gli3 (red), Arl13b (pink, cilia). Panel **H** is merged image of (**D**--**G**). Panel I is 2.5X digital zoom of the boxed cell in panel **H**, and **I'** shows the pink (cilia) channel pixel shifted to permit visualization of colocalized Tbx3 and Gli3 (yellow) within the cilia. White arrowheads highlight Gli3/Tbx3 colocalization. Please see [Figure 7---source data 1](#SD10-data){ref-type="supplementary-material"} for z-stacks. (**J**--**O'**) As above, but MEFS were treated with SAG in DMSO. Please see [Figure 7---source data 2](#SD11-data){ref-type="supplementary-material"} for z-stack. (**P**) Quantitation of Tbx3+ and Gli3+ cilia in MEFS -/+ SAG. SAG treatment causes the majority of cilia to become Tbx3+ and these ciliary Tbx3 signals all colocalize with Gli3.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.033](10.7554/eLife.07897.033)10.7554/eLife.07897.034Figure 7---source data 1.Czi file showing z-stack of wild type MEFs imaged in [Figure 7](#fig7){ref-type="fig"} panel D-I'.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.034](10.7554/eLife.07897.034)10.7554/eLife.07897.035Figure 7---source data 2.Czi file showing z-stack of SAG treated MEFs imaged in [Figure 7](#fig7){ref-type="fig"} panel J-O'.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.035](10.7554/eLife.07897.035)10.7554/eLife.07897.036Figure 7---figure supplement 1.Tbx3 and Gli3 coimmunoprecipitate in whole embryo protein lysates.(**A, B**) Immunoprecipitation (IP) of E10.5 whole embryo protein lysates with antibodies listed at top of panels and immunoblotted (IB) to detect Tbx3 (**A**) or Gli3 (**B**). Black arrowhead indicates IgG. Gli3FL and Gli3R (red arrowheads in **B**) both co-immunoprecipitate with Tbx3. Em, empty lane**DOI:** [http://dx.doi.org/10.7554/eLife.07897.036](10.7554/eLife.07897.036)10.7554/eLife.07897.037Figure 7---figure supplement 2.Tagged Tbx3 does not co-IP with tagged Gli3 in HEK293 cells.Co-IP assay of Myc-tagged Tbx3 and Flag-tagged Gli3 overexpressed in HEK293 cells. IP was performed with antibodies listed at top and immunoblotted for Gli3. Myc-tagged Tbx3 does not interact with tagged Gli3.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.037](10.7554/eLife.07897.037)10.7554/eLife.07897.038Figure 7---figure supplement 3.Tbx3 does not co-IP with Sufu or Spop in mouse embryo lysates.(**A**, **B**) Immunoprecipitations/Immunoblot assaying for interaction between endogenous Tbx3 and Sufu in E10.5 mouse embryo lysates. Sufu did not co-IP Tbx3 (**A**) nor did Tbx3 co-IP Sufu (**B**). (**C**) Immunoprecipitations/Immunoblot assaying for interaction between Tbx3 and Spop in control and *Tbx3^Δfl/Δfl^*E10.5 mouse embryo lysates. No interaction was detected. Note increased Spop in mutant, consistent with previous result in [Figure 4J](#fig4){ref-type="fig"} and data in [Figure 8---figure supplement 1](#fig8s1){ref-type="fig"}.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.038](10.7554/eLife.07897.038)
Immunofluorescence assay of untreated MEFs for Tbx3, Gli3, and Arl13b by triple immunocytochemistry showed that 90% of cilia were Gli3+; Tbx3 colocalized with 66% of the Gli3 signals (N=38 total cilia scored, [Figure 7D--I](#fig7){ref-type="fig"}' [Figure 7---source data 1](#SD10-data){ref-type="supplementary-material"}). Treatment with SAG increased the fraction of Gli3+ cilia to \>95% and all but two of those Gli3 signals colocalized with Tbx3 (N=34, [Figure 7J--O](#fig7){ref-type="fig"}', [Figure 7---source data 2](#SD11-data){ref-type="supplementary-material"}). These results are quantified in [Figure 7P](#fig7){ref-type="fig"}.
Loss of Tbx3 compromises the Sufu/Kif7 complex required for Gli3FL stability and normal processing of Gli3R {#s2-9}
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To obtain further mechanistic understanding into how loss of Tbx3 results in decreased Gli3 proteins, we assayed levels and interactions of members of the Gli3 processing machinery. Gli3FL stability and partial processing versus complete degradation are regulated by opposing actions of Suppressor of fused (Sufu) and speckle-type POZ protein (Spop), respectively ([@bib95]; [@bib98]). In the absence of Smoothened activation, mammalian Sufu recruits GSK3β to phosphorylate Gli3FL downstream of PKA, allowing for its partial processing to Gli3R (which requires Kif7 and intact cilia) ([@bib39]; [@bib88]; [@bib91]). Spop is an adaptor for Cullin3-based E3 ubiquitin ligase that drives complete degradation of Gli3FL in the absence of Sufu, but facilitates its processing to Gli3R when Sufu is present ([@bib35]; [@bib95]; [@bib102]). In contrast to Kif7 and Gli3, we did not detect interactions between Tbx3 and either Sufu or Spop ([Figure 7---figure supplement 3](#fig7s3){ref-type="fig"}). The observation that Gli3FL is virtually undetectable in *Tbx3;PrxCre* anterior mesenchyme ([Figure 4A\'](#fig4){ref-type="fig"}) indicates that despite the increased amount of Sufu ([Figure 4H](#fig4){ref-type="fig"}, [Figure 8---figure supplement 1](#fig8s1){ref-type="fig"}), it fails to prevent degradation of Gli3FL in the absence of Tbx3.
We next tested whether decreased levels of Gli3 proteins in the anterior mesenchyme reflected perturbed interactions between Sufu, Gli3 and Kif7. Limitations in sample quantity made it unfeasible to perform multiple co-IPs on isolated anterior forelimb buds, so we assayed lysates from control and *Tbx3^Δfl/Δfl^* E10.5 embryos. The altered levels of protein expression seen in mutant limb buds were recapitulated in whole embryos ([Figure 8---figure supplement 1](#fig8s1){ref-type="fig"}). As seen in mutant limb buds, the amount of both Gli3FL and Gli3R protein is reduced in *Tbx3^Δfl/Δfl^* embryos ([Figure 8A](#fig8){ref-type="fig"}, lanes 1--4; [Figure 8---figure supplement 1](#fig8s1){ref-type="fig"}; [Figure 8---figure supplement 2A and B](#fig8s2){ref-type="fig"}). Notably, the interaction between Gli3 and Sufu is reproducibly decreased in excess of the decrement in Gli3 proteins; this is evident by quantitating and comparing the ratio of Gli3 proteins detected in control versus mutant to that complexed with Sufu. For example, in the representative experiment shown in [Figure 8A](#fig8){ref-type="fig"}, the Gli3 FL band intensity ratio is \~1.6 fold greater in controls ([Figure 8A](#fig8){ref-type="fig"}, lanes 1 and 3) than in mutants ([Figure 8A](#fig8){ref-type="fig"}, lanes 2, 4), while the amount of Gli3FL protein that was immunoprecipitated with Sufu is 4.6 fold greater in controls than mutants ([Figure 8A](#fig8){ref-type="fig"}, lane 9 versus 10). Mean band intensity ratios of 3 replicate experiments are shown in [Figure 8A'](#fig8){ref-type="fig"}. The decreased Gli3/Sufu interaction in the absence of Tbx3 was also evident when assayed in the opposite direction, that is, by IP of Gli3 and immunoblotting for Sufu ([Figure 8B](#fig8){ref-type="fig"}, panel B' shows relative band intensities for the experiment shown). Sufu/Gli3R interactions were also affected ([Figure 8A](#fig8){ref-type="fig"}'; [Figure 8---figure supplement 2A--B](#fig8s2){ref-type="fig"}). These findings indicate that normal stoichiometry of the interaction of Gli3 proteins with Sufu requires Tbx3.10.7554/eLife.07897.039Figure 8.Altered stoichiometry of interactions between Gli3 and members of its processing/degradation complex.(**A**) Anti-Gli3 immunoblot (IB) on immunoprecipitates (IP) from antibodies listed at top on lysates from E10.5 control (wt) and *Tbx3^Δfl/Δfl^* (ko) embryos. Gli3FL and Gli3R are denoted by red arrowheads, IgG heavy chain with black arrowhead. Note decreased levels of IP'd Gli3FL and Gli3R in mutants compared with controls; the IPs in lanes 1--2 and 3--4 are two independent biologic replicates and the band intensity ratio of control to mutant for both GliFL and Gli3R was \~1.6. The interaction between Gli3 and Sufu is decreased more than can be explained by the overall decrement in Gli3 protein levels: in this representative experiment Sufu co-IPs 4.6X more Gli3FL in controls than in mutants (lane 9 versus 10). (**A'**) Bar graphs show the results of quantitation of band intensities from three3 replicate experiments measured with densitometry and presented as the ratio of signal detected in controls relative to mutants. (**B**) As in A but immunoblot probed for Sufu. Comparison of lanes 1 and 2 confirms decreased interaction between Gli3 and Sufu, despite preserved levels of Sufu in the mutants (lane 10). (**C**) Anti-Gli3 immunoblot with IPs as listed at top. Note increased interaction between Kif7 and Gli3 in mutants (lane 4), despite overall decreased level of Gli3 (lane 8). (**C'**) Quantitation of band intensities from three replicate experiments measured with densitometry and presented as a ratio of signal detected in control relative to mutant. Even though there is less total Gli3 protein, since there is increased interaction between Gli3 and Kif7 in mutants, the Kif7 co-IP control to mutant ratios are \<1. (**D**) Anti-Kif7 immunoblot with IPs as listed at top. Confirms increased interaction between Gli3 and Kif7 in mutants. (**D'**) Quantitation of experiment in **D**. (**E**) Anti-Kif7 immunoblot with IPs as listed at top. The interaction of Kif7 and Sufu is decreased in the absence of Tbx3 (lane 4) despite preserved levels of both proteins (lane 6; [Figure 8](#fig8){ref-type="fig"}, panels **B**, **D** and **F**; [Figure 8---figure supplements 1](#fig8s1){ref-type="fig"} and [2](#fig8s2){ref-type="fig"}). (**E'**) Quantitation of band intensities from two replicate experiments measured with densitometry and presented as ratio of signal detected in control relative to mutant. There is 2 fold less interaction between Kif7 and Sufu in mutants. (**F**) Anti-Sufu immunoblot with IPs as listed at top. The interaction of Kif7 and Sufu is decreased in the absence of Tbx3 (lane 3 versus 4). (**F'**) Quantitation of findings in **F**.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.039](10.7554/eLife.07897.039)10.7554/eLife.07897.040Figure 8---figure supplement 1.Altered protein levels observed in mutant limb buds are also apparent in whole embryos.(**A**, **B**) Immunoblots on protein lysates prepared from E10.5 forelimb buds (**A**) and whole embryos (**B**). Actin is loading control. (**C**) Quantitation of protein levels in A and B comparing amount detected in control to mutant. Note increased levels of Sufu and Spop in mutant limbs and embryos that results in ratios of control to mutant \<1. nd, not detected**DOI:** [http://dx.doi.org/10.7554/eLife.07897.040](10.7554/eLife.07897.040)10.7554/eLife.07897.041Figure 8---figure supplement 2.Replicate experiments confirming altered stoichiometry of interactions between Gli3 and members of its processing complex in Tbx3 mutants.(**A**) Anti-Gli3 immunoblot (IB) on immunoprecipitates (IP) from antibodies listed at top on lysates from E10.5 control (wt) and *Tbx3^Δfl/Δfl^* (ko) embryos. Gli3FL and Gli3R are denoted by red arrowheads, IgG heavy chain by black arrowhead. Note decreased levels of IP'd Gli3R in mutants (lane 4) compared with control (lane 5). (**A'**) Quantitation of IPd proteins detected in A. The interaction between Gli3R and Sufu is decreased more than can be explained by the overall decrement in Gli3R protein levels: in this experiment, \>9 fold more Gli3R co-IPs with Sufu in controls than mutants (lanes 3 versus 4), whereas the decrement in Gli3R is only 3.8 fold (lanes 5 versus 6). (**B**, **B'**) Additional replicate co-IP experiment confirming decreased interaction between Gli3R and Sufu (lanes 7 and 8) in excess of decrement in overall Gli3R level (lanes 5 and 6). (**C**, **C'**) Anti-Gli3 immunoblot (IB) and quantitation of IPs from antibodies listed at top on lysates from E10.5 control (wt) and *Tbx3^Δfl/Δfl^* (ko) embryos. 3.3 fold [more]{.ul} Gli3R co-IPs with Kif7 in mutants (lane 2) than in controls (lane 1), despite an overall 2.9 fold [decrease]{.ul} in Gli3R levels (lanes 5 versus 6). (**D**, **D'**) Anti-Kif7 immunoblot (IB) and quantitation of IPs from antibodies listed at top on lysates from E10.5 control (wt) and *Tbx3^Δfl/Δfl^* (ko) embryos. In this example, the amount of Kif7 that co-IPs with Sufu is decreased in 2.4 fold in Tbx3 mutants, consistent with previous results shown in [Figure 8](#fig8){ref-type="fig"} panels **E** and **F**.**DOI:** [http://dx.doi.org/10.7554/eLife.07897.041](10.7554/eLife.07897.041)
In wild type embryos, we detected only trace interaction between Kif7 and Gli3R assaying by co-IP in either direction ([Figure 8C](#fig8){ref-type="fig"}, lane 3; [Figure 8D](#fig8){ref-type="fig"}, lane 7; [Figure 8---figure supplement 2C](#fig8s2){ref-type="fig"}, lane1). However, there was a robust and reproducible interaction in mutants despite the decrease in the total amount of Gli3 proteins. In the representative experiment shown in [Figure 8C](#fig8){ref-type="fig"}, in which we IP'd for Kif7 and assayed for Gli3, the band intensity ratios of control (lane 3) to mutant (lane 4) were 0.1 for Gli3FL and 0.25 for Gli3R. Quantification of the band intensity ratios from three replicate experiments in which we IP'd for Kif7 and assayed for Gli3 is shown in [Figure 8C](#fig8){ref-type="fig"}'. Furthermore, the increased interaction of Kif7 with Gli3 was confirmed by IP of Gli3 and assay for Kif7, as shown in [Figure 8D](#fig8){ref-type="fig"}: the band intensity ratio of control (lane 7) to mutant (lane 8) was 0.1, indicating a marked increase in interaction in mutants ([Figure 8D](#fig8){ref-type="fig"}' graphs relative band intensities in experiment 8D).
Lastly, the interaction between Sufu and Kif7 was reproducibly decreased when assayed by co-IP in either direction ([Figure 8E](#fig8){ref-type="fig"}, lane 3 versus 4; [Figure 8F](#fig8){ref-type="fig"}, lane 3 versus 4; [Figure 8---figure supplement 2D](#fig8s2){ref-type="fig"}, lane 7 versus 8). Quantification of the band intensity ratios from replicate experiments in which we IP'd for Sufu and assayed for Kif7 is shown in [Figure 8C](#fig8){ref-type="fig"}'. [Figure 8F](#fig8){ref-type="fig"}' graphs relative band intensities in experiment 8F in which we IP'd for Kif7 and assayed for Sufu. Note that this decrease in Sufu/Kif7 interaction occurs despite increased and normal levels of these proteins, respectively. In total, these data indicate that Tbx3 is required for normal stoichiometry and function of the Sufu/Kif7 complex that stabilizes and processes Gli3 as modeled in [Figure 9](#fig9){ref-type="fig"}.10.7554/eLife.07897.042Figure 9.Model of compartment specific functions of Tbx3 in forelimb bud mesenchyme and altered interactions and stoichiometry of the Kif7/Sufu Gli3 processing complex in *Tbx3;PrxCre* mutants.In posterior forelimb mesenchyme, Tbx3 is required for normal levels of Hand2 upstream of Shh. Shh pathway activity and other Tbx3-reponsive factors promote digit 5 formation. In the absence of Tbx3, there is decreased expression of Hand2 and Shh and other digit 5 promoting pathways. In anterior mesenchyme, Tbx3 is in a complex with Gli3 proteins, Kif7 and Sufu and required for the stability of Gli3 FL and Gli3R. In the absence of Tbx3, Sufu and Spop protein levels are increased yet there is decreased interaction between Sufu and Kif7, and Sufu and Gli3. In mutant anterior mesenchyme, Gli3FL is barely detected: it is either degraded or converted to Gli3R. Levels of Gli3R are abnormally low due to a combination of decreased amount of Gli3FL precursor and its processing to Gli3R, and excess degradation. These findings are consistent with decreased function of cilia and Kif7 (required for processing of Gli3FL to Gli3R), and of Sufu (required for stability of both Gli3FL and Gli3R).**DOI:** [http://dx.doi.org/10.7554/eLife.07897.042](10.7554/eLife.07897.042)
Discussion {#s3}
==========
Role of Tbx3 in limb bud initiation {#s3-1}
-----------------------------------
*Tbx3* expression in the LPM is required for normal *Tbx5* expression ([Figure 2A--C](#fig2){ref-type="fig"}) implicating *Tbx3* as one of the earliest limb initiation factors. This is consistent with our finding that Tbx3+ progenitors in the LPM give rise to the majority of limb bud mesenchyme. Determining the fate of these progenitors in the future will help reveal the mechanism for loss of ulna/fibula and digits 2--5 in *Tbx3* null embryos.
Differential sensitivity of the left and right limbs to Tbx3 {#s3-2}
------------------------------------------------------------
Intrinsic differences in anatomically bilaterally symmetric structures have long been suspected: acetazolamide teratogenizes only the right limb in rats ([@bib44]; [@bib100]) and nitroheterocyclics such as valproate also induce unilateral defects ([@bib14]; [@bib18]). Directional asymmetry in limb size in human fetuses was recently reported ([@bib89]). Left/right identity differences in bilaterally symmetric structures such as the limbs may be upstream of secondary patterning events such as dorsal/ventral axis appropriate to body side. Shiratori and colleagues reported asymmetric expression of Pitx2c in the developing mouse limb and postulated functional left/right differences ([@bib81]). Our finding that the left limb is more sensitive to Tbx3 than the right is consistent with this hypothesis and warrants further investigation in humans with UMS and other mouse models.
Discrete functions for Tbx3 in the anterior and posterior limb bud mesenchyme {#s3-3}
-----------------------------------------------------------------------------
A model of molecular mechanisms contributing to the different anterior and posterior limb phenotypes of *Tbx3;PrxCre* mutants is shown in [Figure 9](#fig9){ref-type="fig"}.
Digit 5 formation is exquisitely sensitive to Shh activity and Grem1 ([@bib26]; [@bib80]; [@bib104]; [@bib105]), so the altered expression of these genes in posterior mutant mesenchyme helps to explain loss of this digit. *Shh* null heterozygotes (*Shh*^+/-^) have normal digit number, suggesting that the loss of digit 5 in *Tbx3;PrxCre* mutants is not solely due to decreased *Shh* expression and pathway activity in posterior mesenchyme. Importantly, it is not known if compensatory mechanisms preserve normal Shh protein levels or activity in *Shh*^+/-^ mutants to support normal limb development. Indeed, a \'buffering system\' to modulate polarizing activity by Shh was proposed in the chick limb ([@bib78]).
We found evidence of dysfunction of other digit 5 promoting pathways in posterior mesenchyme of *Tbx3;PrxCre* mutants. Compound *Hand2/Gli3* null mutants have more severe polydactyly than *Gli3* mutants ([@bib23]), indicating that the role of Hand2 in digit formation is not limited to regulation of *Shh* expression. Aberrant expression of other BHLH factors ([Supplementary files 1](#SD12-data){ref-type="supplementary-material"},[3](#SD14-data){ref-type="supplementary-material"}: *Hand1, Bhlhe40, Bhlh*e*41, Bhlh*a*15*) may also contribute to the phenotype because the stoichiometry and interactions of BHLH factors have complex roles in limb development ([@bib20]). Altered levels of BMP responsive targets (*Dkk1, Noggin, Grem1, Grem2*) in *Tbx3;PrxCre* mutants suggest disrupted BMP signaling. Overexpression of *Gata6* decreases *Shh* and *Grem1* expression and causes loss of posterior digits ([@bib40]); we detected increased *Gata6* expression in the posterior mesenchyme of *Tbx3;PrxCre* mutants ([Supplementary file 3](#SD14-data){ref-type="supplementary-material"}). Additional studies are needed to determine if Tbx3 transcriptional or post-transcriptional functions directly regulate these pathways.
Although Tbx3 is also present in posterior mesenchymal cilia, we did not detect any evidence of altered stability or processing of Gli3 in the posterior mesenchyme ([Figure 4A](#fig4){ref-type="fig"}, representative of three replicates). Nonetheless, it is possible that decreased Shh signaling in the posterior of *Tbx3;PrxCre* mutant limb buds creates changes in Gli3 levels/ratios below our ability to detect. If so, decreased Shh activity would be predicted to increase Gli3R, decreasing digit number.
It is notable that decreased expression of *Tbx2* observed in *Tbx3;PrxCre* mutants would be predicted to result in increased *Grem1* expression ([@bib19]) however, *Grem1* expression is decreased ([Figure 3](#fig3){ref-type="fig"}). Decreased Shh signaling and *Grem1* expression in the posterior mesenchyme would both be predicted to result in decreased *Fgf4* and *Fgf8* expression in the posterior AER ([@bib38]; [@bib60]). Rather, the finding that these transcripts are increased in the posterior ([Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}) indicates that loss of Tbx3 in posterior mesenchyme disrupts the Shh-Grem1-FGF signaling loop.
In wild type anterior mesenchyme, our data support the model that Tbx3 is part of a Kif7/Sufu complex that drives processing of the majority of Gli3FL to Gli3R and also prevents complete degradation of Gli3FL by Spop ([@bib95]). Note that our model and data show a marked decrease in Gli3FL (virtually undetectable by western blot in anterior mesenchyme), but residual Gli3R in mutants. This is consistent with the phenotype of Gli3 deficiency, and digit 1 polysyndactyly is also seen with decreased function of Kif7 or Sufu. Residual Gli3R in the mutants is sufficient to prevent the extreme polydactyly seen in complete absence of Gli3, Kif7 or Sufu. In *Tbx3;PrxCre* mutant limb buds, Kif7 and Sufu proteins are present at normal and increased levels, respectively, but their interaction with each other, and that of Sufu with Gli3, are decreased. Because Spop facilitates processing of Gli3FL to Gli3R in the presence of Sufu, increased levels of Spop in mutants drives complete degradation of any Gli3FL not converted to Gli3R, resulting in undetectable levels of Gli3FL in anterior mesenchyme. The decreased levels of Gli3R in the anterior likely result from a combination of decreased levels of Gli3FL precursor, inefficient processing due to altered Kif7/Sufu function, and excess Gli3R degradation, as evident by the lower molecular weight species present in [Figure 3A](#fig3){ref-type="fig"}.
This synthesis is consistent with the large body of published data indicating that anterior digit number is tightly regulated by the balance of Gli3A and Gli3R, in turn controlled by Kif7 and Sufu ([@bib9]; [@bib91]; [@bib94]; [@bib96]; [@bib107]). Kif7^-/-^ and other ciliary mutants maintain robust levels of Gli3FL but have a marked decrease in processing it to Gli3R and their PPD phenotypes are consistent with decreased Gli3R ([@bib12]; [@bib17]). Furthermore, recent studies from Chi Chung Hui's group demonstrate that *Kif7;PrxCre* mutants have anterior PPD and that increasing Gli3R or decreasing Gli activators rescues all but digit 1 duplication ([@bib106]).
*Tbx3* mutant limbs also display evidence of Sufu dysfunction with markedly decreased levels of Gli3R and absent Gli3FL. Levels of Gli2, Gli3FL and Gli3R are all drastically reduced in *Sufu* mutants, even in the absence of cilia, consistent with the fact that Spop is not a ciliary protein and drives degradation of the full length proteins in the absence of Sufu ([@bib10]; [@bib36]; [@bib86]; [@bib95]). However, βTrCP and Spop can only mediate [processing]{.ul} of Gli3FL to Gli3R in the presence of both Sufu and Kif7/intact cilia/intraflagellar transport ([@bib17]; [@bib43]; [@bib51]; [@bib95]; [@bib98]). This processing is believed to occur at the basal body/centrosome ([@bib77]; [@bib93]; [@bib98]; [@bib99]). Thus, our data strongly support that Tbx3 functions at the cilia or basal body, as a part of the complex that regulates Gli3 processing and stability ([@bib77]; [@bib98]).
Loss of Tbx3 could also influence stability and processing of Gli2. The anterior PPD phenotype of *Gli3* heterozygotes is slightly more severe in a *Gli2* null background ([@bib6]; [@bib62]). Gli2 is also expressed in the posterior mesenchyme where it regulates digit patterning but not digit number ([@bib6]). Bowers' study also demonstrated that the role of Gli activators is in AP patterning of the posterior limb, whereas Gli repressors regulate digit number and anterior limb AP patterning. This is consistent with our findings that Tbx3 affects anterior digit number by regulating Gli3 repressor stability in the anterior mesenchyme.
The functional significance of increased interaction between Kif7 and Gli3R that we detect in mutants requires additional study nonetheless, since the anterior phenotype of *Tbx3;PrxCre* mutants is one of Gli3R deficiency rather than absence, we conclude that either there is a pool of Gli3R unbound by Kif7 and/or Gli3R complexed with Kif7 still has repressor function.
In addition to its function as a Gli3R co-repressor, Zic3 also regulates cilia morphogenesis and function ([@bib85]). Since decreasing Zic3 levels in *Gli3^+/-^*mutants rescues their preaxial polydactyly ([@bib74]), *Zic3* overexpression in *Tbx3;PrxCre* mutants may contribute to the PPD phenotype.
Our data exclude ectopic Shh pathway activity as a cause of the PPD in *Tbx3;PrxCre* mutants: using multiple methods at multiple stages, we did not detect anterior *Shh, Gli1,* or *Ptch1* expression in the anterior mesenchyme. Notably, expression of the BMP antagonist *Grem1* is normally increased in PPD associated with ectopic anterior Shh pathway activity ([@bib54]; [@bib103]), but in *Tbx3* mutants, *Grem1* expression is markedly decreased. This is additional evidence in support of unique Tbx3-dependent pathways regulating digit number.
Materials and methods {#s4}
=====================
Mice {#s4-1}
----
Experiments were conducted in strict compliance with IACUC/AALAC standards. The *Tbx3^fl^*allele was detailed in [@bib22]. *Prx1Cre, RARCre* and *Rosa26^LacZ^* were previously reported ([@bib83]; [@bib63]; [@bib53]). Generation of the *Fgf8^mcm^* and *Tbx3^mcm^* alleles will be described elsewhere.
β-Galactosidase detection {#s4-2}
-------------------------
Males bearing *Fgf8^MCM^* or *Tbx3^MCM^*alleles were crossed with *Rosa26^LacZ/+^*females. Females were gavaged with tamoxifen (10mg/gm body weight) at stages stated in text. β−galactosidase activity was assayed using established protocols ([@bib70]).
Skeletal preparations {#s4-3}
---------------------
E15.5 fetuses were fixed in 4% PFA overnight, rinsed in water for 2 days and alcian blue stained for 30 hr, then cleared in BABB. Older specimens were processed as in Moon et al. (2000).
Whole mount RNA in situ hybridization {#s4-4}
-------------------------------------
Digoxigenin-labeled riboprobes were generated according to manufacturer's instructions (Roche). Embryos were processed using a standard protocol ([@bib70]).
RNA isolation and reverse transcription--qPCR analysis {#s4-5}
------------------------------------------------------
Total RNA preparation, cDNA generation and qPCR were carried out as described in [@bib101]. Primer sequences are provided in [Supplementary file 5](#SD16-data){ref-type="supplementary-material"}. All qPCRs were performed on a minimum of three biologic replicates of pooled forelimb buds ([Figure 3](#fig3){ref-type="fig"}) or anterior and posterior forelimb bud segments ([Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}).
Gene expression analyses by microarray {#s4-6}
--------------------------------------
Total RNA was prepared from three pools of dissected E10.25 control (*Tbx3 ^fl/+^*) and *Tbx3;PrxCre* mutant forelimbs using the RNAeasy Micro Kit (Qiagen 74004). The microarray and genomic analysis and bioinformatics core facilities at the University of Utah performed Agilent mouse whole-genome expression arrays and array image data analysis using Agilent Feature Extraction software. Subtle intensity-dependent bias was corrected with LOWESS normalization, with no background subtraction. Statistical analysis of normalized log-transformed data was performed in GeneSifter ([www.genesifter.net](www.genesifter.net)). Differentially expressed transcripts were defined (adjusted for multiple testing using the Benjamini and Hochberg method) as p\<0.05. The results presented in [Supplementary file 1](#SD12-data){ref-type="supplementary-material"} show transcripts that were statistically differentially expressed +/-1.3 fold in the mutant limb buds; yellow highlighting indicates changes that were replicated by RNA-Seq.
RNA-sequencing to detect differential gene expression {#s4-7}
-----------------------------------------------------
Total RNA was isolated from pools of dissected anterior and posterior regions of E11 control (*Tbx3^fl/+^*) and *Tbx3;PrxCre* forelimbs using the RNAeasy Micro Kit (Qiagen). Each pool contained 12 forelimbs and two biologic duplicate pools were assayed. cDNA was generated, sequenced, and raw sequence reads were processed as described in [@bib42]. [Supplementary file 2](#SD13-data){ref-type="supplementary-material"} contains transcripts that are differentially expressed +/-1.3 fold (+/-0.38 in log base 2, column L) in control anterior (CA) compared to control posterior (CP) limb segments. The transcripts listed in [Supplementary file 3](#SD14-data){ref-type="supplementary-material"} were the result of mining the data to detect differential expression +/-1.3 fold (+/- 0.38 in log base 2, column L) in control posterior (CP) relative to mutant posterior (MP) segments. [Supplementary file 4](#SD15-data){ref-type="supplementary-material"} shows the result of mining the data to detect differential expression +/-1.3 fold (+/-0.38 in log base 2, column L) in control anterior (CA) relative to mutant anterior (MA) limb segments.
Note that transcripts that were not differentially expressed +/-1.3 fold are not listed on the tables. The complete unmined dataset is available on GEO.
Immunoblotting {#s4-8}
--------------
Protein lysates were prepared from E10.5 dissected limb buds or embryos or cultured MEFs using Dignam buffer. 50 ug of total protein were then subjected to SDS-PAGE analysis followed by immunoblotting according to standard protocols. Primary antibodies: Sufu (\#2522, Cell signaling), Spop (\#PA5-28522), Myc (\#SC-789, Santa Cruz), Flag (\#F7425, Sigma), E2F1 (\#137415, Abcam), Ubiquitin (\#7780, Abcam), Gli3 (AF3690, R and D systems), Tbx3 C-terminal antibody ([@bib22]); Kif7 (ab 95884, Abcam); β tubulin (Santa Cruz).
Immunoprecipitations {#s4-9}
--------------------
Immunoprecipitations were performed as described in [@bib41] and [@bib42]. Briefly, protein lysates were prepared from limb buds or embryos at E10.5 or transfected HEK293 cells using Dignam buffer C. Cleared protein lysates were obtained by centrifugation at 12,000g for 10 min. Equal amounts of protein lysates were incubated with 5--10μg of respective antibodies over night at 4°C with gentle shaking. Immune complexes were isolated with Protein-G Dynal beads and washed three times with Dignam buffer C. Precipitates were eluted from the beads by boiling in SDS-loading dye for 10 min, and analysed by western blotting by standard procedures using indicated antibodies at a dilution of 1:1000.
Densitometry analysis {#s4-10}
---------------------
Immunoblot signals were quantified by densitometry using ImageJ64 software as per the procedure described by Luke Miller (<http://www.lukemiller.org/ImageJ_gel_analysis.pdf>).
Immunofluorescence of sectioned and intact limb buds {#s4-11}
----------------------------------------------------
Sections: Samples were fixed in 4% PFA at 4°C for 2 hr then washed with 0.3% Triton-X100 (in PBST). Heat retrieval in citrate buffer (Vector Laboratories) was performed for 2 min in a pressure cooker. Slides were washed with PBST and incubated in PBST with 5% serum corresponding to the secondary antibody origin for 1 hr. Slides were incubated with primary antibody in PBST 5% serum overnight at 4^°^C. Primary antibodies and dilutions: Tbx3 C-terminal antibody 1/200 ([@bib22]), Tbx3 N- terminal antibody 1/100 (Abcam ab99302), Tbx3 internal epitope antibody (Santa Cruz A-20), Arl13b 1/100 (USDavis/NIH NeuroMab Clone N259B/66), Gli3 1/100 (R&D systems AF3690), pHH3 (Ser10), Millipore 06--570(1:2000); (1:50); Kif7 (1:200, kind gift from Dr. C-c Hui). After washing in PBST for 15 min, slides were incubated with secondary antibody from either Invitrogen or Jackson Immunoresearch diluted 1/1000 in PBST 2% BSA with Hoechst 33,342 at 1ug/ml for 1h at room temperature. Final wash was with PBST for 15 min and mounted using Fluoromount-G from Southern Biotech. Secondary antibodies: Donkey anti-rabbit Alexa 488; Donkey anti-goat Alexa 488; Donkey anti-mouse Alexa 647; Donkey anti-goat Alexa 594.
Whole forelimb buds: samples were fixed in 4% PFA for 1 hr at room temperature with PBST. Heat retrieval in citrate buffer (Vector Laboratories) was performed for 5 min at 100^°^C followed by washing with PBST. Limb buds were incubated in PBST with 5% serum that correspond to the secondary antibody origin (Goat or Donkey) for 2 hr. Limb buds were incubated with primary antibody in PBST with 5% serum overnight at 4^°^C, then washed with PBST once and incubated in PBST with 2% BSA for 4 hr. Incubated with secondary antibody and Hoechst as above with overnight at 4°C. Prior to imaging, samples were washed with PBST for 4 hrs and incubated in PBS/50% glycerol overnight.
[TUNEL]{.ul}: was performed as described in ([@bib70])
Confocal microscopy and image processing of immunofluorescence on intact limb buds {#s4-12}
----------------------------------------------------------------------------------
Imaging was performed using a Zeiss confocal microscope LSM710 with Zen black imaging software <http://www.zeiss.com/microscopy/en_us/downloads/zen.html>. 100x objective was used.
To generate the Arl13b/Tbx3 colocalization maps, we used Zen to define pixels in each plane of z that exceeded an arbitrary threshold of 0.1 relative intensity in both Arl13b and Tbx3 channels using the imaging calculation subtab in the image processing menu. This was followed by calculation of the maximum intensity projection. To quantitate Tbx3 positive cilia, the 3D object counter in ImageJ ([@bib4]) was used employing the "redirection" option to superimpose Arl13b+ objects into the Tbx3 channel.
Assaying cilia volume and length {#s4-13}
--------------------------------
Cilia volume was assayed by quantitating Arl13b signal on 100x images of sectioned forelimbs from four pairs of mutant and control embryos stained for Arl13b and analyzing resulting in ImageJ. Imaged were Gaussian blurred using radius equal to \'1\' and further 3D object counter was used to measure volumes and surface area with threshold of 900 out of 4000 range. Distributions and parameters of the volume distributions shown in [Figure 4---figure supplement 2A and B](#fig4s2){ref-type="fig"} were calculated in Excel.
We calculated that the surface area and volume of both WT and mutant cilia tightly fit the equation: Surface=8.01 X Volume^0.69^ ([Figure 4---figure supplement 2D](#fig4s2){ref-type="fig"}), which indicates that the cilia shapes are the same in the mutants and controls, and change size proportionally. In this case, the relative change in volume between mutant and control (Volume mutant)/(Volume control)=1.1747, which correspond to a change in length of 6%.
MEF Immunocytochemistry {#s4-14}
-----------------------
MEFs from E10.5 embryos were plated on fibronectin coated Matteks. MEFs were cultured and processed as in [@bib41]; cilia outgrowth was stimulated by culture in 0.5% FBS for 24 hr followed by incubation with 100 nM SAG or 100 nM SHH (Phoenix Pharmaceuticals) for 24 hr. Cells were washed 2x with PBS and fixed in 4% PFA for 20 min on ice, then washed 2X with PBS and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After blocking in 5% donkey serum + 3% BSA for 1 hr, cells were incubated with anti-TBX3 (custom C-terminal or Santa Cruz sc-17871) and anti-Arl13b (1:50 NeuroMAb) at room temperature for 2 hr. Cells were washed 5 X 5 min in PBS and then incubated in secondary donkey anti-goat-488 or anti-rabbit 488 and donkey antimouse -594 (Jackson Immunoresearch). After washing 3 x 5min in PBS, cells were incubated in DAPI for 20 min. Cells were then washed 3X with PBS and mounted in Dabco for imaging. Images were captured on a Zeiss LSM-710 confocal microscope and processed using Zen software as described above.
Assaying protein interactions with transfected, tagged proteins {#s4-15}
---------------------------------------------------------------
HEK-293 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were maintained in 5% C0^2^ incubator at 37°C. Myc- tagged full length TBX3 was generated as previously described ([@bib42]). The Flag-tagged Gli3 construct was obtained from Addgene (51246) and the Flag-tagged Kif7 construct was a kind gift of Kathryn Anderson. Transfections of plasmids were performed with Lipofectamine 2000 reagent (Invitrogen) as per the manufacturer's protocol.
We thank Drs. Chi-Chung Hui, Cliff Tabin and Kathyrn Anderson for generously providing the Kif7 polyclonal antibody, the *Prx1Cre* mouse line, and the Flag-tagged Kif7 construct, respectively. We thank Susan Mackem, Steven Vokes and Brian Harfe for helpful discussions and Diana Lim for creating the graphic art. We thank Peter Gallagher for technical assistance.
Additional information {#s5}
======================
The authors declare that no competing interests exist.
UE, Acquisition of data, Analysis and interpretation of data.
PKP, Acquisition of data, Analysis and interpretation of data.
JMR, Conception and design, Acquisition of data, Analysis and interpretation of data.
BM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.
AF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.
TM, Acquisition of data, Analysis and interpretation of data.
AMM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents.
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols with WCR IACUC protocol number 203-14.
Additional files {#s6}
================
10.7554/eLife.07897.043
###### Differentially expressed transcripts detected by microarray of E10.25 control and *Tbx3;PrxCre* mutant forelimb buds.
Table contains statistically significant differentially expressed genes determined as described in Methods section. Column 1 contains mean processed signal intensity of 3 biologic replicates from control limb, column 2 contains mean processed signal intensity of 3 biologic replicates from mutant limb. Fold changes are shown in Column E (Ratio). Yellow highlight of Gene ID (column N) indicates finding reproduced by RNA-Seq.
**DOI:** [http://dx.doi.org/10.7554/eLife.07897.043](10.7554/eLife.07897.043)
10.7554/eLife.07897.044
###### Differentially expressed transcripts detected by RNA-Seq of E11 control anterior forelimb buds versus control posterior forelimb buds.
Transcripts that are differentially expressed +/- 1.3 fold (+/- 0.38 in log base 2, column L) based on mean FPKM values in control posterior (CP, column O) compared to control anterior (CA, column P) limb segments. Values for each biologic replicate are in columns Q-T.
**DOI:** [http://dx.doi.org/10.7554/eLife.07897.044](10.7554/eLife.07897.044)
10.7554/eLife.07897.045
###### Differentially expressed transcripts detected by RNA-Seq of E11 control posterior forelimb buds versus *Tbx3;PrxCre* posterior forelimb buds.
Transcripts that are differentially expressed +/- 1.3 fold (+/- 0.38 in log base 2, column L) based on mean FPKM values in mutant posterior (MP, column O) compared to control posterior (CP, column P) limb segments. Values for each biologic replicate are in columns Q-T.
**DOI:** [http://dx.doi.org/10.7554/eLife.07897.045](10.7554/eLife.07897.045)
10.7554/eLife.07897.046
###### Differentially expressed transcripts detected by RNA-Seq of E11 control anterior forelimb buds versus *Tbx3;PrxCre* anterior forelimb buds.
Transcripts that are differentially expressed +/- 1.3 fold (+/- 0.38 in log base 2, column L) based on mean FPKM values in mutant anterior (MA, column O) compared to control anterior (CA, column P) limb segments. Values for each biologic replicate are in columns Q-T.
**DOI:** [http://dx.doi.org/10.7554/eLife.07897.046](10.7554/eLife.07897.046)
10.7554/eLife.07897.047
###### qPCR primer sequences.
**DOI:** [http://dx.doi.org/10.7554/eLife.07897.047](10.7554/eLife.07897.047)
10.7554/eLife.07897.048
Decision letter
Akhmanova
Anna
Reviewing editor
Utrecht University
,
Netherlands
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
\[Editors' note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.\]
Thank you for choosing to send your work entitled \"Tbx3 is a Ciliary Protein that Regulates Gli3 Stability to Control Digit Number\" for consideration at *eLife*. Your full submission has been evaluated by Janet Rossant (Senior editor) and three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in *eLife*.
All referees agreed that the part of the paper describing the phenotype of Tbx3 loss in limb development is solid, and thus the comments concerning this part were relatively minor. However, the referees were much more skeptical about the strength of the experimental support for the most novel and interesting claim, the localization of Tbx3 to the cilia and the interaction of Tbx3 with the Hedgehog pathway components such as KIF7. Both the biochemical and subcellular localization experiments would need to be very significantly extended (for example, by using tagged proteins for immunoprecipitation assays), and more thorough controls for these experiments would need to be included. Also the strength and the value of the RNA-seq data presented in the paper were questioned, and these data thus need to be improved. Taken together, it appears that very significant additional work will be necessary to make the story convincing.
*Reviewer \#1:*
This paper describes the role of Tbx3 in limb development and connects this function to cilia-dependent regulation of the Hedgehog pathway. The authors use conditional ablation of Tbx3 to characterize in detail its role in limb formation. The authors also show that Tbx3 localizes to cilia, interacts with Gli3 and Kif7 and affects the formation of protein complexes involved in Hedgehog signaling.
I think that the paper is interesting and potentially suitable for publication in e*Life*. However, while the phenotypic description of Tbx3 loss appears solid to me, the part describing the biochemical interactions and ciliary localization of Tbx3 misses important controls and is therefore less convincing.
1\) [Figure 5A](#fig5){ref-type="fig"}: it is very surprising that Tbx3 precipitates with equal efficiency with Tbx3 and Kif7 antibodies. Anti-Kif7 blot must be included here to show the efficiency of Kif7 immunoprecipitation. In the legend, the% of input loaded must be indicated, so that the efficiency of immunoprecipitation can be estimated.
A similar comment applies to [Figure 6O](#fig6){ref-type="fig"}. A Tbx3 blot must be included in this panel, as well as the input lanes, so that the efficiency of immunoprecipitation can be estimated. The loading of IgG in the control lane seems to be lower than in the two other lanes, which makes the value of this control doubtful. It would be preferable, instead of IgG, to use as a negative control antibodies against another protein, which does not bind to Gli3 or Tbx3.
Similar comments also apply to the blots shown in [Figure 7E, G](#fig7){ref-type="fig"} and H. Arl13 blot should be shown in panel E, Kif7 blot in panel G and Sufu blot in panel H. Are the cut blots in panel E (including input lanes) derived from the same or different gels and do they show the same exposures? The observed differences in immunoprecipitation efficiency are not convincing and therefore need to be quantified using three independent experiments for all data shown in [Figure 7](#fig7){ref-type="fig"}.
2\) Are there any indications that the interactions of Tbx3 with Kif7 and Gli3 might be direct? For example, do these proteins co-immunoprecipitate when overexpressed in cultured cells?
3\) The specificity of the Tbx3 staining in cilia ([Figure 6](#fig6){ref-type="fig"}) must be confirmed using Tbx3 knockout cells.
*Reviewer \#2:*
This is an extensive body of work using a range of genetic tools and molecular approaches to tease apart the function(s) of Tbx3 in the limb.
The first part of the paper describes an analysis of the phenotypes produced following conditional deletion of Tbx3 at different times and places during limb development using a variety of cre lines. I have relatively minor comments on this.
The second, and main part of the paper ([Figures 5](#fig5){ref-type="fig"}--[8](#fig8){ref-type="fig"}) covers all the work from which the title of the manuscript derive. I have several concerns here and in general remain skeptical (but with an open mind) about the data and particularly the interpretation.
In many instances I feel the authors have gone too far in their conclusions without the necessary experimental data to support the *final* conclusions.
First and foremost, immunostaining for Tbx3 and Arl13b on limb sections is interpreted as showing Tbx3 in limb mesenchymal cilia. Immunostaining of Tbx3 is throughout the entire cell and apparent co-expresssion with Arl13b could simply be explained by overlap in stain. Much higher resolution microscopy imaging that can circumvent these potential artifacts is required. The staining on MEFS do not clarify the issue. It is not clear why the Tbx3 staining pattern is so different in these cells from that shown in A-C. (Other issues such as the legend for panels H-K states 25/33 cilia were Tbx3+. I do not see more than 20 cells or so in this panel further compounding the problem) (It can also be argued that any patterns identified in MEFs may not have relevance to the limb.)
I also could not see a clear effect on KIf7 localization form the panels C in [Figure 5](#fig5){ref-type="fig"}. Table E appears to be an extreme way to show an apparent 8% difference on cilia length (I could not see where these numbers came from). This section ends with the conclusion that the data show that Tbx3 is present at baseline in cilia and is trafficked to and within cilia in response to hedgehog signaling. Unfortunately, I remain unconvinced of any of this.
There are similar issues with the data in [Figure 7](#fig7){ref-type="fig"}. Fundamentally I don\'t see clear evidence of co-expression of Tbx3, Gli3 Arl13b although I appreciate that IP data is also included.
The data on effect on GLi3 stability are potentially interesting but perplexing
What is the effect on Gli3 expression in the mutant limbs? It might be expected following reduction of hand2 and Shh expression in the mutant that Gli3 expression would be expanded but the western data would suggest not. Is there more GLi3FLin the MP? It is perhaps surprising that apparently equivalent levels of Gli3R are present in CP and MP.
The Tbx3 cKO is close (but I would not say identical to or a phenocopy of) a Het *Gli3* mutant. This is not conveyed in the final schematic which suggests a critical role of Tbx3 in Gli processing and that this in its absence *all* Gli processing is affected. I would expect a more profound polydactyly if this were the case. The actual \'polydactyly that presents lacks a structure that could be considered a true digit (1H, H\').
A further question raised is that if Tbx3 is playing such a critical role in Gli processing/Shh signalling in the anterior is it or why is it not playing a similar role in the posterior. This is not discussed and does not appear to have been studied.
Some other minor issues from [Figure 1](#fig1){ref-type="fig"} include:
1L: I do not see any loss of Sox9 activity in the region indicated with the red arrow head. This domain looks relatively normal to me.
I do not see clear evidence of a delay in ossification. In the text the authors conclude that this \"reveals a role for Tbx3 in later aspects of Indian hedgehog regulated bone morphogenesis." This has not been investigated to any extent and this type of comments goes far beyond what is actually shown in this paper. Also here the loss of the deltoid tuberosity need not have anything to do a direct effect on bone (cf Blitz, E, 2009).
Comparison of the phenotypes produced using the early acting (RARCre) and later acting (Prx1Cre) appears to show an effect of Tbx3 on the early stages of limb initiation that can be distinguished form later events of limb bud patterning through establishment of Shh in the posterior. A regulatory relationship between Tbx3, Hand2 and Shh has been reported in chick (Rallis, 2005).
Decreased expression of markers detected by RNAseq analysis is interpreted as evidence that fewer cells are undergoing condensation and chondrogenesis in posterior mesenchyme. This would be much better analyzing in a more direct way rather than making inferences from RNA-seq data.
Similarly, 5th digits agenesis is attributed to deceased cell number as opposed to decreased proliferation of digit 5 progenitors. These conclusions are drawn from a broad analysis of effects throughout the limb bud rather than focusing on the cells in question themselves.
The authors have some interesting results and observations. I remain open to the ideas they are suggesting but I do feel strongly that currently the data shown does not support their main conclusions.
*Reviewer \#3:*
The study described the limb phenotype in several Tbx3 conditional and null mutants in careful detail. Most of the conclusions based on whole mount RNA in situs are convincing and new findings. The most novel and surprising finding of the study is that the transcription factor TBX3 is localized to the cilium and interacts with KIF7. HOX proteins have been shown to interact with and inhibit GLI3 in the limb, however cilium localization was not explored. Given that the vast majority of TBX3 protein is in the nucleus ([Figure 6](#fig6){ref-type="fig"}), it seems surprising to me that the tiny amount in the cilium can be detected with IP ([Figure 7E-H](#fig7){ref-type="fig"}). The interaction with GLI3 could be in the nucleus and/or cilium, but KIF7 should not be in the nucleus. To further bolster the most significant claim of the paper, I think that additional experiments are needed. In particular, the specificity of the antibodies must be proven (e.g. using Tbx2 mutant MEFs for the Tbx3 antibody) and ideally some results confirmed using tagged proteins. Tagged GLI3 is available and other proteins could be easily generated. A genetic test would be to determine whether heterozygosity for Kif7 in mice (or knockdown in cells) rescues the defect proposed to be due to increased KIF7. In addition, many of the claims of changes in gene expression based on the RNA-seq data in Tables 2 and 3 do not seem to be significant differences, and some are opposite to what is said in the Results. Thus, the value of the RNA-seq data, is not clear, especially as only two samples were analyzed for each genotype, although I appreciate it is a difficult experiment. Many of the additional points listed below are simple changes that would make the paper more accessible and data more convincing.
[Figure 3A](#fig3){ref-type="fig"}\': The mutant limb looks smaller than the control limb in (A), therefore the domain is expected to be smaller. The text implies the domain is smaller relative to the size of the limb. Which is the case?
\"\[...\]downregulation of posterior genes including Shh, Shh pathway members, Tbx2, Sall1, Dkk1, and Osr1\[...\]\": The decrease is very little (e.g. Sall1 -0.3). More important, why are Grem1, Ptch1 and Gli1 not down in the RNA-seq, which would confirm the data in [Figure 3C, D](#fig3){ref-type="fig"} and address the question of whether the decrease is just due to the limb being smaller?
\"\[...\]up regulation of (AFP, Ttr, Apob, Apoa1, Apoa4, Trf, Ttpa)\[...\](Table 2).": Apob and Ttpa are actually down and the others are barely up in the anterior (Table 2).
Results: \"more single puncta cilia in mutants than controls ([Figure 5E](#fig5){ref-type="fig"} 84% vs 71%, p\<0.001). The levels of Kif7 mRNA\[...\] unaffected\[...\] Tables 2, 3\[...\]\": In Table 3 Kif7 is -0.3, which is greater than many of the changes in expression level claimed elsewhere in the text.
Results: \"The presence of Tbx3 in cilia and response to Hedgehog pathway activation was confirmed with a commercially available anti-Tbx3 antibody and both SAG and Shh stimulation ([Figure 6M, N](#fig6){ref-type="fig"}).\": Tbx mutant fibroblasts should be shown to demonstrate specificity of the antibody.
Tagged proteins should be used to confirm the interactions thought to be detected with commercial antibodies.
[Figure 7C, D](#fig7){ref-type="fig"}: quantification is needed.
Results: \"Consistent with our hypothesis, there was decreased interaction between Gli3FL and Sufu in mutants ([Figure 6E](#fig6){ref-type="fig"}, lane 8\[...\]\": Do the authors mean [Figure 7E](#fig7){ref-type="fig"}? Also, since Gli3 FL and R are reduced, does this experiment say anything?
Discussion: \"Future studies will determine if Tbx3 transcriptional or posttranscriptional activity regulates production of a factor that represses expression or stability of Hand2 mRNA.\": In the posterior limb, wouldn\'t Tbx3 also interfere with processing of Gli2/3 into activators, like in cilia mutants, and this could explain the posterior limb phenotype by the same mechanism as the anterior limb bud?
\[Editors' note: what now follows is the decision letter after the authors submitted for further consideration.\]
Thank you for resubmitting your work entitled \"Tbx3 is a Ciliary Protein and Regulates Gli3 Stability to Control Digit Number\" for further consideration at *eLife*. Your article has been favorably evaluated by Janet Rossant (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:
1\) There are some remaining concerns about the quality of the biochemical data. Specifically, the composition and the order of loading of the lanes is not the same in [Figure 4B, C](#fig4){ref-type="fig"} and [Figure 7A, B](#fig7){ref-type="fig"}, creating the impression that the shown immunoprecipitations are not from the same experiment. Gli3 and KIF7 blots should be included in [Figure 7C](#fig7){ref-type="fig"}. Most importantly, error bars should be included in the plots showing the quantification of immunoprecipitation results in [Figure 8](#fig8){ref-type="fig"} in order to illustrate that the experiment was performed more than once and the observed differences are significant.
2\) Better discussion should be included to explain why the authors insist that TBX3 does not regulate GLI3 function in the posterior limb, and why they have ignored the highly related protein GLI2. If their hypothesis is correct, one would expect the same might be true for GLI2 since it also binds SuFu and requires cilia for proper processing. Also, if whole embryos reveal the same protein interactions as the anterior limb, why would the posterior limb be different? Gli2/3 double limb mutants could well have a worse posterior limb phenotype than Gli3 null or het mutants. Only a relatively late double conditional mutant of Gli2/3 has been published, and it indicated the posterior phenotype is worse than in single Gli3 mutants. Thus, some of the posterior phenotype in Tbx3 mutants could be due to the altered GLI processing and activity in this tissue, rather than just the decreased Shh expression, especially as Shh heterozygous mutants have normal limbs. Finally, it seems to be an over-interpretation of the data to say that the posterior limb is not dependent on cilia. In cilia mutants' production of both activators and repressors is altered (diminished), but obvious phenotypes are only seen where the balance is greatly skewed. It would be nice to extend the Discussion of your paper to address these points.
\[Editors\' note: further revisions were requested prior to acceptance, as described below.\]
Thank you for resubmitting your work entitled \"T-box 3 is a Ciliary Protein and Regulates Gli3 Stability to Control Digit Number\" for further consideration at *eLife*. Your revised article has been favorably evaluated by Janet Rossant (Senior editor) and a reviewing editor. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:
1\) The scan of the Western blot shown in [Figure 7C](#fig7){ref-type="fig"} is not publication quality. Please substitute it for a high-resolution scan. Also, please make sure that in all cases where lanes of Western blots have been left out, a clear separator line is present (this currently seems not to be the case in [Figure 7C, 7C\'](#fig7){ref-type="fig"} and [8E](#fig8){ref-type="fig"}).
2\. Please reconsider the title of your paper. \"T-box3 is a ciliary protein and regulates..\" doesn\'t read well. Furthermore, e*Life* encourages the authors to provide brief explanations for the acronyms used in the title and Abstract. For your paper, mentioning that Gli3 is a player in Sonic Hedgehog signaling would be appropriate.
10.7554/eLife.07897.049
Author response
\[Editors' note: the author responses to the first round of peer review follow.\]
*Reviewer \#1:*
*1) [Figure 5A](#fig5){ref-type="fig"}: it is very surprising that Tbx3 precipitates with equal efficiency with Tbx3 and Kif7 antibodies. Anti-Kif7 blot must be included here to show the efficiency of Kif7 immunoprecipitation.*
We agree; we have repeated the co-IP a total of 4 times: a different experiment is shown (now in [Figure 4B](#fig4){ref-type="fig"}) than in the original submission and another example is in the new [Figure 7A](#fig7){ref-type="fig"}. The anti-Kif7 blot requested by the reviewer reveals that as predicted, Tbx3 is not as efficiently IP'd by anti-Kif7 antibody as Kif7 itself.
*In the legend, the% of input loaded must be indicated, so that the efficiency of immunoprecipitation can be estimated.*
\% input added to the figures and legends as requested.
*A similar comment applies to [Figure 6O](#fig6){ref-type="fig"}. A Tbx3 blot must be included in this panel, as well as the input lanes, so that the efficiency of immunoprecipitation can be estimated. The loading of IgG in the control lane seems to be lower than in the two other lanes, which makes the value of this control doubtful. It would be preferable, instead of IgG, to use as a negative control antibodies against another protein, which does not bind to Gli3 or Tbx3.*
We have made the additions and changes as requested, and used an additional negative control antibody as suggested by the reviewer. These data are now presented in the current [Figure 7](#fig7){ref-type="fig"}.
*Similar comments also apply to the blots shown in [Figure 7E, G](#fig7){ref-type="fig"} and H. Arl13 blot should be shown in panel E, Kif7 blot in panel G and Sufu blot in panel H. Are the cut blots in panel E (including input lanes) derived from the same or different gels and do they show the same exposures? The observed differences in immunoprecipitation efficiency are not convincing and therefore need to be quantified using three independent experiments for all data shown in [Figure 7](#fig7){ref-type="fig"}.*
Because it requires that a much greater amount of protein be loaded to detect these proteins from non- IP'd material, the blots in panel E were from a different gel to avoid exposure and well distortion issues that arise from the different amounts of protein loaded. The experiments originally presented in [Figure 7](#fig7){ref-type="fig"} have now been repeated a minimum of 3 times and examples are shown in [Figure 8](#fig8){ref-type="fig"} and [Figure 8---figure supplement 2](#fig8s2){ref-type="fig"}.
*2) Are there any indications that the interactions of Tbx3 with Kif7 and Gli3 might be direct? For example, do these proteins co-immunoprecipitate when overexpressed in cultured cells?*
We have performed the co-IPs with tagged Kif7 and tagged Gli3 as requested ([Figure 4 C, D](#fig4){ref-type="fig"} and [Figure 7---figure supplement 1](#fig7s1){ref-type="fig"}) in HEK293 cells. We found that Flag-Kif7 and Myc-Tbx3 interact, but Flag-Gli3 and Myc-Tbx3 do not under these conditions.
*3) The specificity of the Tbx3 staining in cilia ([Figure 6](#fig6){ref-type="fig"}) must be confirmed using Tbx3 knockout cells.*
We agree and apologize for not including this critical control in our initial submission. We have now repeated these experiments and used *Tbx3* mutant limb buds ([Figure 5](#fig5){ref-type="fig"}) as well as *Tbx3* mutant MEFs ([Figure 6](#fig6){ref-type="fig"}) and confirmed the specificity of signal in the cilia. Please note that in addition to our custom antibody against the C-terminus of Tbx3, 2 different commercial anti-Tbx3 antibodies detected Tbx3 in cilia ([Figure 5---figure supplement 2](#fig5s2){ref-type="fig"}, Abcam anti-Tbx3 antibody; [Figure 6---figure supplement 1](#fig6s1){ref-type="fig"}, panels C, D SantaCruz A-20 anti-Tbx3 antibody).
*Reviewer \#2:*
*First and foremost, immunostaining for Tbx3 and Arl13b on limb sections is interpreted as showing Tbx3 in limb mesenchymal cilia. Immunostaining of Tbx3 is throughout the entire cell and apparent co-expresssion with Arl13b could simply be explained by overlap in stain. Much higher resolution microscopy imaging that can circumvent these potential artifacts is required. The staining on MEFS do not clarify the issue.*
We agree; please see response to Reviewer 1 above and the new data provided in control and mutant limb buds, including 100X confocal maximum image projections in [Figure 5](#fig5){ref-type="fig"} and the z-stacks provided in Videos 3 and 4.
*It is not clear why the Tbx3 staining pattern is so different in these cells from that shown in A-C.*
We have shown that the staining pattern of Tbx3 is very context/tissue dependent (Frank et al., 2012; Frank et al., 2013, Kumar et al., 2014a *eLife*). In this case, serum starved MEFs are very different than the rapidly proliferating mesenchymal cells in the limb bud. Even within the limb, cells in the AER have a different pattern of Tbx3 staining (mostly cytoplasmic, none in cilia) than those in the mesenchyme (cytoplasmic, nuclear and cilia). In some cell types, such as the dorsal root ganglia, all staining is nuclear. The MEF pattern shown here is very similar to what we demonstrated in MEFs in Kumar et al., 2014a, *eLife*.
*(Other issues such as the legend for panels H-K states 25/33 cilia were Tbx3+. I do not see more than 20 cells or so in this panel further compounding the problem) (It can also be argued that any patterns identified in MEFs may not have relevance to the limb.)*
We apologize for this error; the text was written to describe a larger, lower magnification panel that we subsequently chose to replace with the panel presented. In the revision, we have replaced all of this data with cleaner, higher magnification/better resolution confocal images in [Figures 6](#fig6){ref-type="fig"} and [7](#fig7){ref-type="fig"} that can also be viewed orthogonally and through z-stacks using the free Zen software that we employed to obtain and process the images <http://www.zeiss.com/microscopy/en_de/downloads/zen.html>. In addition to viewing z-stacks through the limb or MEFs, this software also permits the entire stack to be viewed in 3D and orthogonally so that the overlap is visible at the pixel level. We have also employed the image calculator function of this software to objectively quantitate overlap at the pixel level between the Arl13b and Tbx3 signals in control versus mutant. This method is the most sensitive and objective available to us to define true colocalization. It confirms the presence of Tbx3 in cilia and clearly shows Tbx3 immunoreactivity in limb mesenchymal cilia that is virtually absent after Cre recombination in mutants.
*I also could not see a clear effect on KIf7 localization form the panels C in [Figure 5](#fig5){ref-type="fig"}.*
The goal of the panel as shown was to provide the overall picture of a 40X confocal field and to show that there is not an appreciable difference in the number of ciliated limb mesenchymal cells between control and mutant. However, as stated in the legend, the white arrowheads denote the cilia with multiple spots or streak of Kif7 signal. This is quantitated on a much larger scale from many such fields in the bar graph which represents the results from 3 independent reviewers blinded to genotype, scored the Kif7 immunoreactive pattern of the cilia on 20 fields from 3 mutants and 3 controls.
*Table E appears to be an extreme way to show an apparent 8% difference on cilia length (I could not see where these numbers came from).*
We have replaced this Table with more accurate quantitation based on 3D analysis of the cilia, as described in response to Reviewer 1 above; this is in [Figure 4---figure supplement 2](#fig4s2){ref-type="fig"}.
This section ends with the conclusion that the data show that Tbx3 is present at baseline in cilia and is trafficked to and within cilia in response to hedgehog signaling. Unfortunately, I remain unconvinced of any of this.
*There are similar issues with the data in [Figure 7](#fig7){ref-type="fig"}. Fundamentally I don\'t see clear evidence of co-expression of Tbx3, Gli3 Arl13b although I appreciate that IP data is also included.*
Please see response to Reviewer 1 and extensive new, appropriately controlled data in [Figures 5](#fig5){ref-type="fig"}--[8](#fig8){ref-type="fig"}. We are confident that the reproducible immunofluorescence and IP data we have now provided address the reviewer's doubts.
*The data on effect on GLi3 stability are potentially interesting but perplexing.*
We agree that this is an extremely interesting finding and while discovering the exact mechanism whereby Tbx3 stabilizes Gli3 is beyond the scope of this manuscript, the fact that Tbx3 is in a complex with Kif7 and Sufu which regulates processing and stability of Gli3 provides the first step toward understanding this.
*What is the effect on Gli3 expression in the mutant limbs? It might be expected following reduction of hand2 and Shh expression in the mutant that Gli3 expression would be expanded but the western data would suggest not. Is there more GLi3FLin the MP? It is perhaps surprising that apparently equivalent levels of Gli3R are present in CP and MP.*
As shown by the graph of qPCR results in [Figure 3](#fig3){ref-type="fig"}, and the new in situ images provided there Gli3 mRNA levels are increased, as is expected with decreased Shh activity (Wang et al.,2000). We have now included an early microarrary experiment which also showed upregulation of *Gli3* expression in mutants (new Table 2).
In the posterior mesenchyme, the level of Gli3R is very low compared to anterior and this has been published by numerous groups including (Wang et al.,2000). Although Shh signaling is reduced in *Tbx3* mutants, the westerns all indicated that baseline processing of Gli3FL to Gli3R is intact in mutant posterior mesenchyme.
The Tbx3 cKO is close (but I would not say identical to or a phenocopy of) a Het Gli3 mutant. This is not conveyed in the final schematic which suggests a critical role of Tbx3 in Gli processing and that this in its absence all Gli processing is affected. I would expect a more profound polydactyly if this were the case. The actual \'polydactyly that presents lacks a structure that could be considered a true digit (1H, H\').
The phenotype of Gli3 null heterozygotes or Gli3R deficiency varies depending on genetic background and protein level; and others have also reported polysyndactyly of digit 1 (Wang 2007;Hill et al., 2009; Lopez-Rios et al., 2012;Quinn et al., 2012). The polysyndactylous digit of Tbx3;PrxCre mutants has two phalanges, the primary criteria for digit 1 (thumb) identity. Even in our mutants, although the penetrance is 100%, the severity of the duplication varies. *Tbx3;PrxCre* mutants are Gli3 deficient and not completely lacking Gli3R, thus PPD is the observed (and expected) phenotype. In the Discussion we consider other molecular events occurring in these mutants that may contribute to the phenotype, including overexpression of Zic3.
Our schematic model shows that in the anterior mesenchyme, some Gli3FL remains complexed with Sufu, but most is processed to Gli3R (which is also the case in controls); some of this Gli3R is aberrantly degraded which is what our data show. Our model does *not* indicate that all Gli3 processing is affected. The very low levels of Gli3FL detectable in the mutants are consistent with excess degradation of Gli3FL.
*A further question raised is that if Tbx3 is playing such a critical role in Gli processing/Shh signalling in the anterior is it or why is it not playing a similar role in the posterior. This is not discussed and does not appear to have been studied.*
With due respect to the reviewer, this is not discussed because, unlike other regions of the embryo, Shh signaling in the posterior limb mesenchyme is not dependent on cilia or Gli3R. Mouse mutants of Kif7 and ciliary components have anterior PPD; posterior digits are apparently normal. Our data indicate that Tbx3 regulates ligand-dependent Shh signaling in the posterior mesenchyme by affecting the level of *Hand2* expression upstream of *Shh* and its downstream activator functions. In the anterior, we show strong evidence that Tbx3 regulates ligand-independent pathway function by controlling the stability and processing of Gli3. Gli3 processing requires Kif7, Sufu and intact cilia. Although the interactions and processing may not occur within the cilia proper; the literature supports that they occur in the cytoplasm or centrosome/basal body adjacent to the cilia (reviewed in Ryan and Chiang 2012; Ingham and McMahon 2009; and Wang et al., 2013; Wen et al., 2010, Humke et al., 2005; Tukachinsky et al., 2010).
As the reviewers are aware, the cellular compartments in which different functionally relevant interactions are occurring is an ongoing and highly controversial area in the field for all components of the pathway. Elegant work has been done with tagged proteins and overexpression systems. Despite this, these issues have not yet been resolved, especially for endogenous interactions and functions of Kif7, Sufu or Gli3. What is clear is that even if the functionally relevant interactions are occurring in the cytoplasm adjacent to (or even far) from cilia, processing of Gli3 to Gli3R requires the cilia.
*Some other minor issues from [Figure 1](#fig1){ref-type="fig"} include:*
*1L: I do not see any loss of Sox9 activity in the region indicated with the red arrow head. This domain looks relatively normal to me.*
We agree that *Sox9* expression at E10.5 appears normal however, the morphology of the limb is not at this stage and is variable as is apparent from the images in [Figure 3](#fig3){ref-type="fig"} as well. In [Figure 1L](#fig1){ref-type="fig"}, the red arrow denotes abnormal indentation/lack of posterior tissue while the red bracket highlights extra tissue in the anterior, digit 1 forming region. We have clarified the text on this point.
*I do not see clear evidence of a delay in ossification.*
We have now added a label for the ossification center (oc) to the figures and the legends to clarify. Please consider the images of E15.5 skeletal preps in [Figure 1G, H](#fig1){ref-type="fig"} and [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"} panels K-N. There is no ossification center in the humerus of the mutants, whereas all control and the *Tbx3fl/fl;Fgf8^mcm/mcm^*mutant (which have normal limbs) specimens have a well-defined ossification center.
In the text the authors conclude that this \"reveals a role for Tbx3 in later aspects of Indian hedgehog regulated bone morphogenesis." This has not been investigated to any extent and this type of comments goes far beyond what is actually shown in this paper.
We have removed this statement; it was a hypothesis that we did not test.
Also here the loss of the deltoid tuberosity need not have anything to do a direct effect on bone (cf Blitz, E, 2009).
We agree and did not make this statement.
Comparison of the phenotypes produced using the early acting (RARCre) and later acting (Prx1Cre) appears to show an effect of Tbx3 on the early stages of limb initiation that can be distinguished form later events of limb bud patterning through establishment of Shh in the posterior. A regulatory relationship between Tbx3, Hand2 and Shh has been reported in chick (Rallis, 2005).
We apologize for failing to cite this reference and have included it in the revision.
*Decreased expression of markers detected by RNAseq analysis is interpreted as evidence that fewer cells are undergoing condensation and chondrogenesis in posterior mesenchyme. This would be much better analyzing in a more direct way rather than making inferences from RNA-seq data.*
The lack of digit 5 chondrogenesis is shown in [Figure 1](#fig1){ref-type="fig"} panel N and we have now confirmed the RNA-Seq data of decreased expression of Shh activated targets with qPCR ([Figure 3](#fig3){ref-type="fig"} and [Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}.
*Similarly, 5th digits agenesis is attributed to deceased cell number as opposed to decreased proliferation of digit 5 progenitors. These conclusions are drawn from a broad analysis of effects throughout the limb bud rather than focusing on the cells in question themselves.*
We have now specifically quantified proliferation in the posterior region containing d5 progenitors and added this data to [Figure 3](#fig3){ref-type="fig"}.
*Reviewer \#3: The study described the limb phenotype in several Tbx3 conditional and null mutants in careful detail. Most of the conclusions based on whole mount RNA in situs are convincing and new findings. The most novel and surprising finding of the study is that the transcription factor TBX3 is localized to the cilium and interacts with KIF7. HOX proteins have been shown to interact with and inhibit GLI3 in the limb, however cilium localization was not explored. Given that the vast majority of TBX3 protein is in the nucleus ([Figure 6](#fig6){ref-type="fig"}), it seems surprising to me that the tiny amount in the cilium can be detected with IP ([Figure 7E-H](#fig7){ref-type="fig"}). The interaction with GLI3 could be in the nucleus and/or cilium, but KIF7 should not be in the nucleus.*
We and others have shown (using different antibodies) that Kif7 is present in both the cilia and in scattered punctate regions of unknown composition in the cytoplasm and nucleus ([Figure 4](#fig4){ref-type="fig"}; He et al., 2014) We now also provide a confocal 100X maximum image projection showing this in sectioned limb bud and the z-stack ([Figure 4---figure supplement1](#fig4s1){ref-type="fig"} and Video 2).
The relative amounts of Kif7 in these different compartments have not been determined; most immunofluorescence images (including ours in [Figure 4](#fig4){ref-type="fig"}) highlight the signal in cilia. We have previously demonstrated (and do so again here) that Tbx3 is present in the cytoplasm and the nucleus, and that the relative abundance in these compartments is cell/tissue specific.
We do not claim that the IP'd material only reflects an interaction in the cilia and we think it likely that the relevant interactions between Tbx3, Gli3, Kif7 and Sufu are occurring at the ciliary base or centrosome as has been proposed by Ryan and Chiang and others (reviewed in Ryan and Chiang 2012; Ingham and McMahon 2009; and Wang et al., 2013; Wen et al., 2010, Humke et al., 2005; Tukachinsky et al., 2010). As discussed above, dissecting the cellular compartments in which different functionally relevant interactions has been a major, unsolved challenge in the field. What is clear is that processing of Gli3 to Gli3R requires the cilia. Our schematic specifically avoids indicating any localization for the Kif7/Tbx3/Gli3 complex (beyond anterior mesenchyme) because at this time, we can only hypothesize which subcellular compartment is functionally important. Determining this is clearly beyond the scope of this manuscript.
*To further bolster the most significant claim of the paper, I think that additional experiments are needed. In particular, the specificity of the antibodies must be proven (e.g. using Tbx2 mutant MEFs for the Tbx3 antibody) and ideally some results confirmed using tagged proteins. Tagged GLI3 is available and other proteins could be easily generated.*
We have performed these experiments, provide the data in the revised manuscript, and detailed the changes in response to Reviewers 1 and 2 above.
*A genetic test would be to determine whether heterozygosity for Kif7 in mice (or knockdown in cells) rescues the defect proposed to be due to increased KIF7.*
There is not an increase in the level of Kif7; we detected a change in localization in mutants.
*In addition, many of the claims of changes in gene expression based on the RNA-seq data in Tables 2 and 3 do not seem to be significant differences, and some are opposite to what is said in the Results. Thus, the value of the RNA-seq data, is not clear, especially as only two samples were analyzed for each genotype, although I appreciate it is a difficult experiment. Many of the additional points listed below are simple changes that would make the paper more accessible and data more convincing.*
We apologize for the inconsistencies between the text and the RNA-Seq tables; please see detailed explanations below.
*[Figure 3A](#fig3){ref-type="fig"}\': The mutant limb looks smaller than the control limb in (A), therefore the domain is expected to be smaller. The text implies the domain is smaller relative to the size of the limb. Which is the case?*
The text states "*Hand2* transcripts are reduced in E9.5 and E10.5 *Tbx3;PrxCre* mutantforelimb buds ([Figure 2---figure supplement 1D](#fig2s1){ref-type="fig"}; [Figure 3 A,A](#fig3){ref-type="fig"}')". Based on the in situ images, the domain is both smaller and the intensity of the signal throughout the domain is decreased, which is consistent with fewer transcripts. We now provide qPCR data that support this claim ([Figure 3F](#fig3){ref-type="fig"}). The mutant limbs are not in general smaller at E10.5-11.5; as noted above, the morphology and size varies, as it does among somite-matched wild type embryos, even within the same litter.
*\"\[...\]downregulation of posterior genes including Shh, Shh pathway members, Tbx2, Sall1, Dkk1, and Osr1\[...\]\": The decrease is very little (e.g. Sall1 -0.3).*
To address issues on the RNA-Seq data in general. Comments by both reviewers 2 and 3 led us to go back to the *eLife* website where we found that Tables 2 and 3 were duplicated and incorrectly labeled; the Table we referred to in the text was Table 2 when discussing the *posterior* expression analysis, but the table provided to reviewers labeled as Table 2 contained the *anterior* analysis. We sincerely apologize that the reviewers did not have access to files that were correct, and instead had files that were mislabeled relative to how they were discussed/referred to in the text, and that we failed to catch the error before the manuscript was sent for review.
Since initial submission, we have not had the time to generate sufficient animals to repeat the RNA-Seq experiment, but we have done additional validation of the datasets by qPCR ([Figure 3](#fig3){ref-type="fig"}, [Figure 3---figure supplement 4](#fig3s4){ref-type="fig"}). We have now labeled the tables with clear titles, provided methods that precisely describe how the changes contained in the tables were defined. Within the tables, we have labeled the data columns explicitly as mutant or control, and the compartments for the FPKM reads, as well as the pooled sample raw values (2 pools per genotype). We have also included additional data from an early microarray we performed in 2010 which in addition to the phenotypes, initially launched our investigation of the Shh pathway in the posterior and Gli3R in the anterior. This microarray analysis is in the present Table 2; changes that were replicated by the RNA-Seq experiment are highlighted in yellow in this table.
For the comparison of control posterior and mutant posterior (current Table 4), the fold changes in column L are given in log base 2, thus:
*Tbx2* -0.61, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)
*Dkk1* -0.77, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)
*Osr1* -0.89, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)
*Cntfr* -0.93, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)
On the current Table 4, *Sall1* is not listed because it was not changed more than +/-1.3 fold which was the criteria for inclusion on the table.
*\"\[...\]up regulation of (AFP, Ttr, Apob, Apoa1, Apoa4, Trf, Ttpa)\[...\] (Table 2).": Apob and Ttpa are actually down and the others are barely up in the anterior (Table 2).*
Please note, the manuscript paragraph was describing changes in the *posterior* mesenchyme and Table 2 should have been also *posterior*; as noted above, the reviewer was unfortunately evaluating the anterior analysis and thus it is understandable that the reviewer did not agree with our claims.
In the posterior dataset contained in the present Table 4, log base 2, column L:
Apob +2.7 (increased greater than 4 fold and statistically significant)
AFP +2.1 (increased greater than 4 fold and statistically significant)
Apoa4 +1.7 (increased greater than 2.5 fold and statistically significant)
Ttr +1.4 (increased greater than 2 fold and statistically significant)
Apoa1 +1.3 (increased greater than 2 fold and statistically significant)
Trf +0.6 (increased greater than 1.5 fold and statistically significant)
Ttpa +0.5 (increased greater than 1.5 fold and statistically significant)
However, based on the reviewers' comments and the fact that we do not pursue these changes further, we chose not to make any claims about this set of transcripts in the revised manuscript.
More important, why are Grem1, Ptch1 and Gli1 not down in the RNA-seq, which would confirm the data in [Figure 3C, D](#fig3){ref-type="fig"} and address the question of whether the decrease is just due to the limb being smaller?
The mutant limbs are not in general smaller at E10.5-11.5; as noted above, the morphology and size varies, as it does among somite-matched wild type embryos in the same litter.
We performed thein situs showing decreased expression of these transcripts long before performing the RNA- seq experiment (the entire series of experiments took over 7 years to complete) because the phenotype and wealth of published data led us to consider alterations in Shh pathway activity as at least contributory to the loss of digit 5, and because the 2010 microarray detected the decrements in *Shh, Hand2* and *Gli1* transcripts. We cannot explain the failure of the RNA-Seq experiment to detect these differences. As this and the other reviewers noted, RNA-Seq is a screening test and must be validated for specific genes; in the case of *Shh, Grem1, Ptch1, Ptch2, Gli1, Hand2,* and many others, the in situ and qPCR data are consistent and clear.
Despite the failure of the RNA-Seq to detect some bona fide changes, we do not think that these RNA-Seq data have no value to us or to the field: aside from confirming and expanding on previously published anterior/posterior polarity datasets in the wild type limb (which we have now included in a separate Table 3: Differentially expressed transcripts detected by RNA-Seq of E11 control anterior forelimb versus control posterior forelimb buds), many expression changes detected in the control versus mutant datasets have been validated by multiple methods including in situ hybridization, microarray and qPCR and were also validated for effects on alternative splicing in our 2014 Plos Genetics paper (Kumar et al., 2014b, *eLife*).
*Results: \"more single puncta cilia in mutants than controls ([Figure 5E](#fig5){ref-type="fig"}, 84% vs 71%, p\<0.001). The levels of Kif7 mRNA\[...\] unaffected\[...\] Tables 2, 3 \[...\]\": In Table 3 Kif7 is -0.3, which is greater than many of the changes in expression level claimed elsewhere in the text.*
See notes on the mix-up of the tables above; the original Table 3 reviewed contained the posterior gene expression data. In the current *anterior* analysis (Table 5), Kif7 is not listed on this Table because Kif7 log base 2-fold change is only 0.1042 (1.07 fold, not statistically significant) and only transcripts with changes +/- 1.3 fold (0.38 log base 2) are included on the revised tables.
*Results: \"The presence of Tbx3 in cilia and response to Hedgehog pathway activation was confirmed with a commercially available anti-Tbx3 antibody and both SAG and Shh stimulation ([Figure 6M, N](#fig6){ref-type="fig"}).\": Tbx mutant fibroblasts should be shown to demonstrate specificity of the antibody.*
We agree and have responded as noted in response to other reviewers above. The specificity of our custom antibody has previously been published Frank et al., 2012, Frank et al., 2013 and is also shown in [Figure 1](#fig1){ref-type="fig"} panel E/F and now, specifically relevant to ciliary localization in revised [Figures 5](#fig5){ref-type="fig"} and [6](#fig6){ref-type="fig"}.
*Tagged proteins should be used to confirm the interactions thought to be detected with commercial antibodies.*
These data are now provided for the interactions between Tbx3 and both Kif7 and Gli3. The interactions we detected between Kif7 and Sufu and Gli3 proteins are well established in the literature.
*[Fig7C, D](#fig3){ref-type="fig"}: quantification is needed.*
We have now replicated these experiments and quantitated the changes in interactions using densitometry and the appropriate input controls. These data are in [Figure 8](#fig8){ref-type="fig"} and its supplements.
*Results: \"Consistent with our hypothesis, there was decreased interaction between Gli3FL and Sufu in mutants ([Figure 6E](#fig6){ref-type="fig"}, lane 8\[...\]\": Do the authors mean [Figure 7E](#fig7){ref-type="fig"}? Also, since Gli3 FL and R are reduced, does this experiment say anything?*
We agree that the overall decrease in amount of Gli3 proteins in *Tbx3^△fl/△fl^*mutants makes it difficult to detect any decrement in protein/ protein interactions. We have not only assayed the Sufu/Gli3 interaction in both directions, but quantitated the amount of interaction and compared it to the decrease in protein levels (all in current [Figure 8](#fig8){ref-type="fig"} and its supplements). In general, control embryos have \~ 1.6 fold more Gli3R than *Tbx3^△fl/△fl^* mutants ([Figure 8A, A'](#fig8){ref-type="fig"} lanes 1-4, and [Figure 8---figure supplement 2A and B](#fig8s2){ref-type="fig"}). However, the ratio of Gli3R interacting with Sufu in controls ranges from 4.3 to 9.5 fold higher than in mutants ([Figure 8---figure supplement 2A-B](#fig8s2){ref-type="fig"}'). The interaction with Gli3FL is harder to assess reliably/quantitatively because of variable transfer efficiency due to high molecular weight. In the experiment shown in [Figure 8A](#fig8){ref-type="fig"}, the ratio of Gli3FL in controls to mutants is 1.6-1.7, while the ratio of that IPd with Sufu is 4.6 fold greater in controls. When assaying for Sufu that coIPs with Gli3, the change is not as dramatic because it reflects the Sufu that IPs with all GLi3 species ([Figure 8B](#fig8){ref-type="fig"}).
*Discussion: \"Future studies will determine if Tbx3 transcriptional or posttranscriptional activity regulates production of a factor that represses expression or stability of Hand2 mRNA.\": In the posterior limb, wouldn\'t Tbx3 also interfere with processing of Gli2/3 into activators, like in cilia mutants, and this could explain the posterior limb phenotype by the same mechanism as the anterior limb bud?*
As discussed in detail above, Shh signaling in the posterior limb bud is [not]{.ul} cilia dependent; no ciliary mutants described thus far have posterior limb defects. Cilia mutants fail to process Gli3FL into Gli3R but as discussed above and in the manuscript, cilia phenotypes affect anterior digit number. Our data do not reveal any decrease in GliFL in the posterior, Shh responsive compartment.
\[Editors' note: the author responses to the re-review follow.\]
*The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below: 1) There are some remaining concerns about the quality of the biochemical data. Specifically, the composition and the order of loading of the lanes is not the same in [Figure 4B, C](#fig4){ref-type="fig"} and [Figure 7A, B](#fig7){ref-type="fig"}, creating the impression that the shown immunoprecipitations are not from the same experiment. Gli3 and KIF7 blots should be included in [Figure 7C](#fig7){ref-type="fig"}.*
We responded with this query on Jan. 26:
"We agree that it is not ideal that the order of the lanes is not consistent; nonetheless, the IPs were from the same experiment/limb bud lysates. These experiments were repeated at the request of the reviewer from the first submission. Since these IPs require literally hundreds of limb buds, and the results shown in the revision reproduce those originally presented, repeating them for the sake of changing the order of the lanes seems excessively burdensome and time/embryo consuming given that the conclusion is unchanged."
We received this response from *eLife*:
*1) [Figure 4B, C](#fig4){ref-type="fig"} and [Figure 7A, B](#fig7){ref-type="fig"}: Repeating the experiment just to present a consistent order of the lanes is indeed not necessary in this case since the material is difficult to obtain. The authors should state in the main text of the paper (if this is correct) that the lanes shown in these gels are from the same immunoprecipitations.*
We apologize that the order of the lanes is not consistent; the IPs were from the same experiment and that is now stated in the text.
Control blots for Gli3 and KIf7 from the same experiment should be included in [Figure 7C](#fig7){ref-type="fig"}.
Now provided.
*Most importantly, error bars should be included in the plots showing the quantification of immunoprecipitation results in [Figure 8](#fig8){ref-type="fig"} in order to illustrate that the experiment was performed more than once and the observed differences are significant.*
We sent this query on Jan. 26:
"To clarify, [Figure 8](#fig8){ref-type="fig"} shows analysis of each protein-protein interaction performed once in each direction. The quantitation in the accompanying bar graph is for the blot shown and that is why the bar graphs do not have error bars. The repeat experiments showing that the altered interactions are reproducible, and their quantitations, are in [Figure 8---figure supplement 2](#fig8s2){ref-type="fig"}.
When considering these blots, it is important to realize that the IP and transfer efficiency do unavoidably vary from experiment to experiment, making it impossible to combine raw densitometry intensity data from all experiments to obtain the type of statistical analysis the reviewer is requesting. Furthermore, we think that the key point is that the changes in interactions in mutants are reproducible from experiment to experiment and when assayed in both directions.
As the reviewers are aware, it took 7 months to obtain sufficient numbers of embryos to provide the repeat experiments requested in the first review. We appreciate your reconsideration and additional clarification as to what it required for acceptance of our final submission."
We received this response from *eLife*:
*"Quantification of immunoprecipitation results in [Figure 8](#fig8){ref-type="fig"}: It is obvious that to draw any conclusions about protein quantities on Western blots, each experiment should be performed at least twice. It is also obvious that the raw densitometry data cannot be compared between different experiments. However, band intensity ratios, which are currently presented per experiment, should be easy to combine and average between two or more experiments/samples, generating error bars. The authors should also label these plots appropriately (for example, instead of \"ratio wt to mutant, lanes 1:2\" use a label \"Gli3FL, anti-Gli3-IP, ratio wt to mutant\", etc.). Basically, instead of leaving the job of comparing different experiments to the reader, the authors should combine the quantitative outcome of their repeated experiments in one plot and include it in the main figures of the paper* \".
We have now remade [Figure 8](#fig8){ref-type="fig"} and supplements in accordance with these requests. We have separated [Figure 8](#fig8){ref-type="fig"} into sections entitled: Gli3/Sufu Interactions; Gli3/Kif7 Interactions; Kif7/Sufu Interactions
Within each of these sections, the upper blots are representative IP experiments that were repeated 3 times. Adjacent to these blots are bar graphs with the averaged band intensities and standard error of the mean. The lower blots in each section are the complementary IP/western performed in the "other direction"; these complementary experiments were done once, so the bar graphs only show the ratio of band intensities between control and mutant for that experiment.
So, for each interaction, we not only performed replicate experiments (some of which are presented in [Figure 8---figure supplement 2](#fig8s2){ref-type="fig"}), but also the complementary experiments. We hope the reviewers agree that this is a rigorous analysis for a methodology that is difficult to quantitate.
"*Please note that ratio of two bands cannot be negative (because if one positive value is divided by another positive value, the outcome is positive), so the values showing an increase in the mutant should be presented differently".*
Thank you for the correction. We have remedied this in [Figure 8](#fig8){ref-type="fig"} and [Figure 8---figure supplement 2](#fig8s2){ref-type="fig"}.
*2) Better discussion should be included to explain why the authors insist that TBX3 does not regulate GLI3 function in the posterior limb, and why they have ignored the highly related protein GLI2. If their hypothesis is correct, one would expect the same might be true for GLI2 since it also binds SuFu and requires cilia for proper processing. Also, if whole embryos reveal the same protein interactions as the anterior limb, why would the posterior limb be different? Gli2/3 double limb mutants could well have a worse posterior limb phenotype than Gli3 null or het mutants. Only a relatively late double conditional mutant of Gli2/3 has been published, and it indicated the posterior phenotype is worse than in single Gli3 mutants. Thus, some of the posterior phenotype in Tbx3 mutants could be due to the altered GLI processing and activity in this tissue, rather than just the decreased Shh expression, especially as Shh heterozygous mutants have normal limbs.*
We agree, and have considered and investigated this further. While *ShH^+^*/- mutants do not have an abnormal digit phenotype, and they have half as much *Shh* mRNA in their limb buds, the amount of protein is normal (Brian Harfe, personal communication/manuscript in revision) revealing a compensatory mechanism at the translational or post-translational level. Sanz-Ezquerro and Tickle (2000) proposed a compensatory, buffering system to stabilize polarizing activity in chick limb stating: "A buffering system can account for several regulative features of polarising region signalling. It can explain why limbs with normal patterns develop after application of extra Shh polarising region cells (Tickle et al., 1975), Shh expressing cells (Riddle et al., 1993) or Shh beads (Yang et al.,1997) to the posterior margin of chick buds, and why normal patterned limbs also develop after most, but not all, of the polarising region is removed (Fallon and Crosby, 1975; Pagan et al., 1996)."
Additionally, the *ShH^+^*/- genotype is sensitized to other mutations. For example, Zeller's lab showed that rescued digits in Grem1-/-;Bmp4+/- are more sensitive to reduced Shh level (Benazet et al., 2009) and Hui\'s lab showed that Irx3/5 KO (excess HH activity) is improved by Shh dosage reduction. In addition, the Wnt7a mutant (Parr and McMahon, 1995), which affects Shh activation, has loss of digit 5. Mackem's group re-evaluated the Wnt7a mutant (Zhu and Mackem, 2009), and found delayed onset and decreased level of *Shh* expression -- which is consistent with our results.
In consideration of this information and to address the reviewers'suggestions, we have modified the model presented in [Figure 9](#fig9){ref-type="fig"} and added the following paragraphs to the relevant sections of the Discussion.
For the section discussing posterior phenotype and mechanisms:
"Digit 5 formation is exquisitely sensitive to Shh activity and Grem1 (Harfe et al., 2004; Scherz et al., 2007; Zhu and Mackem, 2011; Zhu et al., 2008), so the altered expression of these genes in posterior mutant mesenchyme helps explain loss of this digit. \[...\] Nonetheless, it is possible that decreased Shh signaling in the posterior of *Tbx3;PrxCre* mutant limb buds creates changes in Gli3 levels/ratios below our ability to detect. If so, decreased Shh activity would be predicted to increase Gli3R, decreasing digit number."
For the section discussing anterior phenotype and mechanisms we have added:
"Loss of Tbx3 could also influence stability and processing of Gli2. The anterior PPD phenotype of *Gli3* heterozygotes is slightly more severe in a *Gli2* null background (Bowers et al., 2012; Mo et al., 1997). \[...\] This is consistent with our findings that Tbx3 affects anterior digit number by regulating Gli3 repressor stability in the anterior mesenchyme."
Finally, it seems to be an over-interpretation of the data to say that the posterior limb is not dependent on cilia. In cilia mutants' production of both activators and repressors is altered (diminished), but obvious phenotypes are only seen where the balance is greatly skewed. It would be nice to extend the Discussion of your paper to address these points.
We agree; in the manuscript, we discuss what is known with regard to ciliary function and limb phenotypes but do not exclude the possibility of posterior functions yet to be determined.
\[Editors\' note: further revisions were requested prior to acceptance, as described below.\]
*The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below: 1) The scan of the Western blot shown in [Figure 7C](#fig7){ref-type="fig"} is not publication quality. Please substitute it for a high-resolution scan.*
We have replaced the image in 7C with that exported at 600 dπ from the scanner; we have shown the original image without any alterations to lane order or intensity. Because of the magnification at which the image was originally taken, the Biorad scanner gives a pixelated quality to the over exposed IgG bands and unfortunately, this is the lowest exposure we have of this experiment. We have provided additional experiments showing the Gli3/Tbx3 interaction in the new [Figure 7---figure supplement 1](#fig7s1){ref-type="fig"}.
*Also, please make sure that in all cases where lanes of Western blots have been left out, a clear separator line is present (this currently seems not to be the case in [Figure 7C, 7C\'](#fig7){ref-type="fig"} and [8E](#fig8){ref-type="fig"}).*
For [Figure 7C](#fig7){ref-type="fig"}, the new image shown was exported at 600 dπ from the scanner; we have shown the original image without any alterations to lane order or intensity, thus there are no lane rearrangements requiring separator lines. We have added more obvious separators to [Figures 7C'](#fig7){ref-type="fig"}and [7C"](#fig7){ref-type="fig"} and presented these blots with lane order consistent with that of Panel 7C. We have added the requested separator to [Figure 8E](#fig8){ref-type="fig"}.
2\. Please reconsider the title of your paper. \"T-box3 is a ciliary protein and regulates..\" doesn\'t read well. Furthermore, eLife encourages the authors to provide brief explanations for the acronyms used in the title and Abstract. For your paper, mentioning that Gli3 is a player in Sonic Hedgehog signaling would be appropriate.
We have revised the title to: "T-box 3 is a Ciliary Protein and Regulates Stability of the Gli3 transcription factor to Control Digit Number". We think that including Sonic Hedgehog signaling in the title is too cumbersome and also, inaccurate because its function in the anterior is Shh independent, as stated in the Abstract. We are open to suggestions if the title is still not satisfactory.
We have revised the Abstract as requested to include additional reference to Gli3 as a transcriptional effector in the Hedgehog pathway:
"Crucial roles for T-box3 in development are evident by severe limb malformations and other birth defects caused by T-box3 mutations in humans. \[...\] Remarkably, T-box3 is present in primary cilia where it colocalizes with Gli3. T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing. Thus T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function."
[^1]: These authors contributed equally to this work.
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uming w is positive.
w**(-99/13)
Simplify ((v*v**(6/25))/v)/v*v**21/v assuming v is positive.
v**(481/25)
Simplify (z/(z*z*z**(-2/5)/z))**(-13/8)*(z/(((z/(z*z**(1/2)/z))/z)/z))**(11/8) assuming z is positive.
z**(223/80)
Simplify (t*t**(-1))**(-1/34)*(t*(t/(t**1*t*t))/t)**(1/24) assuming t is positive.
t**(-1/12)
Simplify c**(-1/4)/c*c**2*c**(-1)/c*(c*(c/(c**(-2/7)/c)*c)/c)/c*c*c*c assuming c is positive.
c**(113/28)
Simplify ((r*r**(-2))**31)**(-18) assuming r is positive.
r**558
Simplify (n*n/((n*n*(n**(-8/19)/n)/n*n*n)/n))/n**(-46) assuming n is positive.
n**(901/19)
Simplify w**(-2/37)/((w*w**(-16))/w) assuming w is positive.
w**(590/37)
Simplify s/s**(2/5)*s/s**(-3/17) assuming s is positive.
s**(151/85)
Simplify (t**(-1)/(t/t**(4/9)*t))**(-9) assuming t is positive.
t**23
Simplify (k*((k/(k/k**(-4))*k)/k)/k*k*k**(-4/9)/k*k)**(3/28) assuming k is positive.
k**(-31/84)
Simplify (h/h**(1/13))**41 assuming h is positive.
h**(492/13)
Simplify k/(k/k**(-4/15))*k/(k/(k*k**(-9))) assuming k is positive.
k**(-124/15)
Simplify b**(-10/9)*b*b**20/b assuming b is positive.
b**(170/9)
Simplify (d**(-3/2))**(-46) assuming d is positive.
d**69
Simplify (c*c**(-3/5))/c*c*c**(5/3)/c*c assuming c is positive.
c**(31/15)
Simplify (u/((u/u**(-6))/u)*u**(-2/11))/(u*u**(-2/13)*u**(5/2)) assuming u is positive.
u**(-2439/286)
Simplify (a/(a*a/(a*a**(-3/8)*a)))/a**(-20) assuming a is positive.
a**(165/8)
Simplify w**(-17)*w*w**(-36)*w assuming w is positive.
w**(-51)
Simplify (l**6)**(2/17) assuming l is positive.
l**(12/17)
Simplify (m**(4/5)*m**(4/3)/m)**22 assuming m is positive.
m**(374/15)
Simplify l**(-19)*l**(-19) assuming l is positive.
l**(-38)
Simplify a*a/(a*a/(a**(-1/8)*a)*a)*a*a*a**2*a/a**10*a*a*a/(a**(-3)*a*a) assuming a is positive.
a**(-9/8)
Simplify ((z/(z**(-2/35)*z*z*z))/z)**(-25) assuming z is positive.
z**(515/7)
Simplify r**(-25)/r**(-2/11) assuming r is positive.
r**(-273/11)
Simplify (c*c**6)/c**20 assuming c is positive.
c**(-13)
Simplify y/(y**(-5/2)/y)*y**(-3/7) assuming y is positive.
y**(57/14)
Simplify n/(n*n**(16/7))*n/(n*(n**31/n)/n) assuming n is positive.
n**(-219/7)
Simplify n**(1/9)*n*n/((n**(-15/8)/n)/n) assuming n is positive.
n**(431/72)
Simplify (z*z**(-1)*z**7)**(-16/3) assuming z is positive.
z**(-112/3)
Simplify ((j*j/j**2*j)/(j*j/j**(-2/17)))**(3/2) assuming j is positive.
j**(-57/34)
Simplify ((r**(4/3)/r)/(r/(r/r**(2/17))))/((r*(r*r*r*r*r*r*r**(4/7)*r*r*r)/r)/(r**(-3/5)*r)) assuming r is positive.
r**(-15986/1785)
Simplify (r*r**(20/9)*r)/r**(-39) assuming r is positive.
r**(389/9)
Simplify (f/f**(2/11))**(-2/3)*f**(-2/11)/(f**(1/7)*f*f) assuming f is positive.
f**(-221/77)
Simplify (p/(p*p**(-12)*p))/(p/(p*p**41)) assuming p is positive.
p**52
Simplify c**(-7)/((c**(-1/2)*c*c)/c) assuming c is positive.
c**(-15/2)
Simplify i*(((i**(-3/4)/i)/i)/i)/i*i**10*i**6/i*i*i**9 assuming i is positive.
i**(85/4)
Simplify (x**(2/5))**(4/3) assuming x is positive.
x**(8/15)
Simplify ((l/(((l/l**8)/l)/l))/((l*l**8*l*l)/l*l*l))**0 assuming l is positive.
1
Simplify (k**(-14))**33 assuming k is positive.
k**(-462)
Simplify ((m/(m**(-8)/m))/(m**(-2/3)*m))/(m**(-5)/m**(-5)) assuming m is positive.
m**(29/3)
Simplify (q*q**(5/8)/q*q)**(-5/13) assuming q is positive.
q**(-5/8)
Simplify (s**(1/10)*s*s**(-3))**(3/8) assuming s is positive.
s**(-57/80)
Simplify (h**(1/9)*(h*h**(-3/5))/h)**12 assuming h is positive.
h**(-88/15)
Simplify (n**(-2/27)*n**(-7))/((n/(n**(-2/19)/n))/n*n**(-8)) assuming n is positive.
n**(-92/513)
Simplify (n**(3/8))**28 assuming n is positive.
n**(21/2)
Simplify (c*c*((c*c/((c/(c*c/(c*c*c/c**(2/7)*c*c)))/c)*c*c)/c)/c*c*c*c/(c*c**(-4)/c)*c)/(c*c/c**(6/5)*c*c**(5/2)/c*c) assuming c is positive.
c**(349/70)
Simplify ((i/(i/i**8))/i)/i*i/i**(-24) assuming i is positive.
i**31
Simplify ((((l*l**(1/7)*l)/l)/l*l)/l)**(2/65) assuming l is positive.
l**(2/455)
Simplify (d**(-9/2)*d)/(d*d*d**(-9)*d) assuming d is positive.
d**(5/2)
Simplify ((v*v/v**(-11))**(-3/5))**(-39) assuming v is positive.
v**(1521/5)
Simplify ((g/(g/g**1))/g)**(-39)*g*(g/(g*g*g/(((g*(g/(g**(1/2)*g*g))/g*g)/g*g)/g)*g))/g*g*g*g**(-5) assuming g is positive.
g**(-15/2)
Simplify w**(-4)/(w*w**(-6/13)) assuming w is positive.
w**(-59/13)
Simplify ((((n/n**(-1/6))/n)/n)**(-1/7))**(-43) assuming n is positive.
n**(-215/42)
Simplify (m*m**(-7/2)*m*m)/(((m/(m*m/m**(-2/37)))/m)/m) assuming m is positive.
m**(189/74)
Simplify ((k/k**(2/17))**(21/4))**(2/11) assuming k is positive.
k**(315/374)
Simplify (m/(m/(((m**(-1/4)*m)/m)/m)*m*m))**24 assuming m is positive.
m**(-78)
Simplify (y/(y/y**(-1/13)))/((y*y**(-4/3)*y)/y)*(y*y/(y/(y**0*y))*y)**(2/7) assuming y is positive.
y**(304/273)
Simplify ((h**(-1)*h)**41)**(-18/11) assuming h is positive.
1
Simplify c**(2/13)*((c/(c*c/c**5)*c)/c)/c*(c*c**5)/(((c/c**4)/c)/c) assuming c is positive.
c**(184/13)
Simplify (u**(-2/45)*u)**(2/53) assuming u is positive.
u**(86/2385)
Simplify (u*((u/((u*(u/(u/(u**(-4)/u*u)*u))/u)/u))/u)/u)/u**(-8)*(u*u**(2/7))**12 assuming u is positive.
u**(206/7)
Simplify (t**6/t*t*t**(6/7))**(-17) assuming t is positive.
t**(-816/7)
Simplify (m**(1/18)*m)/m*m*m**(-1/3)*m**(-5)/m*m/m**1 assuming m is positive.
m**(-95/18)
Simplify (j*j*j**1*j)**11/((j/((j**(-1/9)*j)/j))/j*j**(1/13)) assuming j is positive.
j**(5126/117)
Simplify (d*d**(1/4))/(d*d**(2/53)*d) assuming d is positive.
d**(-167/212)
Simplify ((i*i**2)/i)**(5/8) assuming i is positive.
i**(5/4)
Simplify ((q**(-2/5)/q)/(q*q*q**(2/13)*q*q))/(q**(-2/9)/q**(4/5)) assuming q is positive.
q**(-2651/585)
Simplify l**(-3/4)/(l**(-4)/l)*(l**(-2)/l)/l**(2/7) assuming l is positive.
l**(27/28)
Simplify a**4*a**(-5)*(a*a**10)/a*a**3 assuming a is positive.
a**12
Simplify (v/v**(-2/27))/v**(-4)*(v*(v*v*v*(v*v**0)/v)/v)**(18/11) assuming v is positive.
v**(2965/297)
Simplify (g/(g/((g/(g*g**(-17))*g)/g*g)))**(4/5) assuming g is positive.
g**(72/5)
Simplify (q**(-2/9)*q*q**(-3))**(-20/9) assuming q is positive.
q**(400/81)
Simplify (((k**(-16)/k*k*k)/k)/k)/(k*k**29/k) assuming k is positive.
k**(-46)
Simplify x**39*x/(x/(x/x**(3/2)))*x assuming x is positive.
x**(79/2)
Simplify (v**(2/21)*v**(2/35))**(-1/81) assuming v is positive.
v**(-16/8505)
Simplify ((h/(h/(h**(1/6)/h)))/h**(-2))/(h*h/((h/h**1)/h)*h)**(-15) assuming h is positive.
h**(367/6)
Simplify (((p/(p**(-2/5)*p)*p)/p)/(p**(4/5)*p))**19 assuming p is positive.
p**(-133/5)
Simplify (q**(-18)/q)**(2/117) assuming q is positive.
q**(-38/117)
Simplify (g*g*g/(g/(g**(-1)*g)))**35/(g/(g/(g*g**(-1)*g*g*g)))**(1/5) assuming g is positive.
g**(347/5)
Simplify (r**(-3/7))**40 assuming r is positive.
r**(-120/7)
Simplify ((q/(q/((q**(-2/3)*q)/q)))**(-5))**40 assuming q is positive.
q**(400/3)
Simplify (w**(-2/9)/w)/(w**(-17)/w) assuming w is positive.
w**(151/9)
Simplify ((y*y**(-3/8))/(y/(y/y**(-6/11)*y)))**(-6/5) assuming y is positive.
y**(-573/220)
Simplify d**(2/7)*d*d**4*d*d*d**(1/9)*d*d/(d*d**(-1)*d) assuming d is positive.
d**(529/63)
Simplify (z*(z*z/(z/z**(-2)))/z)/(z/z**(2/21)) assuming z is positive.
z**(-40/21)
Simplify (m/(m**7/m)*m**(-16)/m*m)**(-1/31) assuming m is positive.
m**(21/31)
Simplify ((g*g**2)/(g/g**(-2/11)*g))/(g**(-3)*g**(2/11)*g) assuming g is positive.
g**(29/11)
Simplify ((w*w*w**(6/7))/w**(2/3))**(-12) assuming w is positive.
w**(-184/7)
Simplify (q/(q**(2/25)*q))/q*q/(q*q/(q*q/(q**(-2/61)*q)*q)) assuming q is positive.
q**(-72/1525)
Simplify (s**(-3/7))**23 assuming s is positive.
s**(-69/7)
Simplify ((t**(-1/11)*t)/((t*t**2)/t))**48 assuming t is positive.
t**(-576/11)
Simplify ((p*p**(-1)*p*p)**(-2/25))**(11/7) assuming p is positive.
p**(-44/175)
Simplify (t**(-1/11)*t)**(8/5) assuming t is positive.
t**(16/11)
Simplify ((u*u/((u*u/(u*u**(-1/4)))/u))/(u/(u*(u*u**(2/7))/u)*u))**(-22) assuming u is positive.
u**(-319/14)
Simplify (g**(-1/3))**(-2/3) assuming g is positive.
g**(2/9)
Simplify n*n*n**25/n*n/n**(-7/6) assuming n is positive.
n**(169/6)
Simplify t/t**(-6/5)*t**(-7) assuming t is positive.
t**(-24/5)
Simplify ((f/f**(-11))/(f/(f*(f**(1/6)*f)/f*f*f*f)))**36 assuming f is positive.
f**546
Simplify (p**(6/19))**(2/29) assuming p is positive.
p**(12/551)
Simplify j**(-6/13)*j/j**(-4/11) assuming j is positive.
j**(129/143)
Simplify (m*m**(2/47)*m)/(m |
---
title: Video
desc: The QVideo Vue components makes embedding a video like Youtube easy. It also resizes to fit the container by default.
---
Using the QVideo component makes embedding a video like Youtube easy. It also resizes to fit the container by default.
::: tip
You may also want to check our own HTML 5 video player component: [QMediaPlayer](https://github.com/quasarframework/app-extension-qmediaplayer), which is far more advanced than QVideo (which essentially is an iframe pointing to embedded Youtube videos).
:::
## Installation
<doc-installation components="QVideo" />
## Usage
### Basic
<doc-example title="Basic" file="QVideo/Basic" />
### With aspect ratio <q-badge align="top" label="v1.7+" />
<doc-example title="With aspect ratio" file="QVideo/Ratio" />
### Markup equivalent
<doc-example title="HTML markup" file="QVideo/HtmlMarkup" />
## QVideo API
<doc-api file="QVideo" />
|
Shop By
Fabric
Brand new printed quilters cotton fabric has just arrived at Fur Addiction. Perfect for teddy bear paw pads, clothing and accessories. As well as many other craft uses. Prints have been carefully selected to complement our range of fur fabric and patterns. |
Patrick Subrémon
Patrick Subrémon (born 25 November 1947 in Saint-Laurent-d’Aigouze, Gard, Languedoc-Roussillon), is a French civil servant, prefect from 2000.
He is graduate of Institut d'études politiques d'Aix-en-Provence in Aix-en-Provence, Bouches-du-Rhône, PACA.
Career
Patrick Subrémon was from 1982 to 1984 chef de cabinet (principal private secretary) to Édith Cresson minister of Agriculture (1981–1983) and minister of Foreign Trade and Tourism (1983–1984). He is one of the persons behind the project Disneyland Paris. In January 1984, going with Édith Cresson, then minister of Foreign Trade and Tourism into United States, Patrick Subrémon met Ray Watson, president of Walt Disney Productions and Frank Stanek, promoter de Tokyo Disneyland et elaborates with them the general lines of the project of Disney in Europe.
Appointed sub-prefect en 1984, and prefect en 2000, he fulfilled several offices in prefectorial administration or in the Ministry of Interior. Especially he was prefect of Haute-Saône (2000–2003), of Allier (2003–2005)
of Eure-et-Loir (2005–2007)
and of Indre-et-Loire (2007–2009).
He is now inspecteur général de l'administration (inspector general of administration).
See also
References
"Subrémon, Patrick, Edmond" (prefect, born in 1947), pages 2067–2068 in Who's Who in France : Dictionnaire biographique de personnalités françaises vivant en France et à l’étranger, et de personnalités étrangères résidant en France, 44th edition for 2013 édited in 2012, 2371 p., 31 cm, .
Notes
Category:Living people
Category:1947 births
Category:People from Gard
Category:Sciences Po alumni
Category:Prefects of France
Category:Prefects of Haute-Saône
Category:Prefects of Allier
Category:Prefects of Eure-et-Loir
Category:Prefects of Indre-et-Loire |
1. Field of the Invention
This invention relates to a stereomicroscope including an objective lens disposed opposite to an eye, and the first and the second observing optical systems for three-dimensionally observing the eye.
2. Prior Art of the Invention
Heretofore, a stereomicroscope used for a surgical operation, for example, an operation for cataracta and the like has been known.
The stereomicroscope of this type, as shown in FIG. 9, includes an objective lens 2 disposed opposite to an eye 1, the first and the second observing optical systems 3 and 4 for three-dimensionally observing the eye 1 through the objective lens 2, and an illuminating optical system 5 for illuminating the eye 1.
The first and second observing optical systems 3 and 4 comprises zoom lenses 6a and 6b, imaging lenses 7a and 7b, and oculars 8a and 8b, while the illuminating optical system 5 comprises an illuminating light source K, a condenser lens 9, and a mirror 10.
When the eye 1 is illuminated by the illuminating optical system 5, the eye 1 can be three-dimensionally observed at a predetermined magnification by operating the zoom lenses 6a and 6b.
By the way, an operation for cataract is performed in such a manner as to cut out the fore-brain of a crystal body and to take out turbidity. However, when the rest amount of turbidity becomes little, the turbidity is illuminated utilizing a reflected light on the eye fundus. The reason is that if a little amount turbidity is directly illuminated by the illuminating optical system 5, it becomes difficult to observe the turbidity.
However, when the optical axis 105a of the illuminating optical system 105 is different from the optical axis 104a of the observing optical system 104 as shown in FIG. 10 an illuminated area T1 by the illuminating optical system 105 on the eye fundus 1a is displaced from the spot O on the optical axis of the observing optical system 104 as shown in FIG. 11. And a reflected pencil of rays by the illuminated area T1 proceeds toward the illuminating optical system 105. As a result, when the crystal body 1b is observed from the observing optical system 104, the reflected light from the eye fundus 1a scarcely enters to the observing optical system 104, and a pupil area 1c is observed dark as shown in FIG. 12.
Therefore, when the crystal body 1b includes, for example, turbidities A and B, the turbidities A and B are not illuminated by reflected light from the eye fundus and observation of the turbidities A and B becomes difficult. In FIG. 11, the characters U, V, denote pencils of rays to be converged to the turbidities A and B by reflection from the eye fundus 1a. The symbolic character 1d in FIG. 12 denotes an iris.
When the illuminating optical system 105 is approached to the observing optical system 104 and the illuminated area T1 of the illuminating light reaches the spot O including a pencil of rays V as shown in FIG. 13, the peripheral pencil of rays including V among the reflected light by the illuminated area T1 in FIG. 13 enters the observing optical system 104. Owing to the foregoing, the pupil area 1c looked from the observing optical system 104, as shown in FIG. 14 is dark at an area G including the turbidity A and bright at an area Q including the turbidity B.
When the optical axis 105a of the illuminating optical system 105 and the optical axis 104a of the observing optical system 104 are aligned, the wost of the reflected pencil of rays by the illuminating area T1 shown in FIG. 15 enters the observing optical system 15. As a result, the pupil area 1c, when looked from the observing optical system 104, it comes to have a uniform brightness owing to the reflected light from the illuminated area T1 as shown in FIG. 16.
By the way, in the stereomicroscope shown in FIG. 9, since the optical path 5a of the illuminating optical system 5 is different from the optical paths 3s and 4s of the first and the second observing optical systems 3 and 4 on the objective lens 2, the pupil area 1c does not come to have a uniform brightness when the eye 1 is looked from the oculars 8a and 8b of the observing optical systems 3 and 4. That is, as is shown in FIG. 18, a left-hand side area P1 of the pupil area 1c becomes bright in a left view field L and the right-hand side area P2 becomes dark owing to inversion. In the right view field R, as is shown in FIG. 18, the right-hand side area P1 of the pupil area 1c becomes bright and the left-hand side area P2 becomes dark owing to inversion.
In this way, the position of the area P1 which becomes bright and the position of the area P2 which becomes dark in the pupil area 1c are different depending on the left view field L and the right view field R. Because of the foregoing, there arises such a problem as that image fusion of the pair of eyes becomes difficult to obtain and a surgical operation becomes very difficult to perform.
Also, there is another example of the prior art in which a half mirror H is installed as indicated by the broken line of FIG. 9 and the eye 1 is illuminated from an outside position Ha of the first and second observing optical paths 3a and 4a as shown in FIG. 19. However, this prior art does not provide a uniform brightness at the pupil area as in the above-mentioned example. That is, the area P1 which becomes bright and the area P2 which becomes dark in the pupil area 1c are different depending on the left view field L and the right view field R as shown in FIG. 20. Therefore, an image fusion of the pair of eyes becomes difficult to obtain and a surgical operation becomes difficult to performed. |
Public utility companies manage unexpected crises by disseminating usable and understandable information about the incident/crisis to the general public. It is often hard to deal with the general public in a crisis communication situation;...
The number of encounters between black bears (Ursus americanus) and people is increasing in Connecticut. This increase necessitates management planning, which cannot occur until the actual population size of black bears has been determined. A...
The purpose of this study is to compare kindergarten teachers' perspectives regarding literacy education in S. Korea and the U.S.A; the need for literacy education, who drives that need, the level of expected achievement, teaching time, teachers'...
Thesis advisor: Thomas R. King.; "Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Arts in Biomolecular Sciences."; M.S.,Central Connecticut State University,2012.; Includes bibliographical references (leaves 38-39). |
Online:
Click here to visit our secure donation site, managed by Planned Parenthood Federation of America.
Phone:
Call us at 503.280.6156 and someone will assist you with making a secure donation over the phone.
Mail:
Print our Donate by Mail form to include with your check. Please send checks payable to Planned Parenthood Columbia Willamette to:
Planned Parenthood Columbia Willamette
3727 NE Martin Luther King Jr. Blvd.
Portland, OR 97212
Gifts to Planned Parenthood Columbia Willamette are fully tax-deductible as allowed by law. Planned Parenthood Columbia Willamette is a tax-exempt organization under Section 501(c)(3) of the Internal Revenue Code. We do not sell or distribute our donors' names.
Our federal tax ID is 93-6031270. |
/*
Copyright (C) 2000,2004 Silicon Graphics, Inc. All Rights Reserved.
This program is free software; you can redistribute it and/or modify it
under the terms of version 2.1 of the GNU Lesser General Public License
as published by the Free Software Foundation.
This program is distributed in the hope that it would be useful, but
WITHOUT ANY WARRANTY; without even the implied warranty of
MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.
Further, this software is distributed without any warranty that it is
free of the rightful claim of any third person regarding infringement
or the like. Any license provided herein, whether implied or
otherwise, applies only to this software file. Patent licenses, if
any, provided herein do not apply to combinations of this program with
other software, or any other product whatsoever.
You should have received a copy of the GNU Lesser General Public
License along with this program; if not, write the Free Software
Foundation, Inc., 51 Franklin Street - Fifth Floor, Boston MA 02110-1301,
USA.
*/
#ifndef PRO_ALLOC_H
#define PRO_ALLOC_H
#ifdef __cplusplus
extern "C" {
#endif /* __cplusplus */
Dwarf_Ptr _dwarf_p_get_alloc(Dwarf_P_Debug, Dwarf_Unsigned);
void dwarf_p_dealloc(Dwarf_Small * ptr); /* DO NOT USE. */
void _dwarf_p_dealloc(Dwarf_P_Debug,Dwarf_Small * ptr);
void _dwarf_p_dealloc_all(Dwarf_P_Debug dbg);
#ifdef __cplusplus
}
#endif /* __cplusplus */
#endif /* PRO_ALLOC_H */
|
The present disclosure relates to an image forming apparatus.
In general, an image forming apparatus such as a copy machine or a multifunction peripheral is capable of executing a continuous copy process. The continuous copy process is executed during a period from occurrence of a predetermined start event to occurrence of an end event.
For example, the start event is an event in which a predetermined start operation is performed on an operation portion. The end event is, for example, an event in which a sensor provided to a document sheet tray of an automatic document feeder comes to be in a state of not detecting any document sheet, or an event in which a predetermined end operation is performed on the operation portion.
In the continuous copy process, an image reading portion sequentially reads images of a plurality of pages of document sheets, and sequentially outputs a plurality of page image data pieces indicating the respective images of the document sheets. Then, an image forming portion executes a page printing process of forming, on sheets, the images indicated by the plurality of respective page image data pieces.
In addition, it is known that, in a case where the image reading portion continuously reads an image of the same page of the document sheets during the continuous copy process, the image forming apparatus outputs an alert message and stops the copy process. |
Q:
Undefined method for edit page
I am new to Rails. My new.html.erb file works perfect as it shows at http://localhost:3000/signup. However I can't seem to get /edit to work. I receive this error:
undefined method `model_name' for NilClass:Class
Extracted source (around line #3):
1: <h1>Account Information</h1>
2:
3: <%= form_for @user do |f| %>
4: <% if @user.errors.any? %>
5: <div class="error_messages">
6: <h2>Form is invalid</h2>
Here's my edit.html file which is a replica of the new.html that works. I tried removing the error messages code and it still just displayed another error with the page.
<h1>Account Information</h1>
<%= form_for @user do |f| %>
<% if @user.errors.any? %>
<div class="error_messages">
<h2>Form is invalid</h2>
<ul>
<% @user.errors.full_messages.each do |message| %>
<li><%= message %></li>
<% end %>
</ul>
</div>
<% end %>
<div class="field">
<%= f.label :email %><br/>
<%= f.text_field :email %>
</div>
<div class="field">
<%= f.label :password %><br/>
<%= f.password_field :password %>
</div>
<div class="field">
<%= f.label :password_confirmation %><br/>
<%= f.password_field :password_confirmation %>
</div>
<div class="field">
<%= f.label :username %><br/>
<%= f.text_field :username %>
</div>
<div class="field">
<%= f.label :zip_code %><br/>
<%= f.text_field :zip_code %>
</div>
<div class="field">
<%= f.label :birthday %><br/>
<%= f.text_field :birthday %>
</div>
<div class="actions"><%= f.submit %></div>
<% end %>
Here's my users_controller that I'm not sure if you need to look at or not. Maybe I have the def edit part wrong.
class UsersController < ApplicationController
def new
@user = User.new
end
def create
@user = User.new(params[:user])
if @user.save
UserMailer.registration_confirmation(@user).deliver
session[:user_id] = @user.id
redirect_to root_url, notice: "Thank you for signing up!"
else
render "new"
end
def edit
@user = User.find(params[:id])
end
def update
@user = User.find(params[:user])
if @user.update_attributes(params[:user])
flash[:success] = "Account updated"
sign_in @user
redirect_to @user
else
render 'edit'
end
end
end
end
A:
Your code indentation is a tell-tale sign here; you're defining the edit and update methods inside def create; the end immediately prior closes the if @user.save, not the def create.
|
Hampson Hughes Solicitors has warned fast food chains, such as McDonalds, of a possible influx in employees seeking legal action in relation to work related injuries, after recent reports of poor working conditions at their restaurants.
On 4 September, workers from two McDonald’s outlets in Cambridge and Crayford went on strike in the first every UK strike by employees of the world’s largest fast food chain. The strike action was taken after a union ballot over concerns about poor working conditions, low pay, and zero hours contracts.
Two unnamed McDonald’s workers have also appeared in a video created by Momentum, the grassroots political organisation, which was released on social media the day after the strike took place. The video includes the two workers describing and showing images of burns and injuries apparently gained while working at McDonald’s, which they say cannot be avoided when carrying out their normal duties, as no protective equipment is provided by their employer. One worker also claims that she has been given wrong, and potentially dangerous, information from supposedly first-aid trained McDonald’s shift managers on treating a burn she received at work. This, she alleges, was to ensure that she was able to continue her shift immediately, instead of taking time to treat the injury correctly.
Patrick Mallon, a solicitor and head of department (Accidents at Work) at the North West-based personal injury claims firm, said: “These stories are deeply worrying; and judging by the response on social media, there are potentially hundreds of other staff members who have experienced similar avoidable injuries in fast food restaurants across the UK, which is completely unacceptable.
“We believe that major food outlets should be setting an example to others, rather than taking advantage of staff who are too frightened to speak out. Equally concerning are the stories about staff being given incorrect medical advice, by supposedly medically trained managers, after being injured at work, which can result in long-term, or even permanent damage.”
Mallon added that the firm believed that the strike action was “just the tip of the iceberg”.
“We believe it’s only a matter of time before more fast food outlet employees start to speak out about the poor conditions they work under, and we think that legal action from staff who have been injured at work because of employer negligence could quickly follow.
“This should be a warning to all employers in this industry; employees should not, and will not, allow themselves to be silenced from speaking out and taking action when their working conditions do not keep them safe.” |
Q:
How to use Pandas to keep most recent data from Series with overlapping times
I have multiple pandas series, and they have some overlapping times:
In [1]: import pandas as pd
In [2]: cycle_00z = pd.Series(data=[10, 10, 10, 10],
index=pd.date_range('2015-01-01 00', '2015-01-01 03', freq='H'))
In [3]: cycle_02z = pd.Series(data=[20, 20, 20, 20],
index=pd.date_range('2015-01-01 02', '2015-01-01 05', freq='H'))
In [4]: cycle_04z = pd.Series(data=[30, 30, 30, 30],
index=pd.date_range('2015-01-01 04', '2015-01-01 07', freq='H'))
In [5]: cycle_00z
Out[5]:
2015-01-01 00:00:00 10
2015-01-01 01:00:00 10
2015-01-01 02:00:00 10
2015-01-01 03:00:00 10
Freq: H, dtype: int64
In [6]: cycle_02z
Out[6]:
2015-01-01 02:00:00 20
2015-01-01 03:00:00 20
2015-01-01 04:00:00 20
2015-01-01 05:00:00 20
Freq: H, dtype: int64
In [7]: cycle_04z
Out[7]:
2015-01-01 04:00:00 30
2015-01-01 05:00:00 30
2015-01-01 06:00:00 30
2015-01-01 07:00:00 30
Freq: H, dtype: int64
I would like to create another pandas series from these three, which will contain the unique times from these three cycles and the most recent data (when the times overlap). In this case it would look like this:
In [8]: continuous = pd.Series(data=[10, 10, 20, 20, 30, 30, 30, 30],
index=pd.date_range('2015-01-01 00', '2015-01-01 07', freq='H'))
In [9]: continuous
Out[21]:
2015-01-01 00:00:00 10
2015-01-01 01:00:00 10
2015-01-01 02:00:00 20
2015-01-01 03:00:00 20
2015-01-01 04:00:00 30
2015-01-01 05:00:00 30
2015-01-01 06:00:00 30
2015-01-01 07:00:00 30
Freq: H, dtype: int64
Just wondering if there would be a neat way to achieve that using pandas please? I will actually need to implement the technique in xray DataArrays but I guess the idea would be the same. Essentially, always keep data from most recent cycles.
Thanks
A:
One way is to use the combine_first method:
In [39]: cycle_04z.combine_first(cycle_02z).combine_first(cycle_00z)
Out[39]:
2015-01-01 00:00:00 10
2015-01-01 01:00:00 10
2015-01-01 02:00:00 20
2015-01-01 03:00:00 20
2015-01-01 04:00:00 30
2015-01-01 05:00:00 30
2015-01-01 06:00:00 30
2015-01-01 07:00:00 30
Freq: H, dtype: float64
Or, if you are performing the updates in a loop, something like this would work:
In [40]: result = cycle_00z
In [41]: result = cycle_02z.combine_first(result)
In [42]: result = cycle_04z.combine_first(result)
In [43]: result
Out[43]:
2015-01-01 00:00:00 10
2015-01-01 01:00:00 10
2015-01-01 02:00:00 20
2015-01-01 03:00:00 20
2015-01-01 04:00:00 30
2015-01-01 05:00:00 30
2015-01-01 06:00:00 30
2015-01-01 07:00:00 30
Freq: H, dtype: float64
|
About RECEs
Page Content
The training, knowledge, and competencies of early childhood educators are distinct and unique from other professions. The specialized skills of ECEs provide for collaborative opportunities with other regulated professionals.
Practice Settings
RECEs practise in a wide variety of settings:
Advocacy
Government
Post-Secondary Institutions
Full-day Kindergarten & Other Classroom Settings
Children's Services
Family Support Programs
Licensed Child Care
Unlicensed Home Child Care
Recreation Programs
Protected Scope of Practice
The ECE Act defines the practice of early childhood education as "the planning and delivery of inclusive play-based learning and care programs for children in order to promote the well-being and holistic development of children, and includes:
Delivery of programs to children 12 years or younger
Assessment of the programs and of the progress of children in them
Communication with parents or persons with legal custody of children in programs to improve the development of the children
such other services or activities as may be prescribed by the regulations"
Only College members can practice the profession of early childhood education and use the protected titles "Early Childhood Educator" and "Registered Early Childhood Educator" along with the professional designations ECE, RECE and their French equivalents.
The purpose of a protected title is to assure the public that any person who uses it has met the education and other requirements for entry into the profession. The protected title also assures the public that any person who uses it is accountable to practise the profession of early childhood education in accordance with the ethical and professional standards set by the College.
* The College of Early Childhood Educators provides information and communication in an accessible manner when requested. If you require an accessible format and/or communication support, please contact a College staff member or the College at
1 888 961-8558 info@college-ece.ca
. |
School of Health and Exercise Sciences
New research published today in the Journal of Physiology demonstrates that drinking a ketone supplement can lower blood sugar levels and might be a new tool to help diabetics control spikes in blood sugar.
It is with great sadness that we share the news of the passing of Dr Chris Willie. Chris was a Post-Doctoral Fellow in the School of Health and Exercise Sciences at UBCO having also completed his PhD with us in 2014. Chris was an incredibly talented young scientist and educator who had a brilliant mind […]
UBC researcher Danika Quesnel says telling people who are undergoing treatment for an eating disorder to completely abstain from exercise can be detrimental to the patient’s recovery and long-term health.
Partnership sets-up university laboratory downtown Kelowna With world diabetes day just around the corner on November 14, UBC Okanagan associate professor Mary Jung wants to help those living with type 2 diabetes or prediabetes create, and more importantly, maintain healthy habits. The developer and researcher has devoted her research career to investigating what makes healthy […] |
509 A.2d 1160 (1986)
STATE of Maine
v.
Michael GARDNER.
Supreme Judicial Court of Maine.
Argued March 10, 1986.
Decided May 21, 1986.
*1161 Janet T. Mills, Dist. Atty. (orally), South Paris, for plaintiff.
Cloutier, Joyce, Dumas & David, John C. McCurry, (orally), Edward S. David, Livermore Falls, for defendant.
Before McKUSICK, C.J., and NICHOLS, ROBERTS, WATHEN and GLASSMAN, JJ.
GLASSMAN, Justice.
The State appeals from an order of the Superior Court (Oxford County) granting the defendant Michael Gardner's motion to suppress statements claimed to have been taken from him in violation of the fifth amendment of the United States Constitution. The State contends that the suppression justice's reliance on the definition of custody recently articulated in State v. Thibodeau, 496 A.2d 635, 638 (Me.1985), cert. denied, ___ U.S. ___, 106 S.Ct. 1799, 90 L.Ed.2d 343 (1986), was misplaced. Because we agree, we vacate the order.
At the hearing on Gardner's motion to suppress, Corporal Harold Savage and Detective Hubert Carter of the Maine State Police testified about their investigation of the death of David O'Leary on Route # 17 in Roxbury, Maine, on April 6, 1985. In the two days following the accident, their investigation had revealed that on the night of the accident a pedestrian, walking not far from where O'Leary's body was found, identified Gardner's Jeep as one of two vehicles he had seen on the side of the road. At the "Okay Corral," located north of the accident scene, another individual had seen a Jeep and a second vehicle leaving the Okay Corral at approximately the time of the accident. That person provided information as to the driver of the Jeep from which a composite drawing was prepared that bore a striking resemblance to Gardner. Pieces of plastic that appeared to have come from a vehicle and part of a hood latch were found at the scene.
The two officers had learned Gardner was the registered owner of a Jeep before going to his residence on April 9 where they noticed a Jeep in the open garage. When the twenty-five-year-old Gardner answered *1162 the door, the officers, both wearing plain clothes, identified themselves and advised him they were investigating the Roxbury accident.
After a brief conversation, Gardner accompanied the officers to the garage to inspect the Jeep. The Jeep was wet, missing its hood latches, and was dented. Gardner showed the officers the hood latches that he had removed and put in a solvent in the basement of his residence. The officers observed that a piece missing from a hood latch appeared to match that found at the accident scene. Detective Carter then told Gardner he felt they needed to "have a very serious talk about the accident, his vehicle, and [his] involvement" in the accident. The three had just seated themselves at the kitchen table when Gardner's mother arrived. Because she was "very upset" and the difficulty of interviewing either in the presence of the other became obvious, Gardner agreed to accompany Corporal Savage to the police car parked outside the Gardner residence to continue the interview.
On the way to the police car, Gardner was neither handcuffed nor taken by the hand or arm. The four-door unmarked police car had no blue lights, siren, or screen between the front and back seat. Gardner got in the back seat of the car and Corporal Savage the front seat. The car was left unlocked. During the interview that lasted between ten and thirty minutes, Gardner made several incriminating statements.
After the conversation in the police car, Corporal Savage left to obtain a warrant for the Jeep and Detective Carter remained at the Gardner residence to secure the vehicle until Savage returned. During that period of time, Detective Carter overheard Gardner make additional incriminating statements to his mother. In response to Gardner's inquiry, Detective Carter told him there was no reason Gardner could not go outside since he was not under arrest. Gardner was not advised of his rights under Miranda v. Arizona, 384 U.S. 436, 86 S.Ct. 1602, 16 L.Ed.2d 694 (1966). He was never at any time informed he was under arrest or restricted to any area.
Two months later Gardner was indicted for manslaughter (Class B), 17-A M.R.S.A. § 203 (1983 & Supp.1985-1986). Gardner filed a motion to suppress inter alia all the statements made by him on April 9. After hearing, the Court granted Gardner's motion to suppress the statements made by him to Corporal Savage while they were in the police car. The Court found that Gardner was in custody while in the police car and should accordingly have been advised of his Miranda rights. His finding of custody was in reliance upon our definition of custody in Thibodeau, 496 A.2d at 638,[1] where we stated:
"A person is in custody for the purpose of Miranda only when he is deprived of his freedom in some significant way, or would be led, as a reasonable person, to believe he was not free to leave the presence of the police." Bleyl, 435 A.2d at 1358 (citations omitted); see also United States v. Rule, 594 F.Supp. 1223, 1234 (D.Me.1984) (whether a reasonable person in defendant's position would likewise have thought he was not free to go). The latter portion of our definition of "custody" is consistent with the United States Supreme Court's analysis of when a person has been "seized" within the meaning of the Fourth Amendment.
We have never decided that the Maine Constitution requires the warnings provided in Miranda, 384 U.S. at 479, 86 S.Ct. at 1630, be administered to a person undergoing custodial interrogation under penalty of exclusion of the evidence secured. See State v. Bleyl, 435 A.2d 1349, *1163 1358 (Me.1981).[2] Whether Miranda is applicable in any given situation is accordingly a matter of federal constitutional law. Id. We have in several previous cases made clear that even though a decision of the Supreme Court of the United States is the supreme law of the land on a federal constitutional issue, in the interests of existing harmonious federal-state relationships it is a wise policy that we accept, so far as reasonably possible, a decision on a federal constitutional issue rendered by the United States Court of Appeals for the First Circuit. See Littlefield v. State Department of Human Services, 480 A.2d 731 (Me.1984); State v. Knowles, 371 A.2d 624 (Me.1977); State v. Lafferty, 309 A.2d 647 (Me.1973).
On the appeal from United States v. Rule, 594 F.Supp. 1223 (D.Me.1984), vacated sub nom., United States v. Streifel, 781 F.2d 953 (1st Cir.1986), the First Circuit vacated the holding of the district court. That court held the district court "mistakenly thought" that the principal criterion for determining whether Rule's co-defendants, Streifel and Quinn, were in custody for the purpose of Miranda was whether "a reasonable person in defendant's position would have believed he was not free to leave." United States v. Streifel, 781 F.2d at 960. The court recognized that although this was the standard for determining whether a person had been seized within the meaning of the fourth amendment, the issue of custody for the purpose of the fifth amendment should not turn on this factor alone. Rather, it is only one factor among a host of those to be considered in determining whether the "suspect's freedom of action is curtailed to a `degree associated with formal arrest.'" Id. at 961 (quoting Berkemer v. McCarty, 468 U.S. 420, 104 S.Ct. 3138, 3151, 82 L.Ed.2d 317 (1984) (citation omitted)).[3] Until the Supreme Court of the United States decides otherwise, it seems appropriate that we adhere to our policy of deference to the First Circuit, particularly when, as in the instant case, the identical definition of "custody" for the purposes of Miranda was set forth in Thibodeau as was by the district court in Rule. Here the trial court focused on the Thibodeau definition of custody as dispositive of that issue for Miranda purposes, and "it did not make all the findings from which it can be ascertained whether a reasonable person in [the defendant's] position would have believed, not merely that he was not free to go, but that he was actually in custody and `at the mercy of the police.' Berkemer, 104 S.Ct. at 3150." Id. at 961.
Since the Superior Court felt "compelled" to make a finding of custody based on the definition of custody in Thibodeau, we must vacate its order and remand so that the court may make the factual findings necessary in applying the standard articulated herein. On remand, the court should determine whether a reasonable person in Gardner's position would have believed he was "actually in police custody and being constrained to a degree associated with formal arrest." Streifel, 781 F.2d at 962. Because the parties at the time of the suppression hearing may not have appreciated the difference between the standard announced in Thibodeau and that of Streifel, on remand, if requested by either party, the court may take additional evidence.
The entry is:
*1164 Order vacated. Remanded for further proceedings consistent with the opinion herein.
All concurring.
NOTES
[1] In his order on request for findings of fact, the suppression justice concluded:
The facts in this case are sufficiently similar to those in State v. Thibodeau, 496 A.2d 635 (Me.1985), so as to compel this court's conclusion that defendant was in a "custodial setting" while in the police cruiser within the meaning of State v. Thibodeau....
[2] Because the defendant at the suppression hearing did not raise the Maine Constitution and does not challenge the court's finding that his statements were voluntary, we decline to review the issue, raised for the first time on appeal, whether the statements should have been suppressed under article I, section 6 of the Maine Constitution. See State v. Ellis, 502 A.2d 1037, 1038 (Me.1985); State v. Thornton, 485 A.2d 952, 953 (Me.1984).
[3] Among the objective factors to be considered are: 1) whether the suspect was questioned in familiar surroundings; 2) the number of law enforcement officers present; 3) the degree of physical restraint placed upon the suspect; and 4) the duration and character of the interrogation. Streifel, 781 F.2d at 961 n. 13 (citing 1 W. LaFave & J. Israel, Criminal Procedure § 6.6, at 494-99 (1984)).
|
(defproject ╯°□°╯ "0.0.0-SNAPSHOT"
:license {:name "The MIT License"
:url "http://opensource.org/licenses/MIT"}
:dependencies [[org.clojure/clojure "1.7.0-alpha6"]])
|
Q:
AutoHotKey not finding image on a window
I recently discovered AutoHotKey, this scripting language seemed to be amazing !
Unfortunately, I can't manage to make my script find an image on a window (BlueStacks in my case).
Can someone please help me, my script is :
CoordMode, Pixel, screen
CoordMode, Mouse, screen
*ESC::ExitApp
ImgFound := false
while(ImgFound = false)
{
ImageSearch, ButtonX, ButtonY, 0, 0, A_ScreenWidth, A_ScreenHeight, *50 C:\Users\...\Documents\...\test.png
if (ErrorLevel = 2)
{
MsgBox Could not execute script.
ExitApp
}
else if (ErrorLevel = 1)
{
ImgFound := false
}
else
{
MsgBox Found.
click, %ButtonX%, %ButtonY%
ImgFound := true
}
}
A:
Your while loop is unreachable code.
The code execution stops when the first hotkey label is encountered. This is called the Auto-execute Section.
Move your hotkey definition to be at the very bottom.
(All hotkeys always defined by hotkeys labels get always created regardless of being in the auto-execute section or not)
|
Main menu
Breaking
Search form
Opinion
LGBTQ activism has always been an issue close to my heart. Though I am heterosexual, I am a proud and outspoken ally. And that is why me and my twelve loudest friends have chosen to have my bachelorette party at this crowded gay bar during pride weekend.
I, Plain Girl™, have entered into a relationship with the new love of my life and senior problem drinker, Doyle Flannigan. I think what attracted him to me was the fact that I'm not plastic and have a pulse, though I don’t mean to be presumptuous.
Yes, it is I, Victoria van der William Tudor III, the ultimate final woman, here to grace this campus with my humble worldview. After serving an 8-month long sentence at San Quentin Penitentiary (the reason is not important), I have determined that it is prison, and not The Bee, that is the ultimate final club.
Sing, Fates! Sing what will become of those most esteemed members of the Delphic!
The gods on Olympus have blessed our club with the all the will of Zeus himself, that most high and hallowed king before which even the board of the Harvard Financial Analysts Club must kneel. Fortune beyond compare, even to all the gold in the wondrous halls of Crete, is theirs for the taking for those sophomores who do not take issue with using an atmosphere of exclusivity to attract unsuspecting women.
I love my body. I love health. Having relationships, communication, is a healthy thing. When I got back to my room mid-January, though, my floor was deserted and I might as well have been the only person on Earth. But when I popped open the unplugged MicroFridge to find an unopened Chobani I myself had cruelly deserted, then decided to peel back the crusty-curded aluminum and snag a whiff, I knew I had struck an opportunity.
Tweet tweet. That’s right. It’s me, motherfucker. You know every time your mom has told you, “a little birdy told me” followed by some random messed up shit that you’ve done? That’s me, bitch. Big or small, life-threatening or otherwise, I will find out anything and everything that you have done wrong. In fact, my sole purpose in life is to keep your mom updated on all the reasons she should be disappointed in you. Room’s a mess? She knows. C on your midterm? She’s aware. One night stand with that boy Brad from the Alpha Chi Party? She’s on the phone with his mom right now.
CAMBRIDGE, MA -- Listen, I’ve ridden elevators hundreds of times, and never have I given it a second thought. I shuffle in, press the button for the thirteenth floor, stare at my shoes for 45 seconds, and go on my merry way.
But yesterday, in William James, everything changed when I noticed a smoky, disembodied voice coming from the corner -- an amorous timbre that set my heart aflame. It was an uneventful ride down from section until I got to the bottom of the shaft, the doors gave way, and I heard her croon the words every man longs to hear: “First floor. Main lobby.”
It’s no news that queer representation has been an issue in Hollywood. Since the birth of media, the narratives of people like me have been rejected from the big screen in favor of the same love story between a man and a woman being reiterated again and again.
I’m not sure if you’ve heard, but I deleted the apps for Instagram, Snapchat, and Facebook last week, and let me tell you, it was the best decision I’ve ever made. You know why? Because I realized I was spending more time scrolling and liking than living, and also because as soon as I did, my asshole began to shine brighter than a diamond. |
I[NTRODUCTION]{.smallcaps} {#sec1-1}
==========================
The worldwide epidemic of metabolic syndrome (MetS) has led to a striking increase in the number of people afflicted with the diabetes mellitus, and atherosclerotic cardiovascular disease. The MetS or syndrome X is a cluster of interrelated risk factors, which includes central obesity, glucose intolerance, dyslipidemia, and elevated blood pressure. These metabolic abnormalities constitute major risk factors for diabetes mellitus, atherosclerotic cardiovascular disease. At present, five separate definitions for MetS exist the World Health Organization working definition (1999),\[[@ref1]\] the European Group for the Study of Insulin Resistance definition (1999),\[[@ref2]\] the American Association Of Clinical Endocrinologists position statement (2003),\[[@ref3]\] the Adult Treatment Panel III guideline (2005),\[[@ref4]\] and the definition from the International Diabetes Federation (IDF) Consensus Group (2005).\[[@ref5]\] Although there is considerable debate over the terminology and diagnostic criteria of MetS.\[[@ref6]\] Recently, IDF and American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI) representatives held discussions to attempt to resolve the remaining differences between definitions of MetS.\[[@ref7]\] Both sides agreed that abdominal obesity should not be a prerequisite for diagnosis but that it is 1 of 5 criteria, so that the presence of any 3 of 5 risk factors constitutes a diagnosis of MetS.\[[@ref7]\]
For years, the terms impotence and erectile dysfunction (ED) were used interchangeably to indicate the man\'s inability to achieve or maintain erection sufficient for satisfactory sexual intercourse.\[[@ref8]\] The National Institutes of Health Consensus Development Conference advocated that ED be used in place of the term impotence.\[[@ref9]\] ED or impotence was now defined as "the inability of the male to achieve an erect penis as part of the overall multifaceted process of male sexual function." Although ED is not a life-threatening but it is associated with emotional distress can significantly affect important psychosocial factors, including self-esteem and confidence, and damage personal relationships.\[[@ref10][@ref11]\] The association of MetS with cardiovascular disease is well-known, now emerging data have shown a clear relationship of MetS with male sexual dysfunction. The MetS may lead to ED through multiple mechanisms. Atherosclerotic disease, which may be caused by the MetS may lead to ED by affecting the vascular tissues of the penis. Atherosclerosis can also lead to structural damage within the penile tissues.\[[@ref12]\] The MetS leads to endothelial dysfunction also, which leads to a decrease in vascular nitric oxide (NO) levels, with resulting impaired vasodilation. Hypogonadism that may be caused by the MetS can cause ED through altered testosterone (T): Estrogen levels that lead to hypogonadotropic hypogonadism and by decreasing T level, which impair the NO synthesis. Hyperglycemia, induces a series of cellular events that increase the production of reactive oxygen species like superoxide anion that inactivates NO to form peroxynitrite and also increases oxygen-derived free radicals through activation of protein kinase C and other cellular elements.\[[@ref13][@ref14]\] The increase in free radical concentration also leads to atherosclerotic damage to the vascular walls.\[[@ref15]\] Hyperglycemia may also cause glycation of penile cavernosal tissue, that lead to impairment of collagen turnover and later on ED.\[[@ref16]\] Hyperglycemia that associated with a proinflammatory states, which can also lead to endothelial dysfunction by upregulation of E-selectin and altered tumor necrosis factor-α and interleukin-10 ratio.\[[@ref17]\]
The association of MetS and hypertension,\[[@ref18]\] coronary artery disease (CAD),\[[@ref19]\] chronic obstructive pulmonary disease,\[[@ref20]\] polycystic ovarian disease,\[[@ref21]\] thyroid disease,\[[@ref22]\] and psychiatric disorder\[[@ref23]\] is well-described in the literature. Because of the relative paucity of randomized trials on the impact of the MetS on sexual dysfunction, most of the data were collected from cross-sectional or longitudinal studies, where association was observed but not a causation. Gunduz *et al*. first described the association in between MetS and ED and later it was reinforced by Heidler *et al*.\[[@ref24][@ref25]\] In addition, association of MetS and ED was reported in several other studies.\[[@ref26][@ref27]\] Furthermore, investigators still have not completely clarified the effects of the components of the MetS on ED. Furthermore, studies that have focused on the association between MetS and ED in the Indian population are sparse. Therefore, the present study aimed to intensively evaluate the relationship of MetS and ED in Indian men.
M[ATERIALS AND]{.smallcaps} M[ETHODS]{.smallcaps} {#sec1-2}
=================================================
A total of 113 subjects was recruited among those attending outpatient department of Endocrinology and Metabolic Clinic of Lala Lajpat Rai Medical (LLRM) College, Meerut, western Uttar Pradesh between August 2013 and March 2014. Men presented with ED who had three or more criteria to meet the diagnosis of MetS, as recommended by recent IDF and AHA/NHLBI joint interim statement were selected for study.\[[@ref7]\] The study was approved by the institutional committee of ethical practice of our institution, and all of the study participants provided written informed consent. Data on the demographic characteristics (e.g. age, education, and occupation), lifestyle characteristics (e.g. smoking, alcohol consumption, and physical activity), health status, and medical history were collected using a standardized Performa. The anthropometric measurements were performed by trained personnel using a standardized protocol. Body weight was taken by electronic weighing machine and height was measured by stadiometer with the subject barefoot fully erect, with the head in the Frankfurt plane; the back of the head, thoracic spine, buttocks, and both heels together with touching the vertical axis of the stadiometer. The body mass index (BMI) was then calculated formula given by Adolphe Quetelet as weight (in kilograms)/height^2^ (in meters). The waist circumference (WC) was measured midway between the lowest rib and the iliac crest to the nearest 0.1 cm. The blood pressure was measured by trained staff nurses using a mercury sphygmomanometer placed on the right arm of the participants, with the participants in a comfortable sitting position after 5 min of rest.
Any men with chronic renal disease, chronic liver disease, cardiovascular disease, peripheral or autonomic neuropathy, mental illness, history of pelvic trauma and surgery, prostatic disease, use of drugs or alcohol abuse, and smoking (both present and past smoking) were excluded from the study. To avoid the effect of drugs on ED, patients with established diabetes mellitus and hypertension were also excluded from the study. Endocrine causes of ED were also excluded. Finally, a total of 66 men with complete data was included in the study.
Venous blood sample was collected from each participant in between 8 am and 9 am after an overnight fast for laboratory assay. Plasma glucose was measured by a glucose oxidase method. Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides (TGs) were assessed with standard enzymatic spectrophotometric techniques in hospital\'s biochemistry laboratory. Haemoglobin A1c (HbA1c) was measured by high-performance liquid chromatography. Total serum testosterone, prolactin, and fasting insulin were measured with chemiluminescence method by abbott architect i1000SR in endocrinology laboratory. An homeostatic model assessment insulin resistance score (HOMA-IR) was computed with the formula given by matthews *et al*.: Fasting plasma glucose (FPG) (mmol/l) times fasting serum insulin (mU/l) divided by 22.5.\[[@ref28]\]
Assessment of erectile dysfunction {#sec2-1}
----------------------------------
Erectile function was assessed by completing questions one through five of the International Index of Erectile Function (IIEF-5), which is a multidimensional questionnaire consists of 5 questions for assessing ED.\[[@ref29]\] IIEF-5 questionnaire was translated in Hindi for the better understanding. The erectile function score represents the sum of questions one through five of the IIEF-5 questionnaire, with a maximum score of 25; a score \<21 indicates ED. The five questions assess erectile confidence, erection firmness, maintenance ability, maintenance frequency, and satisfaction. The severity of ED is classified into five categories as severe (score 5--7), moderate (score 8--11), mild to moderate (score 12--16), mild (score 17--21), and no (score 22--25) ED. Accordingly, out of 66 men 4, 22, 16 and 24 had mild, mild to moderate, moderate and severe ED, respectively.
Statistical analysis {#sec2-2}
--------------------
For numerical variables, descriptive statistics was performed, and the results were expressed as mean ± standard deviation. Pearson correlation was used for normally distributed variables. A multiple linear regression analysis was carried out with IIEF-5 score as dependent variable and components of MetS FPG, 2 h oral glucose tolerance test (OGTT), TG, HDL, and WC as independent variables. In a multiple linear regression analysis assessing effect on IIEF-5 score age, BMI and WHR were also included as independent variables. In the multiple linear regression analysis, components of MetS FPG, 2 h OGTT, TG, HDL, and WC were used as continuous variables. The Statistical analysis was performed using the Statistical Package for the Social Sciences Version 20 (\[SPSS\] IBM Corporation, Armonk, NY, USA). A *P* \< 0.05 was considered statistically significant.
R[ESULT]{.smallcaps} {#sec1-3}
====================
As [Table 1](#T1){ref-type="table"} shows baseline characteristic of total 66 participants, all the parameters Age, Wt, BMI, Waist, Hip, WHR, FPG, fasting insulin, HOMA-IR, 2 h OGTT, HbA1c, TG, LDL, and HDL were highly variable in all participants. Mean age and BMI of study population were 43.24 ± 6.2 and 27.95 ± 3.27, respectively. Mean WC, FPG, 2 h OGTT, TG, and HDL were 104.05 ± 7.89, 95.80 ± 15.80, 149.65 ± 24.52, 176.15 ± 50.98, and 45.33 ± 8.29, respectively. Mean IIEF-5 score was 10.17 ± 3.76.
######
Baseline characteristics
Correlation between erectile dysfunction and metabolic syndrome components {#sec2-3}
--------------------------------------------------------------------------
By using Pearson correlation analysis, as shown in [Table 2](#T2){ref-type="table"}, IIEF-5 score is statistically significantly correlated with age and correlation is negative. IIEF-5 score is statistically significantly correlated with components of MetS also; BMI (*r* = −0.261, *P* = 0.017); waist (*r* = −0.216, *P* = 0.082); HDL (*r* = 0.434, *P* = 0.000); FPG (*r* = −0.507, *P* = 0.000); 2h OGTT (*r* = −0.542, *P* = 0.000); TG (*r* =−0.426, *P* = 0.000).
######
Correlation between IIEF-5 score and MetS components
Multiple linear regression analysis of various components of metabolic syndrome for erectile dysfunction {#sec2-4}
--------------------------------------------------------------------------------------------------------
Using a multiple linear regression analysis as shown in [Table 3](#T3){ref-type="table"}, we also analyzed the effect on IIEF-5 score in relation to the presence of the MetS and the different components of the syndrome separately. The presence of the various components of MetS was associated with ED and a decrease IIEF-5 score, and this effect was greater than the effect associated with any of the individual components. Of the individual components of the MetS, HDL conferred the strongest effect on IIEF-5 score. However, the overall age had most significant effect on IIEF-5 score.
######
Multiple linear regression analysis of various components of MetS for IIEF-5 score
D[ISCUSSION]{.smallcaps} {#sec1-4}
========================
In the recent years, MetS has received considerable interest because of its association with increasingly common pathophysiologic states such as heart failure, type 2 diabetes mellitus, and ED.\[[@ref30][@ref31][@ref32]\] Multiple studies have shown a direct correlation in between various components of the MetS and ED.\[[@ref33][@ref34][@ref35][@ref36]\] In our study, we also observed independent association with components of MetS and ED. By using data from the Massachusetts Male Aging Study, Kupelian *et al*. have also demonstrated that the presence of MetS is associated with increased ED risk (relative risk = 2.09) even in those with a BMI of \<25.\[[@ref37]\] The cross-sectional analysis of adult participants in the 2001--2004 National Health and Nutrition Examination Survey revealed that poor glycemic control, impaired insulin sensitivity, and the MetS were associated with increased risk of ED.\[[@ref38]\]
Russo *et al*. have also found the insulin resistance as an independent predictor of ED.\[[@ref39]\] Thompson *et al*. confirmed in his study that ED is a sentinel marker and risk factor for future cardiovascular clinical events.\[[@ref40]\] Montorsi *et al*. in the COBRA study observed that in the majority of patients who developed CAD symptoms experienced ED before CAD by an average of 2--3 years.\[[@ref41]\] As the prevalence of the MetS is increasing at an alarming rate throughout the world. So by identifying high-risk asymptomatic individuals with the MetS could lead to improvements in the prevention and treatment of ED and hence risk of CAD. Taskinen has reported MetS, the most important public health threat of the 21^st^ century.\[[@ref42]\] Although multiple studies on western ethnicities investigating the associations between MetS and ED have been published, data from Indian population are still limited, and some doubts have not been completely explained. More importantly, identifying the relationships of MetS and ED will be useful to devise effective, population-based, preventive strategies.
Demir *et al*. observed in his study that IIEF-5 scores significantly decreased as the number of metabolic risk factors increased (*P* \< 0.001).\[[@ref26]\] In our study, we also found that the mean IIEF-5 scores decreased with an increasing number of MetS components. This finding proved that the severity of ED was associated with an increasing number of metabolic risk factors.
Even though the MetS appears to be an important risk factor for ED but still it is uncertain that which component of the MetS is more frequently associated with ED. In our study, we have demonstrated the relationship in between ED and various components of the MetS separately. We found an association between ED and the metabolic risk factors of abnormal fasting blood glucose (FBG) (B - 0.68, 95% confidence interval \[CI\] - 0.118--0.018), abnormal HDL (B - 0.135, 95% CI - 0.044--0.126), abnormal WC (B - 0.034, 95% CI - 0.199--0.131), and abnormal TG (B - 0.002, 95% CI - 0.019--0.015). Demir *et al*. and Lee *et al*. also reported a significant association between ED and abnormal FBG.\[[@ref26][@ref43]\] Yang *et al*. has showed that Diabetes mellitus alone, which is a component of MetS, is associated with presence and severity of ED; therefore, the association in between abnormal FBG and ED is appear to be logical.\[[@ref35]\] In addition, our results were not in line with those from some similar studies. Demir *et al*. reported that an abnormal WC was also a significant risk factor predicting the risk of ED. However, we did not find this relationship in our study, although that might be attributable to the higher mean WC in our study.
Our findings indicate that the MetS and ED are common health problems affecting the male quality-of-life in India.
C[ONCLUSION]{.smallcaps} {#sec1-5}
========================
Thus, it is critical to establish strategies to prevent or control the epidemic trend of the MetS and its consequences. The early identification and treatment of at-risk individuals could help target interventions to improve ED and secondary cardiovascular disease, including weight management, lifestyle interventions, and physical activity. Several studies have shown the efficacy of the intervention. Esposito *et al*. found a Mediterranean-style diet rich in whole grain, fruits, vegetables, legumes, and walnut and olive oil might be effective in reducing the prevalence of ED in men with the MetS.\[[@ref44]\]
The authors thank The Principal, LLRM Medical College for supporting endocrinology laboratory for doing all investigations free of cost. No other potential conflicts of interests relevant to this article were reported.
**Source of Support:** Nil
**Conflict of Interest:** None declared.
|
Q:
Derivative of $\sqrt{\frac{x-1}{x+1}}$
$$f(x)=\sqrt{\frac{x-1}{x+1}}$$
\begin{align}
f'(x) & ={1\over 2} \left(\frac{x-1}{x+1}\right)^{\frac{-1}{2}} \cdot {x+1-(x-1)\over (x+1)^2} \\[10pt]
& =\frac{1}{2}\left(\frac{x-1}{x+1}\right)^{\frac{-1}{2}}\cdot{2\over (x+1)^2} =\left(\frac{x-1}{x+1}\right)^{\frac{-1}{2}}\cdot{1\over (x+1)^2}
\end{align}
Is it valid to write $ (\frac{x-1}{x+1})^{\frac{-1}{2}}= \sqrt{\frac{x+1}{x-1}}$ ?
A:
Notice, you need to apply some constraints for the variable $x$,
for all $x>1$, it will be valid to write $$\left(\frac{x-1}{x+1}\right)^{-1/2}=\sqrt{\frac{x+1}{x-1}}$$
|
/* SPARC code for booting linux 2.6
*
* (C) Copyright 2007
* Daniel Hellstrom, Gaisler Research, daniel@gaisler.com.
*
* SPDX-License-Identifier: GPL-2.0+
*/
#include <common.h>
#include <command.h>
#include <asm/byteorder.h>
#include <asm/prom.h>
#include <asm/cache.h>
#include <image.h>
#define PRINT_KERNEL_HEADER
extern image_header_t header;
extern void srmmu_init_cpu(unsigned int entry);
extern void prepare_bootargs(char *bootargs);
#ifdef CONFIG_USB_UHCI
extern int usb_lowlevel_stop(int index);
#endif
/* sparc kernel argument (the ROM vector) */
struct linux_romvec *kernel_arg_promvec;
/* page szie is 4k */
#define PAGE_SIZE 0x1000
#define RAMDISK_IMAGE_START_MASK 0x07FF
#define RAMDISK_PROMPT_FLAG 0x8000
#define RAMDISK_LOAD_FLAG 0x4000
struct __attribute__ ((packed)) {
char traptable[PAGE_SIZE];
char swapper_pg_dir[PAGE_SIZE];
char pg0[PAGE_SIZE];
char pg1[PAGE_SIZE];
char pg2[PAGE_SIZE];
char pg3[PAGE_SIZE];
char empty_bad_page[PAGE_SIZE];
char empty_bad_page_table[PAGE_SIZE];
char empty_zero_page[PAGE_SIZE];
unsigned char hdr[4]; /* ascii "HdrS" */
/* 00.02.06.0b is for Linux kernel 2.6.11 */
unsigned char linuxver_mega_major;
unsigned char linuxver_major;
unsigned char linuxver_minor;
unsigned char linuxver_revision;
/* header version 0x0203 */
unsigned short hdr_ver;
union __attribute__ ((packed)) {
struct __attribute__ ((packed)) {
unsigned short root_flags;
unsigned short root_dev;
unsigned short ram_flags;
unsigned int sparc_ramdisk_image;
unsigned int sparc_ramdisk_size;
unsigned int reboot_command;
unsigned int resv[3];
unsigned int end;
} ver_0203;
} hdr_input;
} *linux_hdr;
/* temporary initrd image holder */
image_header_t ihdr;
void arch_lmb_reserve(struct lmb *lmb)
{
/* Reserve the space used by PROM and stack. This is done
* to avoid that the RAM image is copied over stack or
* PROM.
*/
lmb_reserve(lmb, CONFIG_SYS_RELOC_MONITOR_BASE, CONFIG_SYS_RAM_END);
}
/* boot the linux kernel */
int do_bootm_linux(int flag, int argc, char * const argv[], bootm_headers_t * images)
{
char *bootargs;
ulong rd_len;
void (*kernel) (struct linux_romvec *, void *);
int ret;
/*
* allow the PREP bootm subcommand, it is required for bootm to work
*/
if (flag & BOOTM_STATE_OS_PREP)
return 0;
if ((flag != 0) && (flag != BOOTM_STATE_OS_GO))
return 1;
/* Get virtual address of kernel start */
linux_hdr = (void *)images->os.load;
/* */
kernel = (void (*)(struct linux_romvec *, void *))images->ep;
/* check for a SPARC kernel */
if ((linux_hdr->hdr[0] != 'H') ||
(linux_hdr->hdr[1] != 'd') ||
(linux_hdr->hdr[2] != 'r') || (linux_hdr->hdr[3] != 'S')) {
puts("Error reading header of SPARC Linux kernel, aborting\n");
goto error;
}
#ifdef PRINT_KERNEL_HEADER
printf("## Found SPARC Linux kernel %d.%d.%d ...\n",
linux_hdr->linuxver_major,
linux_hdr->linuxver_minor, linux_hdr->linuxver_revision);
#endif
#ifdef CONFIG_USB_UHCI
usb_lowlevel_stop();
#endif
/* set basic boot params in kernel header now that it has been
* extracted and is writeable.
*/
ret = image_setup_linux(images);
if (ret) {
puts("### Failed to relocate RAM disk\n");
goto error;
}
/* Calc length of RAM disk, if zero no ramdisk available */
rd_len = images->rd_end - images->rd_start;
if (rd_len) {
/* Update SPARC kernel header so that Linux knows
* what is going on and where to find RAM disk.
*
* Set INITRD Image address relative to RAM Start
*/
linux_hdr->hdr_input.ver_0203.sparc_ramdisk_image =
images->initrd_start - CONFIG_SYS_RAM_BASE;
linux_hdr->hdr_input.ver_0203.sparc_ramdisk_size = rd_len;
/* Clear READ ONLY flag if set to non-zero */
linux_hdr->hdr_input.ver_0203.root_flags = 1;
/* Set root device to: Root_RAM0 */
linux_hdr->hdr_input.ver_0203.root_dev = 0x100;
linux_hdr->hdr_input.ver_0203.ram_flags = 0;
} else {
/* NOT using RAMDISK image, overwriting kernel defaults */
linux_hdr->hdr_input.ver_0203.sparc_ramdisk_image = 0;
linux_hdr->hdr_input.ver_0203.sparc_ramdisk_size = 0;
/* Leave to kernel defaults
linux_hdr->hdr_input.ver_0203.root_flags = 1;
linux_hdr->hdr_input.ver_0203.root_dev = 0;
linux_hdr->hdr_input.ver_0203.ram_flags = 0;
*/
}
/* Copy bootargs from bootargs variable to kernel readable area */
bootargs = getenv("bootargs");
prepare_bootargs(bootargs);
/* turn on mmu & setup context table & page table for process 0 (kernel) */
srmmu_init_cpu((unsigned int)kernel);
/* Enter SPARC Linux kernel
* From now on the only code in u-boot that will be
* executed is the PROM code.
*/
kernel(kernel_arg_promvec, (void *)images->ep);
/* It will never come to this... */
while (1) ;
error:
return 1;
}
|
// --------------------------------------------------------------------------------------------------------------------
// <copyright file="DeblockFilter.cs" company="HandBrake Project (http://handbrake.fr)">
// This file is part of the HandBrake source code - It may be used under the terms of the GNU General Public License.
// </copyright>
// <summary>
// Defines the DeblockFilter type.
// </summary>
// --------------------------------------------------------------------------------------------------------------------
namespace HandBrakeWPF.ViewModelItems.Filters
{
using System.ComponentModel;
using System.Globalization;
using System.Linq;
using Caliburn.Micro;
using HandBrake.Interop.Interop;
using HandBrake.Interop.Interop.HbLib;
using HandBrake.Interop.Interop.Model.Encoding;
using HandBrakeWPF.Model.Filters;
using HandBrakeWPF.Services.Encode.Model;
using HandBrakeWPF.Services.Presets.Model;
using HandBrakeWPF.Services.Scan.Model;
using Action = System.Action;
public class DeblockFilter : PropertyChangedBase
{
public static readonly string Off = "off";
public static readonly string Custom = "custom";
private readonly Action triggerTabChanged;
public DeblockFilter(EncodeTask currentTask, Action triggerTabChanged)
{
this.triggerTabChanged = triggerTabChanged;
this.CurrentTask = currentTask;
this.SetPresets();
this.SetTunes();
}
public EncodeTask CurrentTask { get; private set; }
public object Presets { get; set; }
public object Tunes { get; set; }
public bool ShowDeblockTune => this.SelectedPreset != null && this.SelectedPreset.Key != Off && this.SelectedPreset.Key != Custom;
public bool ShowDeblockCustom => this.SelectedPreset != null && this.SelectedPreset.Key == Custom;
public FilterPreset SelectedPreset
{
get => this.CurrentTask.DeblockPreset;
set
{
if (Equals(value, this.CurrentTask.DeblockPreset)) return;
this.CurrentTask.DeblockPreset = value;
this.NotifyOfPropertyChange(() => this.SelectedPreset);
this.NotifyOfPropertyChange(() => this.ShowDeblockTune);
this.NotifyOfPropertyChange(() => this.ShowDeblockCustom);
this.triggerTabChanged();
}
}
public FilterTune SelectedTune
{
get => this.CurrentTask.DeblockTune;
set
{
if (Equals(value, this.CurrentTask.DeblockTune)) return;
this.CurrentTask.DeblockTune = value;
this.NotifyOfPropertyChange(() => this.SelectedTune);
this.triggerTabChanged();
}
}
public string CustomDeblock
{
get => this.CurrentTask.CustomDeblock;
set
{
if (value == this.CurrentTask.CustomDeblock) return;
this.CurrentTask.CustomDeblock = value;
this.NotifyOfPropertyChange(() => this.CustomDeblock);
}
}
public void SetPreset(Preset preset, EncodeTask task)
{
this.CurrentTask = task;
if (preset == null)
{
this.SelectedPreset = new FilterPreset(HandBrakeFilterHelpers.GetFilterPresets((int)hb_filter_ids.HB_FILTER_DEBLOCK).FirstOrDefault(s => s.ShortName == "off"));
this.CustomDeblock = string.Empty;
this.SelectedTune = null;
return;
}
this.SelectedPreset = preset.Task.DeblockPreset;
this.SelectedTune = preset.Task.DeblockTune;
this.CustomDeblock = preset.Task.CustomDeblock;
}
public void UpdateTask(EncodeTask task)
{
this.CurrentTask = task;
this.NotifyOfPropertyChange(() => this.SelectedPreset);
this.NotifyOfPropertyChange(() => this.SelectedTune);
this.NotifyOfPropertyChange(() => this.CustomDeblock);
this.NotifyOfPropertyChange(() => this.ShowDeblockTune);
this.NotifyOfPropertyChange(() => this.ShowDeblockCustom);
}
public bool MatchesPreset(Preset preset)
{
if (this.SelectedPreset?.Key != preset.Task?.DeblockPreset?.Key)
{
return false;
}
if (this.SelectedTune.Key != preset?.Task?.DeblockTune.Key)
{
return false;
}
if (this.CustomDeblock != preset?.Task?.CustomDeblock)
{
return false;
}
return true;
}
public void SetSource(Source source, Title title, Preset preset, EncodeTask task)
{
this.CurrentTask = task;
}
private void SetPresets()
{
BindingList<FilterPreset> presets = new BindingList<FilterPreset>();
foreach (HBPresetTune tune in HandBrakeFilterHelpers.GetFilterPresets((int)hb_filter_ids.HB_FILTER_DEBLOCK))
{
presets.Add(new FilterPreset(tune));
}
this.Presets = presets;
this.NotifyOfPropertyChange(() => this.Presets);
}
private void SetTunes()
{
BindingList<FilterTune> tunes = new BindingList<FilterTune>();
foreach (HBPresetTune tune in HandBrakeFilterHelpers.GetFilterTunes((int)hb_filter_ids.HB_FILTER_DEBLOCK))
{
tunes.Add(new FilterTune(tune));
}
this.Tunes = tunes;
this.NotifyOfPropertyChange(() => this.Tunes);
}
}
}
|
Apple fears Samsung tablet will 'seduce' customers, court told - bane
http://www.smh.com.au/digital-life/tablets/apple-fears-samsung-tablet-will-seduce-customers-court-told-20110929-1kyl5.html
======
josteink
It must be nice having a legal monopoly upheld for you on a whole market
segment. But by all means, lets ignore Apple banning competition en-masse.
Lets discuss how Microsoft is leeching money of competing platforms instead.
That's just so much better.
I'm seriously looking forward to a future without all this patent bullshit.
Doing any real business in this climate seems borderline impossible. And doing
any kind of innovation seems even harder.
------
wccrawford
Wow. I am totally confused now.
"Apple is suing Samsung for patent infringement arguing the firm "slavishly"
copied its iPad."
"Today in court Samsung clarified that the film is "not relied on as prior art
because look and feel is not an issue [in this case] - it would be relevant to
a design case"."
So... Are they suing because they copied the look and feel or not?
------
SODaniel
Seriously, what the hell is going on with these lawsuits? Is Apple really on a
mission to prove that with enough money you can win any legal battle?
------
teilo
Asking because I do not know. Is Australian law so different that this kind of
an argument is actually persuasive? "You should block our competitor from
selling XYZ, because it is such a nice product that we might lose customers to
them."
------
locopati
Didn't Apple already lose this fight?
[http://en.wikipedia.org/wiki/Apple_Computer,_Inc._v._Microso...](http://en.wikipedia.org/wiki/Apple_Computer,_Inc._v._Microsoft_Corporation)
------
CubicleNinjas
These quotes are taken out of context. When displayed later in the piece they
paint the full picture – Apple believes the Galaxy Tab 2 is infringing on its
creative work in hopes to steal customers.
Boring article that relies on hyperbole for clicks. I feel used.
|
##### suite/funcs_1/include/tb3.inc
#
# This auxiliary script is used in several Trigger tests.
#
# If the table need data than the file std_data/funcs_1/memory_tb3.txt
# could be used.
#
--disable_warnings
drop table if exists tb3;
--enable_warnings
--replace_result $engine_type <engine_to_be_used>
eval create table tb3 (
f118 char not null DEFAULT 'a',
f119 char binary not null DEFAULT b'101',
f120 char ascii not null DEFAULT b'101',
f121 char(50),
f122 char(50),
f129 binary not null DEFAULT b'101',
f130 tinyint not null DEFAULT 99,
f131 tinyint unsigned not null DEFAULT 99,
f132 tinyint zerofill not null DEFAULT 99,
f133 tinyint unsigned zerofill not null DEFAULT 99,
f134 smallint not null DEFAULT 999,
f135 smallint unsigned not null DEFAULT 999,
f136 smallint zerofill not null DEFAULT 999,
f137 smallint unsigned zerofill not null DEFAULT 999,
f138 mediumint not null DEFAULT 9999,
f139 mediumint unsigned not null DEFAULT 9999,
f140 mediumint zerofill not null DEFAULT 9999,
f141 mediumint unsigned zerofill not null DEFAULT 9999,
f142 int not null DEFAULT 99999,
f143 int unsigned not null DEFAULT 99999,
f144 int zerofill not null DEFAULT 99999,
f145 int unsigned zerofill not null DEFAULT 99999,
f146 bigint not null DEFAULT 999999,
f147 bigint unsigned not null DEFAULT 999999,
f148 bigint zerofill not null DEFAULT 999999,
f149 bigint unsigned zerofill not null DEFAULT 999999,
f150 decimal not null DEFAULT 999.999,
f151 decimal unsigned not null DEFAULT 999.17,
f152 decimal zerofill not null DEFAULT 999.999,
f153 decimal unsigned zerofill,
f154 decimal (0),
f155 decimal (64),
f156 decimal (0) unsigned,
f157 decimal (64) unsigned,
f158 decimal (0) zerofill,
f159 decimal (64) zerofill,
f160 decimal (0) unsigned zerofill,
f161 decimal (64) unsigned zerofill,
f162 decimal (0,0),
f163 decimal (63,30),
f164 decimal (0,0) unsigned,
f165 decimal (63,30) unsigned,
f166 decimal (0,0) zerofill,
f167 decimal (63,30) zerofill,
f168 decimal (0,0) unsigned zerofill,
f169 decimal (63,30) unsigned zerofill,
f170 numeric,
f171 numeric unsigned,
f172 numeric zerofill,
f173 numeric unsigned zerofill,
f174 numeric (0),
f175 numeric (64)
) engine = $engine_type;
|
Vladimir Yezhurov
Vladimir Raimovich Yezhurov (; born 24 March 1960) is a Russian football manager and a former player. He also holds Tajikistani citizenship. He is an assistant manager with the Under-21 squad of Rubin Kazan.
External links
Category:1960 births
Category:Living people
Category:Soviet footballers
Category:Russian football managers
Category:FC Yenisey Krasnoyarsk managers
Category:FC Belshina Bobruisk managers
Category:Association footballers not categorized by position |
Shelley Weinberger had spent three days and nights listening to the pitiful meows of a cat, stuck more than 15 metres up a tree and unable to come down.
But as the Qu'Appelle, Sask., resident craned her neck to look at the scrawny black stray on Wednesday afternoon, she had a feeling that things would turn around yet.
"I'm actually feeling really hopeful," she said. "I think we're going to do it. I have no idea how, but I'm confident the cat's not going to spend another night in the tree."
Over the course of the afternoon, Weinberger's Qu'Appelle neighbours stop by to take a look and see if they have any ideas on how to bring a cat, stuck high in the tree, down.
Weinberger had first heard the meows of a cat on Sunday evening outside her home but it wasn't until Tuesday before she found the source of the sound, a small black blob far up a tree.
"How do you ignore something like that?" said Weinberger, who describes herself as an animal lover with two cats of her own. "I just can't turn my head away."
Over the next day, she began posting pleas for help and before long there were community members stopping by to offer suggestions.
But what would be effective? Put a bright blanket down with food atop it? Set up scaffolding? Shake the tree and put out a net to catch it?
At that point, it was a head-scratcher.
The cat sat on a branch more than 15 metres off the ground on Wednesday afternoon. Here Jeromy Desjarlais attempts the climb to reach the stray. (CBC News)
The town and fire department didn't have the right equipment to reach the cat, and an electric company that stopped by her house had a bucket lift that reached 12 metres high, still well short of the hungry and distressed animal.
Will rescue cats, in exchange for gas
Things were looking bleak for the stray until Jeromy Desjarlais saw a Facebook post asking people for help.
Desjarlais, owner of Jeromy's Moving and Delivery, handles tree removals and is a skilled climber. He offered to come out and help Weinberger, as long as she could pay for his gas to come from Regina, 53 kilometres away.
On Wednesday afternoon, once he looked up at the slender tree, he hitched up his shorts and gave a brief nod.
"Once I seen it, I knew it was a dangerous one but I knew I could do it."
Jeromy Desjarlais extends his 10-metre ladder along a tree, assessing how much farther he has to climb. (CBC News)
Desjarlais set up his 10-metre ladder against the slender tree, and moved to the top. From there, he climbed farther, snapping branches and harnessing himself to the tree along the way. When he was within an arm's reach of the cat, he held out a hand and waited.
The handful of onlookers below watched with bated breath, until the cat took a tentative step forward, closed the final distance, and went into Desjarlais' arms. Desjarlais made his way down gingerly, with a biting and clawing cat, to cheers.
The cat was happy to get a few morsels of food after it had been rescued. (CBC News)
"I had to climb down with only one arm, and that wasn't fun," he said, looking down at the scratches on his chest and joking, "I'm glad I didn't put him in my shorts."
The cat will visit a vet for treatment, with Weinberger hopeful she can find it a permanent home.
She gave credit to Desjarlais for his "skill, courage and big heart" for the rescue.
"I think that really speaks to people wanting to do good, and come together, help each other out —and he really did." |
Q:
Why I get this 0 as answer for (1/10) in Python?
I am trying this in Python 2.7 Interpreter.
>>> print 1/10
0
>>> print float(1/10)
0.0
>>> print 1/5
0
>>> a = 1/5
>>> a
0
>>> float(a)
0.0
>>>
I want a value in floating point when I divide. Any idea on the logic behind this zero out put and please let me know how to do it correctly.
A:
The float conversion is happening after the integer division. Try this:
>>> print(1.0/10)
0.1
A:
In Python 2.x, by default / means integer (truncating) division when used with two integer arguments. In Python 3.x, / always means float division, and // always means integer division. You can get the Python 3.x behaviour in 2.x using:
from __future__ import division
See PEP 238 for a full discussion of the division operators.
A:
You need to do something like:
print (1.0/10)
When you do:
print 1/10
It is evaluating it as 0 because it is treating it as an integer.
And when you do:
print float(1/10)
You take the stuff inside the float() and evalute that first so in essence you get:
print float(0)
Which would be 0.0
|
/*
Copyright (c) 2004-2010, The Dojo Foundation All Rights Reserved.
Available via Academic Free License >= 2.1 OR the modified BSD license.
see: http://dojotoolkit.org/license for details
*/
/*
This is an optimized version of Dojo, built for deployment and not for
development. To get sources and documentation, please visit:
http://dojotoolkit.org
*/
if(!dojo._hasResource["i18nTest.legacyModule"]){ //_hasResource checks added by build. Do not use _hasResource directly in your code.
dojo._hasResource["i18nTest.legacyModule"] = true;
dojo.provide("i18nTest.legacyModule");
window.i18nTest.legacyModule = function(){
var legacyBundle = dojo.i18n.getLocalization("i18nTest", "legacyBundle"),
result = [];
if(legacyBundle.rootValueOnly!="rootValueOnly"){
result.push('legacyBundle.rootValueOnly!="rootValueOnly"');
}
switch(dojo.locale){
case "ab":
case "ab-cd":
if(legacyBundle.legacyBundle!="legacyBundle-ab"){
result.push('legacyBundle.legacyBundle!="legacyBundle-ab"');
}
if(legacyBundle.abValueOnly!="abValueOnly"){
result.push('legacyBundle.abValueOnly!="abValueOnly"');
}
break;
case "ab-cd-ef":
if(legacyBundle.legacyBundle!="legacyBundle-ab-cd-ef"){
result.push('legacyBundle.legacyBundle!="legacyBundle-ab-cd-ef"');
}
if(legacyBundle.abValueOnly!="abValueOnly"){
result.push('legacyBundle.abValueOnly!="abValueOnly"');
}
if(legacyBundle.abCdEfValueOnly!="abCdEfValueOnly"){
result.push('legacyBundle.abCdEfValueOnly!="abCdEfValueOnly"');
}
break;
default:
if(legacyBundle.legacyBundle!="legacyBundle"){
result.push('legacyBundle.legacyBundle!="legacyBundle"');
}
}
return result.length==0 ? true : result.join(";");
};
}
dojo.i18n._preloadLocalizations("i18nTest.nls.legacyModule", ["ROOT","ar","ca","cs","da","de","de-de","el","en","en-gb","en-us","es","es-es","fi","fi-fi","fr","fr-fr","he","he-il","hu","it","it-it","ja","ja-jp","ko","ko-kr","nb","nl","nl-nl","pl","pt","pt-br","pt-pt","ru","sk","sl","sv","th","tr","xx","zh","zh-cn","zh-tw"]);
|
INTRODUCTION {#s1}
============
Liposarcoma is one of the most common soft tissue sarcomas. It can be classified into four biological groups \[[@R1], [@R2]\]: well-differentiated liposarcoma, myxoid and round-cell liposarcoma (MRCL), pleomorphic liposarcoma, and de-differentiated liposarcoma. Among them, MRCLs are predominant accounting for nearly 33% of all cases \[[@R3]\]. In general, morphologically round-cell liposarcomas are more aggressive than pure myxoid tumors, and a high percentage of round cells is often correlated with poor prognosis. MRCLs are characterized by unique chromosomal translocations and fusions, commonly harboring the t(12;16) (*FUS-DDIT3*) fusion; however, the t(12;22) (*EWSR1-DDIT3*) fusion can also be present. These gene fusions exist only in MRCLs, indicating a high specificity for diagnosis. Clinically, primary MRCL tumors usually arise in the limbs, and rarely from retroperitoneal or subcutaneous tissues like other types of liposarcomas. These tumors commonly metastasize to serosal membranes (peritoneum, pleura and pericardium, and abdominal cavity), distant soft tissues, and bone \[[@R4]\]. Surgical resection remains the most common treatment for MRCL. However, local recurrence and early distant metastasis significantly reduce patient quality of life and long-term survival, and traditional chemo- and radiation-based therapies show limited therapeutic effects.
PI3K is activated by tyrosine kinase growth factor receptors; this leads to phosphorylation of downstream mediators such as Akt and mTOR. Those two factors then promote phosphorylation of targets such as S6 ribosomal protein \[[@R5]\]. The abnormal activation of PI3K/Akt is thought to be an essential step in the initiation and survival of many human tumors. Inhibitors of PI3K signaling have proven to be beneficial for the treatment of many malignancies including breast cancer and non-small cell lung cancer, among others \[[@R6], [@R7]\]. *PIK3CA* encodes the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), and mutations often activate PI3K signaling. Such mutations have been reported to occur in 18% of MRCLs \[[@R8]\], and are associated with round cell transformation and poor clinical outcomes \[[@R9]\].
To date, the biological behavior and pathophysiology of MRCL are poorly understood, and as such, pre-clinical drug trials for this disease are rare. One of the major limitations is the lack of animal models that are comparable to human MRCL. To our knowledge, three MRCL animal models have been reported. All of these models were established using a mesenchymal stem cell line or a MRCL cell line harboring the FUS-DDIT3 gene fusion \[[@R10]--[@R12]\]. It is well established that the FUS-DDIT3 chimeric protein is associated with human liposarcoma. These models were valuable for studies on the *in vivo* function of FUS-DDIT3. However, they did not fully recapitulate the features of human liposarcoma or the cell-cell interactions that occur in human tumors; this limited their value for studies on targeted drug treatments.
Patient-derived xenograft (PDX) models have shown utility in recent years. For these, a tumor mass from a patient is directly implanted into the subcutaneous tissue of a severe combined immunodeficiency (SCID) mouse, which is followed by tumor growth. Upon reaching a sufficient mass, tumors are explanted, divided into smaller samples, and re-implanted for subsequent passage in mice. Therefore, a small piece of tumor can be propagated to achieve a tumor mass many-fold greater than that of the original tumor. Using this principle, the original tumor mass can be reproduced after many passages. Comprehensive genome-wide gene expression analyses have shown that tumors from PDX models at an early passage have genomic expression profiles that are very close to those of the original tumors \[[@R13]\]. Using PDX models, physicians have selected specific therapeutic agents to utilize for patient tumors. Theoretically, this approach could thus be used for personalized patient treatment, especially when the traditional standard of care fails. Researchers have tried to identify key pathway components that are suitable for targeted drug development \[[@R14]\]. However, the establishment of a PDX model is very expensive, requires a high level of technical support, and is associated with a low success rate, thus limiting its application. To our knowledge, no MRCL PDX model has been successfully established.
In the current study, we attempted to establish a PDX model of MRCL. Key features of the model were validated after passaging by assessing histological morphology and by confirming the presence of the FUS-DDIT3 gene fusion. Serially passaged tumors were compared to the original tumor sample from the patient. In addition, our patient tumor sample was positive for a *PIK3CA* mutation. We therefore utilized the PI3K inhibitor PF-04691502 to further validate our PDX model of MRCL.
RESULTS {#s2}
=======
Patient information {#s2_1}
-------------------
The donor sample used to establish the PDX model came from a 47-year-old man. The tumor was first found on the right thigh of the patient in 2006; after complete resection, it returned in 2009 both in the original location and in the retroperitoneum. The tumor was excised again, but reappeared twice in the retroperitoneum until complete surgical resection was impossible in 2013. Before that, no chemo or radiation therapy was administered. Histological analysis of the tumor samples showed that they were consistent with MRCL. The sample used to establish the PDX model in the current study was obtained from the second-to-last operation in July 2012.
PDX model of MRCL {#s2_2}
-----------------
The primary MRCL tumor (P0) was propagated for five passages (P1--P5). We randomly examined xenograft mice from each passage. Based on gross morphology, all xenografts showed preserved features of the original tumor (P0) (Figure [1](#F1){ref-type="fig"}).
{#F1}
Tumor histology and the presence of the DDIT3 gene were examined using samples from P1, P3, and P5. These samples were compared to the patient tumor sample.
Hematoxylin and eosin (H&E) staining showed that P1, P3, and P5 tumor samples were round cell liposarcoma with signet ring cells in the mucous background. These characteristics are specific for MRCL, as shown in the patient tumor sample (Figure [2](#F2){ref-type="fig"}).
{#F2}
Fluorescence *in situ* hybridization (FISH), specific for the FUS-DDIT3 gene fusion, showed that P1, P3, and P5 tumor samples had similar positivity to the patient sample (Figure [3](#F3){ref-type="fig"}).
{#F3}
When comparing tumor-specific gene mutations between P5 and the original patient tumor (P0) samples using whole exome sequencing (WES), we found that the two were nearly identical (Table [1](#T1){ref-type="table"}). According to WES results, a *PIK3CA* mutation was detected in both the P5 model tumor and the original patient tumor, Sanger sequencing was used to confirm the *PIK3CA* mutation in the original patient tumor (Figure [4](#F4){ref-type="fig"}).
###### Shared mutations of myxoid and round cell liposarcoma (MRCL) patient-derived xenograft (PDX) model and the original patient tumor sample
Gene Symbol Original patient tumor sample Corresponding PDX model sample
---------------------------------------- ---------------------------------------- ----------------------------------------
PIK3CA Exon 20: cDNA: G3145C, protein: G1049R Exon 20: cDNA: G3145C, protein: G1049R
TET2 Exon 3: cDNA: T320A, protein: L107H Exon 3: cDNA: T320A, protein: L107H
Exon 7: cDNA: C3809T, protein: T1270I Exon 7: cDNA: C3809T, protein: T1270I
KAT6A Exon 18: cDNA: G3937A, protein: D1313N Exon 18: cDNA: G3937A, protein: D1313N
Exon 17: cDNA: G3937A, protein: D1313N Exon 17: cDNA: G3937A, protein: D1313N
FGFR2 Exon 6: cDNA: A877G, protein: K293E Exon 6: cDNA: A877G, protein: K293E
Exon 6: cDNA: A868G, protein: K290E Exon 6: cDNA: A868G, protein: K290E
Exon 7: cDNA: A868G, protein: K290E Exon 7: cDNA: A868G, protein: K290E
Exon 7: cDNA: A946G, protein: K316E Exon 7: cDNA: A946G, protein: K316E
KDM5A Exon 21: cDNA: C3091T, protein: R1031C Exon 21: cDNA: C3091T, protein: R1031C
BRIP1 Exon 5: cDNA: G430A, protein: A144T Exon 5: cDNA: G430A, protein: A144T
Note: The table exhibits the alteration and location of the mutations detected by whole exome sequencing (WES). For example: Exon 20: cDNA: G3145C, protein: G1049R means the 3145th base sequence in exon 20 has mutated from G to C, and the corresponding protein\'s 1049th amino acid has mutated from glycine to arginine. Due to limited space, we have only listed some tumor related, nonsynonymous mutations on the table.
{#F4}
Short tandem repeat (STR) analysis of P1--P5 specimens showed that after detecting repetitive sequences in 10 STR sites, the similarity between P1 and P3 was 100%, P3 and P4 was 80%, and P4 and P5 was 100% (Table [2](#T2){ref-type="table"}).
###### Short tandem repeat (STR)-analysis of P1--P5 at 10 genetic sites
Genetic Site P1 P2 P3 P4 P5
-------------- ---------- ---------- ---------- ---------- ----------
Amelogenin X, Y X, Y X, Y X, Y X, Y
CSF1PO 9, 11 9, 11 9, 11 11, 13 11, 13
D13S317 8, 8 8, 8 8, 8 8, 11 8, 11
D16S539 11, 12 11, 12 11, 12 9, 12 9, 12
D5S818 13, 13 13, 13 13, 13 13, 13 13, 13
D7S820 11, 12 11, 12 11, 12 8, 11 8, 11
THO1 7, 7 7, 7 7, 7 7, 7 7, 7
TPOX 8, 10 8, 10 8, 10 8, 10 8, 10
vWA 17, 19 17, 19 17, 19 17, 19 17, 19
D21S11 30, 32.2 30, 32.2 30, 32.2 30, 32.2 30, 30.2
Note: The number of the samples shown for each genetic site is the repeat count of the repetitive sequence.
PF-04691502 Treatment {#s2_3}
---------------------
We used the PI3K/mTOR inhibitor PF-04691502 to test our MRCL PDX model. The trial began when the average tumor volume in the PF-04691502 treatment group was 130.00 ± 29.28 mm^3^ and that in the vehicle-only control group was 128.25 ± 32.78 mm^3^. After a 29-day treatment, the average tumor size in the PF-04691502 group was 492.62 ± 652.80 mm^3^, whereas that in the control group was 3303.81 ± 1480.79 mm^3^. Thus, the tumors in the treatment group were significantly smaller than those in the control group (*P* \< 0.001) (Figure [5](#F5){ref-type="fig"}).
{#F5}
Mice were found to tolerate PF-04691502 very well. Average weight loss in the treatment group mice was below 10% (Figure [6](#F6){ref-type="fig"}).
{#F6}
Furthermore, H&E staining showed that PF-04691502 treatment resulted in significantly fewer viable cells compared to that with vehicle treatment, and Ki-67 positivity was significantly lower in the PF-04691502 group than in the control group (1.40 ± 0.89 vs 3.20 ± 1.10, respectively, *P* = 0.022) (Figure [7](#F7){ref-type="fig"}).
{#F7}
Immunohistochemistry showed that the expression of pAkt, pS6, and p4EBP1 was significantly lower in the PF-04691502 group than in the control group (pAkt: 3.00 ± 1.58 vs 7.40 ± 2.07, respectively, *P* = 0.005; pS6: 3.00 ± 0.71 vs 10.20 ± 2.17, respectively, *P* = 0.001; p4EBP1: 3.40 ± 1.34 vs 10.80 ± 2.86, respectively, *P* = 0.001) (Figure [8](#F8){ref-type="fig"}).
{#F8}
Western blotting showed no difference in the expression of PPARgamma and no band was observed for ap2 or adipsin in either the PF-04691502 or control group (data not shown).
DISCUSSION {#s3}
==========
Palliative chemotherapy, including doxorubicin combined with ifosfamide, is usually required for MRCL patients \[[@R15]\]. However, when this fails, there is no effective secondary treatment. Furthermore, chemotherapy is often associated with considerable toxicity. Thus, new and effective targeted therapies are desperately needed for MRCL treatment. However, the lack of a proper animal model has hampered advances in creating and testing such therapeutics.
In our study, we successfully established a PDX model of human MRCL. We validated our model by histological analysis, FISH, WES assays, and STR analysis. We demonstrated that our model tumors have identical features to the original patient tumor, and these features are preserved until at least P5. Our PDX model of human MRCL is believed to be the first of its kind.
WES results showed that our patient tumor sample had a *PIK3CA* mutation, and this was also true of the PDX model. Jordi et al. \[[@R8]\] found that 18% of MRCLs harbor *PIK3CA* mutations, which are believed to be responsible hyperactivation of PI3K/mTOR signaling. Guo and colleagues \[[@R16]\] also found that 22% of liposarcomas have *PIK3CA* mutations, and these all proved to be MRCLs. Demicco et al. \[[@R9]\] showed that activation of the PI3K pathway might induce round cell transformation of MRCLs. We demonstrated that tumor samples from the donor patient and those from the mouse model (P5 as representative) were both positive for a *PIK3CA* mutation. This finding not only validated our PDX model, but also indicates that this animal model is suitable for testing potential treatments for liposarcoma.
We chose the PI3K/mTOR dual inhibitor PF-04691502 to test using our PDX model. PI3K/mTOR pathway plays a key role in regulating cell growth, cell proliferation, synthesis of proteins, and transcription. As predicted, PF-04691502 significantly inhibited tumor growth in P5 PDXs. It also inhibited expression of pAkt, pS6, p4EBP1, and Ki-67, indicating that PF-04691502 probably functions by inhibiting the PI3K/mTOR pathway, consequently suppressing tumor cell proliferation, probably through inhibition of Ki-67 expression. Martin et al. \[[@R17]\] reported that increased Ki-67 staining in prostate cancer is associated with higher PI3K immunohistochemistry scores. In our study, the tumor micromorphology appeared to undergo hyalinization with cell nuclei devitalization after treatment with the PI3K/mTOR inhibitor; this is similar to that seem with radiation, trabectedin, and doxorubicin therapy, as reported by others \[[@R18]\]. This type of change in histologic pattern is probably a common pathological even in MRCL when the therapy is effective. More importantly, side effects were tolerable as weight loss in the mice was never more than 20% with treatment. These results indicate that a PI3K/mTOR inhibitor could have great value for the treatment of MRCL tumors, especially those that are *PIK3CA* mutation positive or those with a high percentage of round cells. Our model could also be used to investigate the interaction between *PIK3CA* and FUS-DDIT3. Recently, de Graaff et al. \[[@R19], [@R20]\] reported the first spontaneously immortalized myxoid liposarcoma cell line and generated xenograft, which is a big step for MRCL drug research. Unfortunately, this cell line was deteremined to be wildtype for *PIK3CA*, thus could not be used to validate the effect of PI3K/mTOR inhibitors in our study.
Kathleen et al. \[[@R21]\] showed that inhibition of the PI3K pathway could increase tumor lipid content, conferring a more differentiated state, in dedifferentiated liposarcoma xenograft models. Herein, PF-04691502 did not have any observable effect on tumor differentiation as the expression of PPARgamma, ap2, and adipsin was not different between the treatment group and patient tumor sample. This likely indicates that the inhibitory effect of PF-04691502 on MRCL is not due to altering cell differentiation, but is rather based on inhibiting cell proliferation.
In our previous study, we identified many miRNAs that are differentially expressed in each subtype of liposarcoma and normal adipose tissues \[[@R22]\]. Using the model described herein, we also validated the function of these miRNAs and identified novel targets for the treatment of MRCL.
In conclusion, we have successfully established the first PDX model of MRCL, which could be used in future research. We showed that inhibitors targeting the PI3K/mTOR pathway could be effective in treating *PIK3CA* mutation positive MRCL. Our results should be further validated using a larger number of samples.
MATERIALS AND METHODS {#s4}
=====================
The patient from whom the tumor was derived gave informed consent, and this research was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (Ethical approval number B2012-022).
PDX establishment {#s4_1}
-----------------
The donor specimen was from a patient known to have MRCL. The patient had multiple surgical excisions and tumor histology signatures were well documented. Several equal-size fragments of tumor tissue from the 4^th^ excision (PA) were carefully harvested from the surgical specimen. These were implanted subcutaneously above the right shoulder blades of 6--8 week-old female BALB/c athymic mice (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai). When tumor masses grew to 2000 mm^3^ (P0), the P0 tumor was explanted and divided into equal sized specimens and implanted subcutaneously into mice as previously stated. These tumors were defined as P1. In the same manner, P2--P5 generations were established \[[@R23]\]. Tumors were measured using digital calipers (Cal Pro, Sylvac, Switzerland). Tumor volumes were estimated using the formula 0.5 × length × width^2^ (mm^3^).
Tumor fragments of 200 mm^3^ from each passage were submerged into special solution (10% DMSO, 20% FBS, and 70% RPMI 1640 medium) and stored in liquid nitrogen for the establishment of future xenografts. Additional pieces between 500 and 1000 mm^3^ from each passage were either snap-frozen in liquid nitrogen or used for histology.
Histological analysis {#s4_2}
---------------------
Tumor samples from PDX models (P1, P3, P5) and patient PA were fixed in neutral buffered formalin and embedded in paraffin blocks. Samples were sectioned into 5-μm specimens, mounted on slides, and prepared for histological analysis. Slides were first stained with H&E using a standard protocol.
FISH {#s4_3}
----
We used the *DDIT3* Break Apart FISH Probe Kit (Abbott Vysis) to test the existence of a *DDIT3* gene fusion. The procedure was performed according to the manufacturer\'s recommendation.
Genomic studies {#s4_4}
---------------
Snap-frozen tumor tissues from each passage were used for genomic analysis. DNA was isolated using a QIAamp DNA mini kit (Qiagen) in accordance with company recommendations. The concentration was quantitated using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). DNA samples with A~260/280~ ratios between 1.8 and 2.0 and A~260/230~ ratios above 2.0, and with quality confirmed by gel electrophoresis, were used for WES and SNP 6.0 array analyses.
Whole exome sequencing {#s4_5}
----------------------
One microgram of each DNA sample, isolated from P5 xenograft tumors, was used for library construction using TruSeq DNA sample preparation kits (Illumina, San Diego, CA, USA). Five hundred nanogram-libraries of DNA from each sample were pooled for exome capturing using the TruSeq Exome Enrichment kit (Illumina). Sequencing was then performed with paired-end 2 × 100 base reads on the Illumina HiSeq 2000 platform (Illumina). Raw. fastq files were first processed by a proprietary algorithm to filter out mouse sequence contamination. We have previously shown that this filter step does not affect human SNP detection \[[@R24], [@R25]\]. After mouse sequences were removed, data were aligned to the human reference genome hg19/GRCh37 by BWA 0.6.1 and processed using variant calling by GATK 1.6.
STR analysis {#s4_6}
------------
Genomic DNA was extracted from frozen P1 to P5 tumor samples from the PDX model. All samples, together with positive and negative controls, were amplified using the GenePrint 10 System (Promega). Amplified products were then processed using the ABI3730xl Genetic Analyzer. Data were analyzed using GeneMapper4.0 software and then compared to the ATCC, DSMZ, or JCRB databases for reference matching. Ten genetic sites were chosen for detection: CSF1PO, TPOX, TH01, VWA, D5S818, D7S820, D16S539, D13S317, D21S11, and Amelogenin. The similarity rates for each sample were calculated using the equation 2M/N; M was defined as the number of the same repetitive sequence in each site between two groups, and N was the number of all sites chosen for detection (which was 40).
*PIK3CA* genotyping {#s4_7}
-------------------
DNA was extracted from two 20-μm-thick formalin-fixed paraffin-embedded tissue blocks. One block was from the patient tumor, the other three MRCL tumor samples were chosen as controls. A QIAamp DNA mini kit DNA isolation kit (Qiagen) was used for DNA isolation. Polymerase Chain Reaction (PCR) was used with primers PIK3CA9F 5′- TG CTTTTTCTGTAAATCATCTGTGA-3′, IK3CA9R 5′-CAT GCTGAGATCAGCCAAAT-3′, PIK3CA20F 5′-ATGATGC TTGGCTCTGGAAT-3′, and PIK3CA20R 5′-CCTATG CAATCGGTCTTTGC-3′, with appropriate controls. PCR products were detected by gel electrophoresis using 2% agarose gels and amplified bands were purified using the QIAquick gel extraction kit (Qiagen).
Compounds and therapeutic assays {#s4_8}
--------------------------------
After confirming genomic consistency, P5 mice were used to further validate our model. A total of 20 mice were used. Treatment group mice (*n* = 10) were gavaged with the PI3K/mTOR inhibitor PF-04691502 (10 mg/kg) \[[@R26], [@R27]\] once per day, for 29 days. Control group mice (*n* = 10) were given the same volume of vehicle, once per day, for 29 days. PF-04691502 was suspended in 0.5% methylcellulose. Tumor sizes from both groups were measured twice per week. Tumor volumes were calculated using the modified ellipsoidal formula: V = 0.5 × (length × width^2^) mm^3^ (length = the greatest longitudinal diameter; width = the greatest transverse diameter). All measurements were obtained while mice were conscious. Animals were also weighed twice per week. The mice were euthanized when tumor volumes exceeded 3000 mm^3^ to ensure their wellbeing.
All procedures related to animal handling, care, and treatment were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec following guidance from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The animals were checked daily for any negative effects from tumor growth or treatments, based on normal behavior including mobility, food and water consumption, body weight gain/loss, eye/hair matting, and any other abnormal effects. Death and observed clinical signs were recorded.
Immunohistochemistry (IHC) {#s4_9}
--------------------------
Ten tumor blocks were randomly selected from the two groups (five per group). Tumor slides were immunostained for phosphorylated AKT (pAKT), phosphorylated S6 (pS6), phosphorylated 4EBP1 (p4EBP1), and Ki-67. Briefly, after de-paraffinization and antigen retrieval, the slides were incubated with antibodies against pAKT (1:300, \#3787, Cell Signaling), pS6 (1:300, \#2215, Cell Signaling), p4EBP1 (1:300, \#2855, Cell Signaling), and Ki-67 (1:500, ab66155, Abcam) overnight at 4°C. The slides were then washed and examined using the REAL™ EnVision™ Detection System, Peroxidase/DAB+, Rabbit/Mouse (\#K5007, DAKO).
Western blotting {#s4_10}
----------------
Western blotting was performed as described previously \[[@R28]\]. Antibodies used were as follows: anti-ap2 (1:50, SC-18661, Santa Cruz Biotechnology), anti-adipsin (1:50, SC-12402, Santa Cruz Biotechnology), and anti-PPARgamma (1:1000, \#2435, Cell Signaling).
Statistical analysis {#s4_11}
--------------------
IHC results were evaluated by two pathologists from Zhongshan Hospital. The data were quantitated according to the range and deepness of color; a score was assigned for each range. A range of 80--100%, was scored as 4; 60--79% was scored as 3, 40--59% was scored as 2, and \< 39% was scored as 1. The scores then were multiplied by the depth of color. The depth of the color was divided into three levels, namely, 1, 2, and 3. All analyses were performed using SPSS 16.0 software. The association between experimental and control groups were examined using the independent-samples *t*-test after a normality test; *P* \< 0.05 was considered statistically significant. The data depicted are represented as mean ± SD, and graphically represented as mean ± SEM.
The authors are grateful to Zhixiang Zhang, Baoyuan Zhang, and Quan Tang of WuXiAppTec for excellent technical support for this work.
**CONFLICTS OF INTEREST**
None.
**GRANT SUPPORT**
This research was funded by Science and Technology Commission of Shanghai Municipality (124119A3500).
|
PHOENIX (January 7, 2020) — Phoenix Rising Football Club, in partnership with FC Tucson, announced today the official 2020 Visit Tucson Sun Cup schedule.
A total of nine matches will be played from Feb. 15-22 at Kino North Stadium in Tucson, Ariz., and Casino Arizona Field in Scottsdale, Ariz. The 2020 Visit Tucson Sun Cup includes Major League Soccer clubs Columbus Crew SC, Houston Dynamo, New York Red Bulls, Real Salt Lake, and Sporting KC, plus USL Championship side Phoenix Rising FC.
The Visit Tucson Sun Cup winner will be determined by total points earned in three matches. Teams will earn three points for a win, one point for a draw, and zero points for a loss.
Fans can purchase their tickets for the Visit Tucson Sun Cup now by going to FCTucson.com. Tickets for the three Phoenix Rising matches can be purchased by clicking here.
Full 2020 Visit Tucson Sun Cup Schedule (Phoenix Rising matches in bold)
Saturday, February 15, 2020
Real Salt Lake vs Sporting KC @ 4:00 PM
Kino North Stadium (Tucson, Ariz.)
Saturday, February 15, 2020
New York Red Bulls vs Houston Dynamo @ 6:30 PM
Kino North Stadium (Tucson, Ariz.)
Saturday, February 15, 2020
Columbus Crew SC vs Phoenix Rising FC @ 7:00 PM
Casino Arizona Field (Scottsdale, Ariz.)
Wednesday, February 19, 2020
Houston Dynamo vs Real Salt Lake @ 4:00 PM
Kino North Stadium (Tucson, Ariz.)
Wednesday, February 19, 2020
New York Red Bulls vs Columbus Crew SC @ 6:30 PM
Kino North Stadium (Tucson, Ariz.)
Wednesday, February 19, 2020
Phoenix Rising FC vs Sporting KC @ 7:00 PM
Casino Arizona Field (Scottsdale, Ariz.)
Saturday, February 22, 2020
New York Red Bulls vs Sporting KC @ 2:00 PM
Kino North Stadium (Tucson, Ariz.)
Saturday, February 22, 2020
Houston Dynamo vs Columbus Crew SC @ 5:00 PM
Kino North Stadium (Tucson, Ariz.)
Saturday, February 22, 2020
Phoenix Rising FC vs Real Salt Lake @ 7:00 PM
Casino Arizona Field (Scottsdale, Ariz.)
About Phoenix Rising FC
Phoenix Rising FC is the highest-level professional soccer franchise in Arizona’s history and the 2019 USL Regular Season Title Winners. The club also holds the record for the longest win streak in American professional soccer history at 20 matches. The club is owned by legendary Chelsea and Ivory Coast striker, Didier Drogba, Advantage Sports Union CEO, Alex Zheng, club governor Berke Bakay, and an impressive collection of business leaders and international celebrities. Established in 2016, Phoenix Rising FC is applying for Major League Soccer (MLS) expansion and currently plays in the USL Championship, the largest Division 2 professional league in the world. The team also owns FC Tucson, which brings USL League One professional soccer to Southern Arizona. For season tickets or more information call 623-594-9606 or visit PHXRisingFC.com. You can also follow the team on Facebook (PHXRisingFC), Twitter (@PHXRisingFC), Instagram (@PHXRisingFC) and YouTube (PhoenixRisingFootballClub) or by downloading the Official Team App in the App Store or Google Play. |
Yen Wen-chang
Yen Wen-chang (; born 1947) is a Taiwanese politician.
Yen was elected to the sixth Legislative Yuan as a member of the Democratic Progressive Party representing Kaohsiung County in December 2004. During his legislative tenure, Yen took an interest in the Kuomintang's assets, and the use of ractopamine in pork imported from the United States. Yen lost reelection in 2008, to Kuomintang candidate Chung Shao-ho.
References
Category:1947 births
Category:Living people
Category:Members of the 6th Legislative Yuan
Category:Kaohsiung Members of the Legislative Yuan
Category:Democratic Progressive Party Members of the Legislative Yuan |
Safety of cordocentesis under ultrasound guidance for fetal blood sampling.
In the present study, we attempted to assess the safety and the risk of cordocentesis under ultrasound guidance for fetal blood sampling. Forty-five cordocenteses in 43 cases were carried out. These forty-three cases included 23 nonimmunologic hydrops fetalis, 12 congenital fetal malformations, 3 chromosome aberrations, 3 Rh isoimmunization, and 2 idiopathic thrombocytopenic purpura. Gestational age when cordocentesis was performed ranged from 17 to 39 weeks of gestation. In 42 (93.3%) out of 45 procedures, pure fetal blood was obtained. Postpuncture bleeding was observed in 17 cases (37.8%). In 12 (70.6%) out of 17 cases, bleeding stopped within 2 minutes. In only one case, bleeding continued for 11 minutes and cesarean section was performed due to fetal distress. Cardiotocogram obtained after cordocentesis revealed no ominous signs in the other 44 procedures. The other complications, including premature rupture of the membranes, premature labor, and intrauterine infection, did not occur during or after this procedure. There were no cases in which any kind of injury to the placenta or umbilical cord was observed. |
{
"extends": "../../tsconfigbase.test",
"include": ["src/*", "test/*"],
"references": [
{
"path": "../apputils"
},
{
"path": "../codeeditor"
},
{
"path": "../codemirror"
},
{
"path": "../coreutils"
},
{
"path": "../observables"
},
{
"path": "../rendermime"
},
{
"path": "../rendermime-interfaces"
},
{
"path": "../services"
},
{
"path": "../translation"
},
{
"path": "../ui-components"
},
{
"path": "."
},
{
"path": "../../testutils"
},
{
"path": "../apputils"
},
{
"path": "../codeeditor"
},
{
"path": "../codemirror"
},
{
"path": "../coreutils"
},
{
"path": "../observables"
},
{
"path": "../rendermime"
},
{
"path": "../rendermime-interfaces"
},
{
"path": "../services"
},
{
"path": "../translation"
},
{
"path": "../ui-components"
}
]
}
|
Q:
Too many arguments provided error in NUnit
I have a NUnit test that should run with 11 params but, it gives me error when I am trying to debug.
Error: Too many arguments provided, provide at most 11 arguments. I have tried to send these params as list in method but, it also couldn't work. What should i do ?
I changed my variable names because of the information security.
[Test]
[TestCase("11111111111", "5355553355", 0, 0, 0, "1", "11111.11111", 0, "INTERNET", null, 1, "abc*@dfg")]
public void FlowTestv2(string a, string b, decimal c, decimal d,
decimal e, string f, string g, decimal h,
string m, string j, string k)
{
FlowRequest(a, b, c, d,e, f, g, h, m, j, k);
Assert.AreEqual(LimitInfo.ErrorMessage, "EndPointMethodNotFound:GetInfo");
}
A:
In your Code Example the method's signature has 11 parameters but your [TestCase] has 12 defined. One too much.
For each attempt to run a test you have to define a own Attribute as exampled in the documentation.
[TestCase(12,3,4)]
[TestCase(12,2,6)]
[TestCase(12,4,3)]
public void DivideTest(int n, int d, int q)
{
Assert.AreEqual( q, n / d );
}
But every TestCase must match the method's signature.
|
/**
* Licensed to jclouds, Inc. (jclouds) under one or more
* contributor license agreements. See the NOTICE file
* distributed with this work for additional information
* regarding copyright ownership. jclouds licenses this file
* to you under the Apache License, Version 2.0 (the
* "License"); you may not use this file except in compliance
* with the License. You may obtain a copy of the License at
*
* http://www.apache.org/licenses/LICENSE-2.0
*
* Unless required by applicable law or agreed to in writing,
* software distributed under the License is distributed on an
* "AS IS" BASIS, WITHOUT WARRANTIES OR CONDITIONS OF ANY
* KIND, either express or implied. See the License for the
* specific language governing permissions and limitations
* under the License.
*/
package org.jclouds.openstack.nova.ec2.services;
import org.jclouds.ec2.domain.KeyPair;
import org.jclouds.ec2.services.KeyPairClient;
import org.jclouds.javax.annotation.Nullable;
/**
*
* @author Adrian Cole
*/
public interface NovaEC2KeyPairClient extends KeyPairClient {
/**
* Imports the public key from an RSA key pair that you created with a third-party tool. Compare
* this with CreateKeyPair, in which AWS creates the key pair and gives the keys to you (Nova
* keeps a copy of the public key). With ImportKeyPair, you create the key pair and give Nova just
* the public key. The private key is never transferred between you and Nova.
*
* <p/>
* You can easily create an RSA key pair on Windows and Linux using the ssh-keygen command line
* tool (provided with the standard OpenSSH installation). Standard library support for RSA key
* pair creation is also available in Java, Ruby, Python, and many other programming languages.
*
* <p/>
* <h4>Supported Formats</h4>
* <ul>
* <li>OpenSSH public key format (e.g., the format in ~/.ssh/authorized_keys)</li>
* <li>Base64 encoded DER format</li>
* <li>SSH public key file format as specified in RFC4716</li>
* </ul>
* DSA keys are not supported. Make sure your key generator is set up to create RSA keys.
* <p/>
* Supported lengths: 1024, 2048, and 4096.
* <p/>
*
* @param region
* region to import the key into
* @param keyName
* A unique name for the key pair. Accepts alphanumeric characters, spaces, dashes, and
* underscores.
* @param publicKeyMaterial
* The public key
* @return imported key including fingerprint
*/
KeyPair importKeyPairInRegion(@Nullable String region, String keyName, String publicKeyMaterial);
}
|
---
abstract: 'Optimal data detection in multiple-input multiple-output (MIMO) communication systems with a large number of antennas at both ends of the wireless link entails prohibitive computational complexity. In order to reduce the computational complexity, a variety of sub-optimal detection algorithms have been proposed in the literature. In this paper, we analyze the optimality of a novel data-detection method for large MIMO systems that relies on approximate message passing (AMP). We show that our algorithm, referred to as individually-optimal (IO) large-MIMO AMP (short IO-LAMA), is able to perform IO data detection given certain conditions on the MIMO system and the constellation set (e.g., QAM or PSK) are met.'
author:
- 'Charles Jeon, Ramina Ghods, Arian Maleki, and Christoph Studer [^1] [^2] [^3] [^4]'
bibliography:
- 'confs-jrnls.bib'
- 'publishers.bib'
- 'VIP.bib'
title: Optimality of Large MIMO Detection via Approximate Message Passing
---
[^1]: An extended version of this paper including all proofs is in preparation [@JGMS2015].
[^2]: C. Jeon, R. Ghods, and C. Studer are with the School of Electrical and Computer Engineering, Cornell University, Ithaca, NY; e-mail: [jeon@csl.cornell.edu]{}, [rghods@csl.cornell.edu]{}, [studer@cornell.edu]{}.
[^3]: A. Maleki is with Department of Statistics at Columbia University, New York City, NY; e-mail: [arian@stat.columbia.edu]{}.
[^4]: This work was supported in part by Xilinx Inc. and by the US National Science Foundation (NSF) under grants ECCS-1408006 and CCF-1420328.
|
Barriers to early detection of breast cancer among women in a Caribbean population.
The purpose of this descriptive study was to identify and describe barriers to early detection of breast cancer, as well as current breast cancer screening behaviors and attitudes regarding the disease, among women aged 20 and older on the Caribbean island of Tobago. Tobago is the smaller of the two islands that make up the nation of Trinidad and Tobago. Between February and June 1996, 265 women fitting the age criteria completed a structured survey questionnaire. Women of African descent made up 89% of the respondents. In terms of age, 48% of those surveyed were between 20 and 39, 40% were between 40 and 59, and 12% were 60 or older. Barriers to early detection identified were a low level of breast self-examination, infrequent clinical breast examinations as part of regular care, unavailability of mammography services on Tobago, cost of screening, and difficulty of traveling to Trinidad for mammography. Furthermore, only a minority of the study participants had ever attended early detection or public awareness programs. The results were nearly the same for individuals with a family history of breast cancer, who would have a higher risk of occurrence of the disease. In addition, the majority of the respondents reported what can be considered a cultural barrier to early detection practices, a belief that no matter what they did, if they were to get breast cancer, they would get it. The authors recommend that mammography services be made available on Tobago. The authors also intend to use the findings to help develop an appropriate, culturally sensitive breast cancer awareness and early detection program for women on the island. |
using System.Reflection;
using System.Runtime.CompilerServices;
using System.Runtime.InteropServices;
// アセンブリに関する一般情報は以下の属性セットをとおして制御されます。
// アセンブリに関連付けられている情報を変更するには、
// これらの属性値を変更してください。
[assembly: AssemblyTitle("setTimeZero")]
[assembly: AssemblyDescription("")]
[assembly: AssemblyConfiguration("")]
[assembly: AssemblyCompany("")]
[assembly: AssemblyProduct("setTimeZero")]
[assembly: AssemblyCopyright("Copyright © 2012")]
[assembly: AssemblyTrademark("")]
[assembly: AssemblyCulture("")]
// ComVisible を false に設定すると、その型はこのアセンブリ内で COM コンポーネントから
// 参照不可能になります。COM からこのアセンブリ内の型にアクセスする場合は、
// その型の ComVisible 属性を true に設定してください。
[assembly: ComVisible(false)]
// 次の GUID は、このプロジェクトが COM に公開される場合の、typelib の ID です
[assembly: Guid("5d104536-ca2e-40d8-8310-adf2312b1e2d")]
// アセンブリのバージョン情報は、以下の 4 つの値で構成されています:
//
// Major Version
// Minor Version
// Build Number
// Revision
//
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When you live in a small city or town in Wyoming, you feel a
responsibility to build up businesses, create jobs and increase opportunities
for everyone.
When you succeed,
just about everyone benefits. It is a very good feeling.
After working
in economic development for 48 years, though, sometimes the thought of going to
more meetings can make you a little bit weary.
When I asked
John Davis of Worland what he thought about the recent news stories about how
Idaho was the fastest growing state and Wyoming was the slowest, he replied:
“An interesting discussion, but one that feels like déjà vu all over
again. This has been a recurring situation all of my life.”
John and I are
about the same age and, yes, it does seem like we have been trying to build our
communities our entire adult life.
And, yet, we
plod along.
This column is
part 2 of an earlier discussion about Idaho and Wyoming compare. Here are some
comments from folks around the state:
Foundation CEO Patrick Henderson of
Sheridan weighs in: “When I first graduated from college – I moved to Pocatello,
Idaho. Nice community – friendly folks, diverse economy, lots of outdoor
recreation and a great college. I have
family that lives in Boise, both in the education field with Boise State.
“One thought is that Idaho is just
a lot warmer to live in than Wyoming and has very little wind.
“Idaho
has lots to offer with fishing, skiing, hunting and climbing opportunities. Idaho
is attractive, but I still prefer my Wyoming!”
One of the best-informed (and
highly-opinionated) guys around is economist Jonathan Schechter of Jackson, who
bemoans Wyoming’s worship of King Coal and finds it similar to the country’s
worship of “King Trump.” He says: “Put more succinctly, Wyoming is putting a
profound amount of energy into denying two basic realities: market forces and
scientifically-grounded truth. The former is especially ironic given our
alleged embrace of said forces.
Schechter continues: “This is
essentially the same phenomenon as is occurring nationally, and in both cases
the process is abetted by an utterly credulous media, which lacks the
intelligence, imagination, courage, and/or ability to act in ways that would enlighten
its audience. In that sense, the media is little more than a fixed part
of the Kabuki dance Wyoming`s legislature is leading, where the script and all
roles are completely rote, leaving no room for change, initiative, or the
like. Switching metaphors, an Emperor`s New Clothes phenomenon.”
Retired
teacher Dennis Coelho of Cheyenne says: “I grew up in southwestern Idaho, on my
grandfather’s homestead, about thirty miles south of Nampa. I have been
living in Cheyenne for almost forty years, and I have often thought of
comparisons between our fair state and our neighbor to the west.
“I know that recently a similar
essay comparing the states has received national recognition.
“I think a comparison has to start
with geography and geology. My grandfather’s farm was at 2,200 feet,
while here it is 4,000 feet higher. Southern Idaho is a-slosh in
water. While grandfather’s place was on the Snake River, The actual water
was in a canyon 400 feet below and useless for farming in our area. Most
of southwestern Idaho draws irrigation from a dam on the Boise River, a project
started around 1900 and the impetus for settlement in the area.
“When I was a lad, circa mid
fifties, Boise was about the size that Cheyenne is now, i.e. about 50,000
people. Tree lined streets and quiet avenues filled with craftsman
houses.
“The Boise area really began to
grow when a couple of tech companies, Micron for example, made a commitment to
build factories and research centers in the area. To some extent, they
chose Boise because there was a two-year college with ambitions to become a
full university, since the state university was in a very inconvenient setting
several hundred miles north in Moscow, where it dominates a small town,
difficult to get to at any time but especially so in winter.
“The economic spark started by Micron is still
growing as more and more people come to the Boise area. Real estate booms,
housing values and development increase. The demographics show solid
growth in that 20-40 age group similar to that in Ft. Collins.
“No arguing with the impacts of energy
development in Wyoming, but Idaho has had its own industrial impacts and
problems especially in the hard rock gold and silver mining areas in the northern
part of the state.
Coelho concludes: “The thing I like
most about Boise is the river running through the city. Wish we had that. But I
am not moving.”
Sunday, January 21, 2018
1804 - Torrington and Lusk are nice Wyoming towns
My recent tour of eastern Wyoming has been among the most
fun experiences of a near half-century in the state.
Nestled
between Devils Tower on the north end and Laramie Peak on the south end and the
rugged hills and buttes of western South Dakota and Nebraska, is a very special
place, stretching from up north to Hulett down to Pine Bluffs on the south. One
of our recent trips involved two wonderful towns, Torrington and Lusk.
It is hard to
find a small city in Wyoming that is more diversified that Torrington.
It has a
thriving Ag community including a big sugar mill plus a community college plus
a large home for children and the state’s medium security prison.
One the town’s
biggest annual events is the 2-Shot Goose Hunt and we were there for the annual
victory banquet Saturday, Dec. 9.
Gov. Matt Mead
was the biggest celebrity at the event, which he told me he enjoys very
much.Former Gov. Dave Freudenthal also
competed this year.And future governor
wanna-be Mark Gordon (our current state treasurer) also competed.
Hunters
compete in teams of two. One year Gov. Mead and his wife Carol were a
team.They camped out in their blind and
saw nary a bird. Mead later quipped at the banquet that night that they had
nothing else to do, so they repeated their marriage vows.
Bob Mayor gave
us a tour of the St. Joseph’s Children’s Home, which was started as an
orphanage some 87 years ago. Today, they serve young boys and girls who usually
are sent to the home by the courts. They usually stay about six months.
The home is
impressive.Its grounds are beautiful
and it has a solemn, beautiful chapel.Its museum is one of the more distinctive in the state.The home was founded by Bishop Patrick
McGovern of Cheyenne.
Our friends
Bryan and Donna Cay Heinz showed us around the area, including some fantastic
old homes.These old homes had crow’s
nests on the roofs where presumably you could watch for hostile Indians or just
check on things for quite a distance.
It was fun
visiting the Torrington Telegram and
meeting publisher Rob Mortimore and Editor Andrew Brosig.I have too much ink in my blood not to just
love the smells and sounds of the local newspaper.And the Telegram
is a darned good one.
The 2-Shot and
other events were held in some of the impressive County Fair buildings.Hard to imagine a town as small as Torrington
having an indoor arena of such size. They host national roping events and you
can see why. It is both enormous and impressive.
Another big
thing in this small town is the Torrington Livestock Exchange. It is one of
three biggest livestock auction barns in the country.Hard to imagine the number of cows that go
through that place each year.
Eastern
Wyoming College is going through a building boom, which we saw courtesy of one
of the students.President Leslie Lanham
Travers is a Lander native, whom I had watched growing up in my town.John Hansen, the director of institutional
development, has a number of impressive projects underway.
The college is all-in when it comes to the trades with a
massive welding teaching complex and an ample cosmetology facility.
As a student
of Wyoming history, it has always been easy to assume that the only major
railroad in the state is the Union Pacific, which runs across the southern tier
of counties.
But the
eastern side of the state was literally also built of towns nestled next to the
railroad, which includes Torrington, Lusk, Newcastle and onward north.
Our friends
Gene and Carol Kupke of Lusk hosted us during our visit to that town.Enjoyed seeing sites like the new overpass,
which was washed out by a flood not that long ago.
For a quarter
of a century, my wife Nancy and I owned a newspaper in Winner, S. D. and often
drove through Lusk on U. S. 20 on our way there from Lander.Always liked the town and always stayed at
the Covered Wagon. They have a terrific little newspaper, which is capably
managed by Lori Himes.
During my
stops in eastern Wyoming we also visited Jeff Rose at the Rose Brothers
Implement Store in Lingle.Last time I
saw him, he was climbing Devils Tower with his daughter.Now he is talking about climbing Gannett
Peak.Good luck on that!
Tuesday, January 9, 2018
1803 - Never forget Leslie Blythe
In her own words, Leslie Blythe could see a time into the
future when she would gather with a few of her girl friends, all of whom were
now over 100 years old, and they would sip cocktails at 4 p.m. Her drink of choice
would naturally be a Cosmopolitan.
All four gals
would be accompanied by their service dogs, which, not unexpectedly, were
Golden Retrievers.
She always
touted her Cosmo because it featured cranberry juice, which contains
antioxidants and prolongs life.
And who could
argue with her peek into the future? Leslie Blythe was a strong, lively young
woman when we had this conversation last year.She was in tip-top shape.She literally
had more friends than anyone anywhere. There is no non-politician in Wyoming who
could match her Rolodex.
Leslie died
from complications of the flu at the age of 58 on Jan. 5 at the Casper hospital.She was ubiquitous.She was the Energizer Bunny. She was a force
of nature. And I am finding it impossible to believe she is really gone.
I met Leslie when she took her
first media job as an intern at our newspaper in Lander.She was probably about 21 and worked with
some well-known Wyoming journalists such as Bruce McCormack (retired Cody
Enterprise publisher) and Milton Ontiveroz of the University of Wyoming’s
publications department.
She graduated
from UW and ended up working with media but doing it primarily through Pacific
Power, which morphed into Rocky Mountain Power. She had a 30-year career and
celebrated that anniversary with delight.
On her
Facebook page back on Dec. 1, she marveled that she had worked for the same
company for so long: “Thirty years ago today, I was blessed to embark on an
amazing journey with a great company. Fresh out of graduate school and just a
pup, I joined the ‘CY Avenue’ gang Dick Brown, Bob Tarantola, Bert Leonard,
Carl Ertler, Debbie Roman, Bill Miller, Bill Edwards, Linda Eckes Evans, Mary
Karantzas, Jane Drake, Jane Chatman, ‘Baughman,’ Ivan Bassart and so many
other wonderful co-workers throughout this state and elsewhere at what was then
called Pacific Power. So many fabulous opportunities over the years, and so
many remarkable adventures these past three decades.
“I feel
fortunate every day to call Rocky Mountain Power my home thank you! And, the
best part is being a member of this team. All the phenomenal people with whom
I`ve had the pleasure to work, as well as all the friends I`ve made over the
years in this great state and elsewhere. Thirty years! Don`t they go by in a
blink! Here`s to many more!”
Leslie and I always
stayed in touch and in recent years we were the two Wyoming representatives on
the Mountain West AAA board.She had a
huge impact on that group and represented Wyoming AAA members well.
It was at one
of our meetings in March 2017 when she got the news that her godson, Aidan
McCroskey, had died.Poor Leslie was
inconsolable. We all felt so helpless, as we tried to help her get through this
tragedy. Now, we are the ones who are inconsolable.
We will be celebrating her life at
a service this summer.
I cannot write
about Leslie without mentioning her love of Golden Retrievers, of which she and
her husband Mark Wilkinson were breeders of champions. They loved those dogs. She
was a champion showman and an officer in the national organization.
Leslie had a
great sense of humor. Her last Facebook post was a funny mention Dec. 30 when
the Quick Lane in Casper, which has a time and temperature sign, showed a
temperature of minus 196. “OK, I don`t want to hear any whining about the ‘cold
weather’ where you live try Casper Wyoming today! Certainly hope this is a sign malfunction, but
I wouldn`t be surprised if not, it`s so cold.”
But the post
that brought tears to my eyes came from her husband who wrote Jan. 5:“This is from Mark. My heart is breaking
knowing I have to inform you of my wife`s passing. Thank you all for the best
wishes and prayers over the past couple of days. Leslie was friend to all and I
know it may seem crass to post here, but I would rather you hear from the
horse`s mouth. Leslie knew so many people that I can`t make all those calls. I
will miss you my love, my wife, my friend.”
Monday, January 8, 2018
1802 - Tater heads outpacing Wyoming?
Far be it for me to refer to Wyoming as a
“hole,” but that was the unique position our state held during the worst bust
in its history.
During
the 1980s and 1990s, our state languished as the rest of the Rocky Mountain
states thrived.The syndrome was
referred to as “the donut hole.” The states around Wyoming make up the donut
and our state, located in the middle, being the “hole.”
This economic situation was blamed on our
state’s singular reliance on energy commodities, leading to countless calls for
Wyoming to diversify.These other states
all grew during those decades while our state lost jobs and state revenues
plummeted. Our economy was stagnant. There were few successes.This was also the time when a chunk of our working
middle class gave up and headed to more friendly economic climes.
The Democratic Party took a hit during
this time as a lot of union members left.The Democrats never really recovered from that exodus, but that is
another story for another time.
Following
that bust, we boomed from 2002 to 2014 as coal surged and oil and gas boomed. I
recall, in 2013, a long-time banker in Casper saying our economy was the best
he had ever seen – perhaps the best ever. But it did not last long.Now the state has fallen into a near-bust situation,
which was graphically pointed out in a widely circulated article in the Washington Post recently.
That
article stated that Idaho was the fastest-growing state in the country in 2017
and Wyoming was the slowest, finishing last. This prompted pundits and
concerned citizens alike to question what we were doing wrong and what on earth
were our neighboring potato-heads doing that was right?
The
numbers, in reality, were not spectacular.Idaho grew 2.2 percent and Wyoming dropped 1.0 percent. When ranked
against the other states and District of Columbia, we looked terrible and Idaho
looks brilliant.
I
emailed this story to some of the smartest people in the state and here are
some of their replies:
“1.
Idaho has a busy cityBoiseas state capital, good airport, university in
downtown area and several Fortune 500 companies and strong corporate giving. Boise
downtown has vibrancy and that has been a steady progression over past 30
years; Coeur d’Alene is their Jackson, but not to our scale. Nearby Spokane
feeds it and nearby lakes provide recreation and tourism year round. An
impressive inclusiveness reach from Boise to the rest of the state.”
“2.
A steady Ag base due to Snake River irrigationpotato farming
“3. In the panhandle there is great
scenery, skiing in winter and hiking/boating in the summer, along with big
spender income in Coeur d`Alene.
“4.
Less severe weather. You don`t hear about interstate closures like Interstate 80
here. Boise gets about 60 days more milder weather than Cheyenne.
Former long-time Wyomingite Bart Smith,
who is currently publisher of the Greeley
Tribune, says: “It’s no surprise that resource-dependent states ride that wave up and
down and have for many, many years. I’m sure a few years ago as oil
boomed and coal was strong that Wyoming was one of the fastest growing states
and all politicians took credit.
“I guess my main thought is this is nothing new at all — it has been
the case for decades, and a Washington
Post writer just now discovered it. That’s my take anyway!”
Cody Beers of Riverton says: “Having just driven through Idaho going to and from the Idaho
Potato Bowl, this story hit the media during the trip.
“Bottom line, our economy is very reliant
on minerals, and Idaho is much closer to the West Coast and Utah. Boise has had
large numbers of people move in from California, and this has occurred in the
northwest corner of the state, too. It`s cold (here) in the winter and the wind
blows, too.
“We spent time with friends in Pocatello.
They like Wyoming, but they love Idaho. Utah is close, and the other reasons
(health care, shopping, etc.).
“I`ve
lived here my whole life, and the people I talk to about Wyoming love coming
here to visit. They just don`t think Wyoming has much to offer, such as
shopping, hospitals, restaurants, etc. Borrowing from the movie Wind River, Wyoming is ‘the land of no
backup.’”
(This ends part 1 on this serious topic.
Check this space soon for part 2.)
Tuesday, January 2, 2018
1801 - The gem of the Wyoming Black Hills
During the past five years, we have made an effort to visit just
about every city and town in Wyoming. We have given talks and been involved
with other authors in book signings.
Lately, I had
not been having very good luck with Wyoming roads, but back on Nov. 30, the
roads were as dry as mid-summer all the way on our 306-mile, one-way journey to
Newcastle.
We drove by
Teapot Dome north of Casper and recalled how this massive oil field was the source
of one of the biggest scandals in American political history.Author Laton McCartney of Dubois wrote an
informative book about that scandal a few years ago. Ironically, the pending
scandal called Uranium One is located about 30 minutes from that site,
according to Tom Lubnau, Gillette.
The towns of
Midwest, Edgerton and Wright are along a route that takes you from Interstate
25 over to Highway 59.The imposing
Pumpkin Buttes loom to the north. The buttes is an area that I have always
wanted to explore.
You head east from Wright and go
through the massive Thunder Basin coalmine complex.If coal is dying, it does not look like it there.
Later, we met
members of the Wright family, who have a big ranch in the area. The town was
named for them.
We passed through the vast Thunder
Basin National Grassland.One rancher
told me three Triceratops fossilized skeletons have been found on their
ranch.That particular critter is the
official state dinosaur for Wyoming.
Newcastle knows
how to celebrate Christmas. They staged a big downtown celebration on Dec. 1,
which was topped off by the 15th annual Pinnacle Bank Festival of Trees.
The senior center was jammed full with people of good cheer, raising money to
charities.
Newcastle
appears to me to be a successful mix of folks from all different kinds of
employment persuasions. Some are coal miners who are bused daily to the Thunder
Basin mine.I ran into former Landerite
Paul Piana, who now works there. Paul is one of the state’s premier mountain
climbers. His wife Deb is mayor of Newcastle.
There is a big
oil refinery in the middle of the Weston County seat that is running at
capacity.Coal trains pass through the
town all day long.
The coolest
building in the town (and one of the most unique in the state) is the county
courthouse.You have to see it to
believe it.It was recently refurbished.It, alone, is worth a trip to Newcastle.
Newcastle is
nestled in the Wyoming Black Hills and is just eight miles from South Dakota.Tourism appears to be a huge opportunity for
growth.
There are big
ranches in the area and an abundance of oil and gas wells.Folks appear to be doing well, although some
business people complained the economy has tightened up in recent years. The
town has a new motel under construction.
My host was
local publisher Bob Bonnar of the News
Letter Journal.He is very energetic
about promoting the town.
Bob is a
former president of the Wyoming Press Association, and he has worked hard for
years pushing newspaper interests across the state. Not sure he has received
the credit he is due for his hard work.
Bob had also lined
me up to talk about Wyoming history with 49 fourth graders on Nov. 1, which was
so much fun.Our future is in good hands
if all young people are as energetic and anxious to learn as that bunch.
Newcastle is
one of Wyoming’s oldest towns.It
originally was a coal-mining hub, hence the name Newcastle, which is the name
of one of Great Britain’s greatest coal mining regions.
Their Wyoming
coal was in an area called Cambria, which was mined back in 1889, before
Wyoming became a state.
One of the early train masters for the CB&Q railroad
at Cambria was a chap by the name of Carl Kugland; he worked there from
1895-1903, and later became a Weston County commissioner, mayor of Newcastle,
and owned an insurance agency there.
He has two granddaughters who live in Wyoming, Jean Denham,
Cheyenne and Kate Brown, Wheatland. |
Former minor leaguer Morris Buttermaker is a lazy, beer swilling swimming pool cleaner who takes money to coach the Bears, a bunch of disheveled misfits who have virtually no baseball talent. Realizing his dilemma, Coach Buttermaker brings aboard girl pitching ace Amanda Whurlizer, the daughter of a former girlfriend, and Kelly Leak, a motorcycle punk who happens to be the best player around. Brimming with confidence, the Bears look to sweep into the championship game and avenge an earlier loss to their nemesis, the Yankees. Written by Rick Gregory <rag.apa@email.apa.org> |
Generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against SARS coronavirus using XenoMouse.
Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be an effective therapy against severe acute respiratory syndrome coronavirus (SARS-CoV). Utilizing the human immunoglobulin transgenic mouse, XenoMouse, we produced fully human SARS-CoV spike (S) protein specific antibodies. Antibodies were examined for reactivity against a recombinant S1 protein, to which 200 antibodies reacted. Twenty-seven antibodies neutralized 200TCID(50) SARS-CoV (Urbani). Additionally, 57 neutralizing antibodies were found that are likely specific to S2. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the RBD, aa 318-510. However, two S1 specific mAbs reacted with a domain upstream of the RBD between aa 12 and 261. Immunoglobulin gene sequence analyses suggested at least 8 different binding specificities. Unique human mAbs could be used as a cocktail that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants. |
Burying Borders - 3 Investor Lessons
What a pleasure it has always been to stop by my local Borders and leisurely browse through books that, admittedly, I would then order from Amazon. It seems those days are soon to be over, but there is still an important lesson here for investors.
Borders has a rich history dating back to the opening of a rental library in 1933, later becoming Borders Group, Inc. (BGP) when it made its public debut in 1995. It displaced the mom and pop local bookstores by its ability to offer customers many times the number of titles. Along with Barnes and Noble, Borders altered the landscape of how books were sold.
Within a couple of years of going public, shareholders were rewarded with the stock price tripling. It was a stock darling. What could possibly go wrong?
We all know the ending to this story. Amazon.com and the eBook rendered the model obsolete much faster than Borders did the same to the local bookstores.
Lessons learned
Using hindsight bias, we can clearly see how flawed their model was, and that they should have entered the e-space much sooner. But, trust me, it wasn't that obvious back then. Here are three key investment lessons we can take away from Borders' fate.
#1 No company is bullet proof. As Apple Inc. was setting a new record, and many analysts were declaring Apple will be the most valuable company on the planet, it was as important then as it is now to remember to always be aware of any company's vulnerability. Only a few years ago, Nokia sat on top of the mobile phone world. State of the art today is ready for a museum a year or two later.
#2 The obvious isn't so obvious. Today it's obvious that Borders should have entered the internet and ebook space much faster than they did. Just as it should have been obvious that Kodak was too late in realizing film was about to take a major backseat to digital. But this perspective comes via hindsight bias, as the unexpected twists and turns of competition and technology weren't quite so obvious at the time.
#3 Diversification is key. Understand your crystal ball is broken. Companies that currently dominate the game may soon be warming the bench. We don't actually know what companies will still be around in 20 years, much less which ones will prosper.
I come to bury Borders, not to praise it.
I'm very saddened by the death of Borders, and even more so that their 11,000 employees will soon be losing their jobs. Capitalism can be a cruel and capricious beast. However, technology and competition bring both lower prices and more convenience and features to all consumers.
And though my index fund lost money as Borders blundered, it gained even more as Amazon prospered.
Allan S. Roth is the founder of Wealth Logic, an hourly based financial planning and investment advisory firm that advises clients with portfolios ranging from $10,000 to over $50 million. The author of How a Second Grader Beats Wall Street, Roth teaches investments and behavioral finance at the University of Denver and is a frequent speaker. He is required by law to note that his columns are not meant as specific investment advice, since any advice of that sort would need to take into account such things as each reader's willingness and need to take risk. His columns will specifically avoid the foolishness of predicting the next hot stock or what the stock market will do next month. |
Q:
Adaptation of sum of arrival times of Poisson process
Let $\{N_t\}_{t\geq0}$ be a Poisson process and $\{F_t\}_{t\geq0}$ be its nautral filtration so that $\{N_t\}_{t\geq0}$ is adapted. $T_i$ be the $i$th arrival time of Poisson process of arrival rate $\lambda$, given $t>0$, prove if $\sum_{i=1}^{N_t} T_i$ is also adapted to $\{F_t\}_{t\geq0}$?
A:
Let $L_t=\sum\limits_{i=1}^{N_t}T_i$, then $L_t=\int\limits_0^t(N_t-N_s)\mathrm ds$. Since $(N_s)_{0\leqslant s\leqslant t}$ is $F_t$-measurable, this proves that $L_t$ is $F_t$-measurable.
To prove the key-formula used above, fubinize $L_t$, that is, using the identity $[s\lt T_i]=[N_s\lt i]$, write $L_t$ as
$$
L_t=\sum\limits_{i\geqslant1}\mathbf 1_{i\leqslant N_t}\int_0^\infty \mathbf 1_{s\lt T_i}\mathrm ds=\int_0^\infty \sum\limits_{i\geqslant1}\mathbf 1_{N_s\lt i\leqslant N_t}\mathrm ds=\int_0^\infty(N_t-N_s)^+\mathrm ds.
$$
|
More from Karl Connor, spokesman at Sellafield: "The shutdown has nothing to do with the work here at the site. This is purely a safety issue because of the high winds and snow, we want our 8,500 workers to go early and get home safely."
More from Karl Connor, spokesman at Sellafield: "The shutdown has nothing to do with the work here at the site. This is purely a safety issue because of the high winds and snow, we want our 8,500 workers to go early and get home safely." |
Q:
Returning a Secure "Winning Code" to User
This is probably a very basic/introductory thing, but let me paint a scenario:
Let's say I'm using jQuery to do some sort of mini quiz game on my site. The user clicks the correct answer, and then a window pops up with a secret "winner's code" that they can redeem elsewhere.
How can I generate the winner's code so that it doesn't just appear in the HTML and in a way that users cannot reverse-engineer it (at least without considerable effort)?
I mean, if I just generated an encoded string containing their username and some sort of additional information only I know, that would work, right? Or MD5 hash or something, but how do I make it so the winner's code itself doesn't appear in the HTML, and only when the correct answer is chosen?
Thank you for any suggested reading/tutorials/assistance/advice you can offer.
A:
There are a couple of questions there.
1) How to generate a secure code:
if you are implementing a 'winner code' you could use an hashed iv + secret + user information, or some signing mechanism. You could also implement a code expiry time on the server so that you could further raise the bar, if necessary.
2) Getting the code to the winning user:
If you don't want the code to appear in the html, then you want to use ajax to get it. Then inject the code into the DOM where you want to display it. Further, you should be using a secure SSL channel to do this so that you guard against sniffing. Even further, consider some kind of 'one-time' token so that a man-in-the-middle cannot repost your code request and receive the same win code.
Hope this gives you something to consider.
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Whipping the Pussy Riot Grrrl Act
“Riot Grrrl, in a conscious response to second-wave feminists’ rejection of the word “girl,” reclaimed it with pride — and also in parody. Songs, performances, and fashion statements mocked the depictions of feminine innocence and compliance served to us in the face of discrimination, exploitation, and endemic sexual abuse.” —My Riot Grrrl by Johanna Fateman
Powerful words that reverberate inside cemented walls where two Pussy Riot members were imprisoned for 21-months, released in what appeared to be a pre-Sochi Olympic “publicity stunt,” and which culminated in a Cossack “pussy whipping” that went viral in a music video the very next day (February 20, 2014).
I won’t lie. When I watched the actual footage of the Cossacks accosting Pussy Riot, I cried — but then got enraged at the horrific absurdity of the Russian Police pepper spraying young women and beating them with whips for singing a song of protest! But it hasn’t stopped there. Recently, two members, Nadezhda Tolokonnikova and Maria Alyokhina sustained chemical burns and head injuries after an attack at a McDonald’s by men (wearing patriotic symbols) who threw trash and shot green paint using syringes.
Women in Russia hold a dual role straight out of the TV series Mad Men — at once carrying the nation on a day-to-day basis while being relatively absent from public life. Outside the home, young women exist to look pretty. They are expected to have babies and cook and clean. They are not supposed to speak up. They are definitely not there to usher in change. By masking their faces, occupying public spaces and raising their voices, Pussy Riot is challenging the entire social order — not just the men to whom their anger has been directed.
Clip from the film The Punk Girl by Siri Anderson
These demeaning and barbaric acts against Pussy Riot remind me of just how gut-wrenchingly spot on Kathleen Hanna of the punk girl band Bikini Kill was when she shouted out to her audiences:
“All girls to the front! I’m not kidding. All girls to the front. All boys be cool, for once in your life. Go back… Back. Back.”
A flip (off) to the script, her words speak loudly and proudly of female empowerment and to the right of female safety while also calling out male privilege. In fact, the necessity of her words is even more apparent when you listen to a later exchange in the 2013 documentary, The Punk Girl, directed by Sini Anderson:
Guy in audience:
“I don’t think it’s a problem, because most of the girls ask for it.”
Kathleen Hanna:
“How do they ask for it?”
Response from guy in audience:
“The way they act… the way they, uh, I can’t say the way they dress, ’cause that’s their own personal choice. Some dumb hoes, these butt rocker bitches walking down the street… They’re asking for it. They deny it but it’s true.”
The attacks against Pussy Riot inspired me to reflect back on this film, and to also consider the wider impact of the Riot Grrrl Movement instigated by Bikini Kill in the early 90’s via their political lyrics, zines and confrontational live shows.
It also triggered memories about my art school days, immersed in the feminist art of Barbara Kruger, Guerrilla Girls, Karen Finley, Jenny Holzer and Kathy Acker – all of whom made work that showed me the power of words and the female body as a vehicle for activism and political protest. But there is a safety issue here that still needs more addressing.
It’s rather frightening to consider that feminist artists — even today — might be afraid to create or do whatever the hell they want in the public sphere. Hopefully, what happened to Pussy Riot makes us want to keep fighting, speaking up and challenging the status quo. Just imagine if all the artists listed above were active in Russia today. Not only would they be risking prosecution for “Hooliganism,” but also risking themselves as potential targets for angry or violent men. It still holds true that when pushing back against male privilege the world gets a bit uglier and a lot more unsafe.
In an interview by Pitchfork in 2012, Hanna explicitly cites Pussy Riot as the most important band of the decade. On her website this comment pretty much says it all:
Her words resonate far outside the physical parameters of a jail cell, and evoke images of the metaphorical and societal cages women continue to be placed in — our beauty, sexuality and gender roles. In another clip, Hanna belts out more girl rage on stage:
“Liberated from the cage… That was a metaphor. I know they have go-go dancers in this cage sometimes… Well, fuck you.”
Power to the voice. In 1989, Hanna travelled to Seattle to meet Kathy Acker, an experimental punk author who wrote brilliantly and unabashedly about abuse, incest, and other forms of sexual extremity. After bluffing her way into an interview, Hanna explained that she was interested in spoken-word performance and in writing. Acker bluntly told her that if she wanted to be heard she needed to perform in a band.
“MAQUILLAJE” by Ana Teresa Barboso Gubo, 2008
While Acker’s words inspired the making of a punk band, two decades later, Bikini Kill and the Riot Grrrl scene would greatly influence the aesthetics of Pussy Riot’s confrontational style, as well as on their prankster performance art. As their moniker suggests, the band has drawn proudly from provocative precedents such as Hole, Dickless, and Honey Bear. Ironic girl humour that mocks conventions of female sexuality and identity while inserting insult and aggression into the semiology. Brilliant, beautiful and brash.
There is also a similarly disturbing, yet comical, incongruity in seeing Pussy Riot lined up in an array of brightly coloured dresses, topped with knitted balaclavas, and wildly acting out atop iconic churches or on the ground in Red Square. It is delightfully subversive (even devilish) for a girl punk band to wear knitted masks during raucous performances. It brings to mind the pink knitted land mines of Barbara Hunt or Barbosa Gubo’s hand-stitched images of women putting on make-up or sewing themselves closed. Donning a dazzling array of girlish domesticated garb, Pussy Riot transforms their collective into a colourful legion of punk girl warriors.
Barb Hunt, antipersonnel, approx. 50 knitted sculptures
Hanna has spoken about the power of Pussy Riot members donning ski masks — a leaderless feminist performance art project that conjures up thoughts of the Guerrilla Girls who hid their faces so as not lose their jobs or ruin their status in the art world (as low as their status probably was). For Pussy Riot, it was to avoid jail and violence. Asserting themselves as “loud and proud” Russian feminists has also meant risking their safety, their voices and their freedom.
“Katya, Masha and I are in jail but I don’t consider that we’ve been defeated… The cost of taking part in creating history is always staggeringly high for people. But that taking part is the very spice of human life.”
— Excerpt from Pussy Riot’s Closing Statements in Court
Ironically, in 2013, The Punk Girl: A film about Kathleen Hanna, premiered at SXSW in Austin alongside a documentary on Pussy Riot and a film called Spring Breakers directed by Harmony Korine. A cheesy parody, the story is about four hipster chicks who cut loose on their spring break adventure by holding up a restaurant and stealing a car — all clad in neon bikinis, pink balaclavas with unicorns and automatic Glocks. In his usual style, Korine recasts, perverts, and transforms an American coming-of-age story. But let’s get real. It’s hipster sexism wrapped in gratuitous satire. A movie that literally comes from the same perverted view as the Girls Gone Wild videos.
In a conversation with The Winehouse Mag, Director Siri Anderson talks about how Hanna appears in her film [in footage from the 90’s] wearing a ski mask when doing a media blackout. Twenty years later, Pussy Riot, puts on knitted masks for the sole purpose of performing political art inspired by the Riot Grrrl movement. It’s a striking tribute that clearly shows the arc and power of feminist artists over a couple of decades — and thankfully, they both exist in stark contrast to the sexism dripping off the screen in Korine’s film — which by the way — received a rave review from the New York Times. “Oh em gee! That is, like, totally gross!”
Great article! It was not enough that they were charged with ‘hooliganism’ Putin had to have them arrested for theft during the Olympics with a few journalists and activists as a BIG warning. But he shot himself in the foot with that one as his image has suffered greatly. The opposition was laughing at him on twitter of all places.
Nadezhda Tolokonnikova and Maria Alyokhina are no pussies and they keep standing up to Putin. You have to admire them. It is one thing for the Dixie Chicks to criticize their President and go on with their lives but these chicks are risking their freedom for their beliefs.
It’s a better image than showing up on an international dating site to try to leave the country at all costs.
Hooliganism is such a bogus charge that needs to be taken off the law books — forever.
I hadn’t heard about the opposition laughing at him on twitter, haha, thank you for sharing that. Good one.
You are spot on Lise with your comment on dating sites. I quite admire these courageous creative women too, and actually, this whole process of writing just made me realize how many kick-ass feminist artists there are in the world that should be thanked!
Interesting overview of historical/artistic context and of how women put their traditional attractive/alluring qualities in a rebelliously grotesque and provocative context.
I was just twittering last night saying that Jane Velez-Mitchell and her HLN colleagues have done more to perpetuate the “war on women” than many other groups, yet she and Nancy Grace glibly cite women’s persecution in the context of abductions/murders of ALWAYS pretty women. It is noticeable that none of these harpies have touched Pussy Riot because the concepts they espouse and the symbolism of their visual representation is way too profound for these muck-raking mavens to understand, never mind the dearth of coquettish “feminine” photos with which to sell the story. I can just imagine JVM featuring the “BEAUUUUTIFUL” members of Pussy Riot who have been unjustly detained/attacked – cut to head shot of garishly coloured knitted balaklava helmet – no posed tilting head of blonde with perfect teeth……. Oh dear…..
Those two “women” (I hesitate to refer to them as women at all) are horrible creatures. They are doing so much damage by perpetuating the very myths, stereotypes and demeaning attitudes that encourage violence against women. Damn hypocrites. They wouldn’t dare touch Pussy Riot with a ten-foot pole! In fact, all the work these feminist artists do and have done are focused on getting to the very root evil of these trashy media portrayals. The documentary on Kathleen Hanna is excellent … in one section it discusses how often the media tried to pit her against other female punk artists. She refused. |
Intraspecific variation in the rainbow trout mitochondrial DNA genome.
Four complete mitochondrial DNA genomes for rainbow trout, Oncorhynchus mykiss, were sequenced and compared to the previously reported complete mitochondrial sequence. Analyses revealed 13 additional bp and 31 insertion/deletion (indels) sites between the four new sequences and the previously published sequence. Twenty-eight indels were single base pairs while the remaining were multiple base pair insertions, two in the ATPase6 gene, one of three bases and one of nine bases, and a two base insert in the control region. Indels in the new genomes were compared to a wide range of salmonid species and corresponded with sequences in closely related species, indicating that errors were likely incorporated into the previously published sequence. Although variable nucleotide positions were observed throughout the newly sequenced genomes, non-synonymous amino acid substitutions were observed in only six of seven ND genes. While there were only 14 amino acid substitutions between genomes, K(a)/K(s) ratios ranged between 0 and 1. A single amino acid deletion in the ND1 coding region unique to rainbow trout was also detected. Using complete mitochondrial genomes for all sequenced Salmonidae species, a phylogenetic analysis was performed to establish a salmonid mitochondrial phylogeny revealing support for Salvelinus, rather than Salmo, as the sister taxa to Oncorhynchus. |
When David Letterman announced his retirement last week everyone starting buzzing about his replacement. Of course, picks turn to those that already have a talk show many thought Ellen DeGeneres would be a great candidate to fill the "Late Show's" empty desk.
And while she was flattered with the proposal, the "Finding Nemo" star said that she loves her spot on daytime television. |
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A week after recreational marijuana was allowed for sale in Colarado, stores say they have already begun selling out.
Sales of the drug, which is available to anyone aged 21 or older, have already hit $5 million.
Erin Phillips, owner of state dispensary STRAINISE, has seen a "vast demographic" of people visiting the store.
She told BBC Radio 5 live's Up All Night: "The demand has just been huge... some of my competitors have already run out of products. It's amazing." |
Adjust highmem offset to 0x10000000 to ensure that all kmalloc allocations
stay within the same 256M boundary. This ensures that -mlong-calls is not
needed on systems with more than 256M RAM.
Signed-off-by: Felix Fietkau <nbd@openwrt.org>
---
--- a/arch/mips/include/asm/mach-generic/spaces.h
+++ b/arch/mips/include/asm/mach-generic/spaces.h
@@ -44,7 +44,7 @@
* Memory above this physical address will be considered highmem.
*/
#ifndef HIGHMEM_START
-#define HIGHMEM_START _AC(0x20000000, UL)
+#define HIGHMEM_START _AC(0x10000000, UL)
#endif
#endif /* CONFIG_32BIT */
|
Above: Anders Breivik, who killed 77 at a Norwegian Social Democrat youth camp last year.
It’s always unwise and often downright irresponsible to jump to conclusions about the motives and political profile of terrorists. When 77 people were picked off at a social democratic youth camp in Norway last year, there was a widespread assumption that this was the work of Islamists. It turned out, of course, that Anders Behring Breivik was a far-right anti-Muslim, probably acting alone. Exactly the opposite mistake has now been made by mainstream and left-wing commentators on the tragic events in Toulouse. Fiachra Gibbons wrote a piece in the Guardian that managed to get just about everything not just wrong, but wrong by 180 degrees:
Police are a long way yet from catching, never mind understanding, what was going through the head of someone who could catch a little girl by the hair so he wouldn’t have to waste a second bullet on her. But some things are already becoming clear. He shouted no jihadist or anti-Semitic slogans, going about his grisly business in the cold, military manner oddly similar to Anders Behring Breivik, the Norwegian gunman who massacred 77 people at a social democrats summer camp last summer.
As with Breivik, politicians will be quick to the thesis of the lone madman. Another lone madman influenced by nothing but his own distorted mind, like the lone gang of neo-Nazis who had been quietly killing Turks and Greeks in Germany for years unbothered by the police, who preferred to put the murders down to feuds or honour killings.
What could be the link, they ask, between Jewish children and French military personnel? The link is they are both seen – and not just by a far-right fringe – as symbols of all that has sabotaged la France forte, to borrow Sarkozy’s election slogan. Confessional schools, be they Jewish or an informal weekend madrassa, are seen as actively undermining the secular Republic by activists of groups like the Bloc Identitaire and the Front National, as well as some members of Sarkozy’s UMP, and even some on the left.
Now, I have no idea who Mr Gibbons is, and he is certainly not the only commentator to get this incident badly wrong, but I’ll bet you that he is on the “multicultural”/liberal/”left” and so predisposed to assume that the killings were the work of the white European far-right. His easy dismissal of the possibility that the killer might have been an Islamist jihadist (“He shouted nojihadist or anti-Semitic slogans“) suggests a predetermined inability to even entertain such a possibility (the article was published on 20 March, one day before the identity of the killer became known).
And that possibility was always there and fairly obvious. Just because the killer had struck at Muslim servicemen did not make it impossible that he was himself a Muslim, as anyone who has studied the form should know. As Nick Cohen notes in today’s Observer:
Breivik’s [and, as it turns out, Mehrah’s – JD] mentality matched that of Parviz Khan, a bloodthirsty fanatic from Birmingham. At his trial in 2008, the police provided tapes of Khan saying that he would behead a British Muslim soldier like “you cut a pig”. Then he would “put it on a stick and say, this is to all Muslims, [you] want to join the kuffar army, this is what will happen to you”. In his study of the case, Shiraz Maher of King’s College London said that most terrorists spread fear indiscriminately. Khan and his fellow plotters were different. They aimed to terrify Muslims who choose to integrate, identify themselves as British and serve British institutions; to let them know that it was an act of “betrayal” to support their own country.
The first lesson from all of this is not to jump to conclusions before sufficient facts are known. The second is that it is not just unwise and distasteful, but also irresponsible, to seek to use tragedies like this to bolster your own political preconceptions. The third is that vulnerable minorities (Muslims in Europe, especially) are now at greater risk than ever, and we must all weigh our words carefully.
Just because it turns out that the killer was not a member of the far-right (actually, I’d argue that he was, but that’s another matter) does not make the nationalist racism of Sarkozy, as he attempts to steal votes from the fascist Front National, any less criminal. It just means that the simple cause-and-effect link to the killings that we on the left wanted to prove, has not been the case. That fact should not be a cause of pleasure or satisfaction to anyone, and it doesn’t invalidate our condemnations of Sarkozy. But, undoubtably the electoral gainers will be the Front National (who are, predicatably, cashing in already) and Sarkozy himself.
But we on the left – and, especially, that section of the left that was inclined to put the killings down to the “political context” – now have some explaining to do. As the simplistic “It is no coincidence thatSarkozy’s racism has been followed by one of the worst racist attacks in France in a generation” explanation has been blown out of the water, we are now obliged to offer our more considered analysis and explanation, in the light of what we now know.
I am not the first to note that when a terrorist is a white neo-Nazi, the liberal-left will focus on his ideology, beliefs and any evidence of a supportive mainstream discourse. However if a terrorist is an Islamist, the same people focus exclusively on his grievances and deprivations. Here’s a particularly crass example, all the more unpleasant because it doesn’t even mention antisemitism as a factor in the equation. Note, also, that the (non) “explanation” given in this dreadful little piece of hackery and insult-to-the-intelligence, could have been wheeled out just as well, had the perpetrator been a member of the white far-right.
The problem with much of the “left”, when it comes to Islamist terrorism, is that they (the “left”) deny any autonomy to the perpetrators. Unlike white far-right terrorists, Islamists are not (it would seem) thinking individuals, autonomous actors, motivated by any coherent ideology. They’re merely victims who react to external forces – racism, “islamophobia,” alienation, poverty, imperialism, etc, etc. The “left” (or at least, a large part of it) effectively infantilises these people, denying them even the perverse dignity of being responsible for their own actions, and of having their own internally coherent political agenda. And that is, ultimately, a form of racism in itself.
I’ll leave the last words to Nick Cohen, who in today’s Observer nailed down many of the points I was mulling over prior to posting this piece:
For conservatives, opposition to radical Islam and indulgence of the white far right allows them to ignore the persistence of racism, most notably in France. They want to comfort their voters by telling them that whatever charges their critics throw at them, they are not as misogynist, homophobic or anti-Semitic as their Islamist enemies are. For leftists, opposition to the white far right and indulgence of radical Islam allows them to hide the descent of their programme of identity politics into squalor and shame, most notably in Britain. As long as they have the British National Party and English Defence League to fight, leftists can forget about their failures to help liberal Muslims and ex-Muslims in their struggles against theocratic power.
Many on both sides will not admit that the motives and targets of totalitarian movements are often identical. After what Europe went through in the 20th century, their ignorance is beyond disgraceful. It is astonishing.
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32 Comments
Jimmysaid,
You were supposed to respond to Toulouse then the white supremacist fascist Breivik pops up. Breivik killed 77 the Islamic fascist killed 7. Maybe the Islamist nutter needed help from Breivik on how to do a better job. You did redeem yourself in the last sentence. How true.
Jimmy: I don’t think the numbers are the crucial issue: its the intention and motivation that counts. After all, the perpetrators of 9/11 clearly hoped and intended to (and nearly succeeded in) kill(ing) tens of thousands. The fact that, as it turned out, they “only” killed three thousand is of no political consequence whatsoever.
skidmarxsaid,
I have no idea who Mr Gibbons is, and he is certainly not the only commentator to get this incident badly wrong, but I’ll bet you that he is on the “multicultural”/liberal/”left”
Didn’t know you went so far as to lament the advent of multiculturalism.
skidmarxsaid,
I don’t think it’s spelled “Kanan Malik”. Do all these funny names look the same to you? Sarah AB will never see her dream fulfilled of seeing him become prome minister if his name isn’t spelled right.Do you want him as PM too?
[Actually while I’m on the subject, I noticed that Sarah AB linked to an Ursula Le Guin story on the Ken Livingstone post at HP. Are you supposed to do that to living authors, even anarchists?
Flugmaschinensaid,
Of course German makes the usual error of evidence free reasoning, imploring the reader to believe that this is not an isolated hate crime, which it is, like most murders are.
She makes no attempt to actually explain how SHE knows that this nutcase scumbag because of course she doesnt. Mindless windbaggery of the worst sort or worse, the most vile aplogist for antisemitic psychopathy.
German also still sticks with the half witted fallacy of conflating racism with religion how many time have these imbeciles been told!!!
And of course as was explained very patronising insofar as ignoring the fact that the gunman could have been acting as a free agent pursuing his own vile agenda then seeking ex post facto justifications (Palestine, hijab ban etc.) when he new his number was up.
German never really found the plot so it is virtually impossible to suggest at this time that she has lost it
Tall Labansaid,
Mod-ghost: I know this is a matter you feel strongly about, and I respect your opinion, but simply don’t agree. I am not, as a matter of principle, in favour of “no platforming” racists – as opposed to fascists. We’ve had this discussion before and I think we both know exactly where the other stands.
The problem, as I’ve explained to you before, is that antisemitism is so widespread and deep-rooted on the Brit “left” (largely, but not just, thanks to Tony Cliff) that a refusal to engage with “left” antisemites would mean we had virtually no-one on the far-“left”, at least, we could speak to. I think that would be counterproductive.
We do ban the likes of Skidmarx and SteveH from time to time, but not because of a “no platform for antisemites” policy but because they’re pains in the arse.
The sad fact is that antisemitism is so engrained in the Brit-“left” that a lot of people think calling for the destruction (or demise) of Israel, referring to “rich” Jews, etc etc, is not an aberration, but part-and-parcel of being “left-wing”. I know this blog has only limited influence, but I also know that by engaging with misguided people, we have convinced one or two. Certainly, the AWL has.
Meanwhile, it has to be admitted that one “Dr Paul”, commenting at Dave’s Part, has a point:
Jim D: ‘I don’t respond to antisemites.’
Well, that doesn’t leave poor Jim many people to whom he can respond, seeing that his group considers that anyone calling for a single-state solution (however democratic) to Palestine and Israel is promoting an anti-Semitic standpoint; indeed, I have personally been accused of that by Sean Matgamna himself.
modernity's ghostsaid,
You are basically putting four arguments here (and please correct me if I summarise them wrong):
1. That antisemitism is widespread and deep-rooted on the British Left.
2. That antisemitism is ubiquitous on the British Far Left.
3. Therefore, if you were to ignore or avoid interacting with people that you consider to be Left antisemites then you would have no one to interact with, given its so prevalent.
4. That you put up with Left antisemites because you wish to interact with others on the British Left, in particular the Far Left.
Is that it? A fair representation of your views?
Please, re-read and let me know if they tally with your beliefs, but do re-read them carefully.
modernity's ghostsaid,
I happen to disagree with him on this, but I wondered if he or the AWL could defend their position outside of their organisation, in an open debate.
I know Trots don’t like being told they are wrong (probably explains why there are so many *ex*-trots), but if you believe something sincerely and with vigour then you should be able to justify it rationally.
I fully appreciate that doesn’t happen very much in politics, but surely those who argue against superstition, the mundane, the beliefs of yesteryear and for rationality, should be able to defend their views with logic and reason?
SteveHsaid,
From the very beginning I knew this was an attack by someone reacting to the mass killing fields the West have produced in the Middle East. The only surprise is that we don’t see more of this kind of thing,
I think this may have something to do with the upbringing of Muslims, who tend to value education and good behaviour higher than our ‘secular culture’ does.
blerehgc ocmaomnetareyrsaid,
MoraLLty BKLRKLEGGGHYH sez ‘if you believe something sincerely and with vigour then you should be able to justify it rationally.’ wOt Moraltity BlerereggvcJHJV means here is ‘I demand U jUMMP thro all of my hoops until U geT bored or exacerbated eNUFF at my STooPID as FcuK interogatION trOLLING and Give up – tHEN i deCLARE myseLF the winneR’. WElL DONe fuCKwIT, haVE a COOKieE.
skidmarxsaid,
Is that deluded fuckwit still here? You might have thought he’d be interested in actual anti-Semitism, like this:
neil_mcgowan_in_moscow
Today 09:16 AM
It’s the end of the line for the Miliband nutters. Britain’s not going to take orders from yankee fascists or Zionist madmen up Labour’s Back Passage any longer.
We are taking Britain back.
But that was in the Telegraph, not a left publication, so it’s not really important to the anti-anti-zionists, and it is increasingly clear that “genuinely concerned about anti-Semitism” actually means for mo’bullshit pretending that the left’s anti-Zionism is antisemitism.
modernity's ghostsaid,
Let me make this clear, for those pampered Oxbridge illiterates who can’t understand the basics:
I do not believe that anti-Zionism is equal to antisemitism.
However, in my experience many non-Jewish anti-Zionists have an unfortunate habit of:
1) being purblind to anti-Jewish racism
2) will ignore and downplay violence against Jews
3) will push sites which contain anti-Jewish racism
4) will push individuals who advance anti-Jewish racism (Atzmon ring any bells)
5) will never admit they’re wrong
6) will use inflammatory language that makes them sound like racists
etc etc
That’s not an exhaustive list, but astute readers will see the problem.
I differ from Jim and the AWL (as best as I can understand their arguments, and I wish they would put them more clearly and more directly), in that I do not believe that the British Left is full of antisemites.
As far as I can see the evidence doesn’t indicate a preponderance of active Jew haters, which is why certain individuals stick out, the David Ellis’s of the world, SteveH, John Wight and Skidmarx, when you examine their postings with a critical eye.
They stick out from the rest of the Left because of their obsessions, because of their rantings and their blindness to anti-Jewish racism.
In that they are very similar to other types of racists, those who whenever a particular topic comes up mounts their hobbyhorse and starts ranting on about “immigrants”, “foreigners” and “blacks”, etc
The behaviour is very similar they become excessively animated when the animus of their hate appears or comes up in discussion.
That’s why they stand out and why their racism is so noticeable, it has the traits and characterisation of other forms of racism, be the target of that racism, Jews, Afro-Caribbeans, the Roma, the Irish, etc.
Finally, for petty bourgeois illiterates, I do not believe that anti-Zionism is the same as antisemitism, to believe so is to ignore 1) mountains of historical evidence 2) the political discourse within the Jewish diaspora and 3) seek simplistic answers to complex political phenomena 4) not to engage with the historiography or theoretical works, etc
skidmarxsaid,
As far as I can see the evidence doesn’t indicate a preponderance of active Jew haters, which is why certain individuals stick out, the David Ellis’s of the world, SteveH, John Wight and Skidmarx, when you examine their postings with a critical eye.
So you are saying that we are “active Jew haters”, so you can point to examples of my employment of anti-Semitic hate speech? No? Then fuck off, you lying little shit, and don’t bother peddling this bollocks ever again.
modernity's ghostsaid,
I am saying that, the David Ellis’s of the world, SteveH, John Wight and Skidmarx are peculiar.
Their animosity towards Jews varies in intensity, they will often mask their aggressive language towards Jews using euphemisms such as “Zionists”, etc
Now anyone familiar with the activities of the Far Right will see the linguistic seepage there.
Further, despite making the point that they are antiracists, they rarely ever see any anti-Jewish racism.
No matter how stark it is, no matter how patiently it is explained.
As with Skidmarx, Bob from Brockly originally believed he could demonstrate the nature of anti-Jewish racism to him.
Eventually, I think Bob concluded that nothing would enlighten Skidmarx to anti-Jewish racism, he details it here:
“In this blog, I have detailed dozens, possibly hundreds, of examples of prejudice and discrimination characterising anti-Israeli campaigning. This is not to say that all, or even most, anti-Israel campaigning is racist.
But the examples of the slippage between Jewish lobby and Israel lobby, Weir’s blood libel, Atzmon and Shamir’s Holocaust revisionism, the support and defence for the likes of Weir and Shamir from all sorts of anti-Zionists, mutterings about the Lehman Brothers in UCU meetings, the circulation of David Duke material by anti-Zionists and any number of others constitute a very substantial body of evidence for the infection of anti-Israel campaigning by antisemitism.
This body of evidence cannot be refuted because all of the anti-Israel campaigners you’ve come across are nice, smart people. That is analogous to Brian of London in that other thread saying “I’ve met the EDL and they are very nice and not fascist at all”.
When evidence of prejudice from people’s own experience is presented (as in Falastin’s account of leftist racial prejudice),
Skid dismisses it as not evidence, but somehow his own experience of not hearing any antisemitism is taken to show that it doesn’t exist.
Falastin, as a Muslim woman, might be taken as a more reliable guide to whether there is prejudice about Muslim women in the left than a white male – this is connected to the MacPherson issue we discussed before, about valuing victims’ experiences of racism over the experiences of the “I’m not racist but” and “Some of my best friends are” types.
So, again, we see the persistence of the refusal to take racism seriously, and a refusal to see the facts. Again, when this happens once or twice we could see it as missing the point or ignorance. Its repetition over and over again in the face of refutation becomes complicity with racism. ”
I will leave it to readers to consult Bob’s site and make up their own minds, but people like Skidmarx are an exception, in my experience, which is where I differ from Jim & the AWL’s position (as I understand it)
modernity's ghostsaid,
On Weir, I see no flaw in Adam Holland’s posts whose urls Miles provided.
Skid, however, writes: ‘I don’t find Adam Holland convincing.
He claims that she is making allegations of ritual murder, she’s not as far as I can see.
And when he says: “Jewish opposition to the blood libel” or “dismissing them as merely the result of a Jewish conspiracy of silence”, he seems to dishonestly claim his views as those of all Jews.’
Weir’s August 2009 Counterpunch article denied the obvious truth that the allegation that Jews use Christian blood for ritual purposes is a libel; to do so she lied in several large and small ways; and Adam Holland’s first post shows this beyond any reasonable doubt whatsoever.
Denying this seems perverse to me (altho Moddy would provide a compelling alternative explanation, which is, I think, what motivates his no platform question).
Even more perverse is Skid’s claim that Holland ‘dishonestly’ claims his views are those of all Jews, simply because he uses words like “Jewish opposition”, as if that means “the opposition by every single Jew”, a palpably absurd reading.
Note that when Holland uses that phrase, in his second post, he is talking about Weir’s source Israel Shamir, by whom Skid claims to be not impressed. The charges we’re talking about are that Mendel Beilis and Dreyfus were guilty; Jewish opposition to those claims long pre-dates Holland’s existence. ” [My emphasis]
skidmarxsaid,
Still the bullshit comes. Still not about my views, just that I’m insufficiently condemnatory of others. And as I showed on the Assad thread here, I went on to say something like “Every time I read the Alison Weir piece it looks worse.”
And I’m not impressed by Israel Shamir!
You’ve found nothing that I’ve said that is anti-Semitic. Fuck off, you lying little shit. |
The present application is directed to a method for laminating a plastic web onto the surface of an extruded plastic substrate. The plastic web may be a facade strip having a textured appearance and the extruded plastic substrate may be cut into segments after lamination for use in making prefabricated window systems.
An example of a prior art window system having extruded members will now be described with reference to FIG. 1. The window system includes a rectangular mainframe having a top frame portion 10, a bottom frame portion 12, and side frame portions 14 which connect portions 10 and 12. Frame portions 10-14 are made from extruded polyvinyl chloride (PVC) and all have the same cross-sectional configuration, except for features such as drainage channels which are fabricated after extrusion. Because they have the same cross-sectional configuration, frame portions 10-14 can be cut from a long substrate (not shown in FIG. 1) of extruded PVC. Frame portions 10-14 are joined at their corners by edge welds 16 and lateral welds 18 (which are also present on the side of the window system not shown in FIG. 1). The term "weld" in this context means that the corners have been joined by molten PVC which, when it cools, seals one frame portion to an adjacent frame portion along a smooth seam.
The side frame portions 14 provide channels for guiding a screen member 18 and for guiding window units 20 and 22 (which have sashes made from segments cut from an elongated substrate of extruded PVC, not illustrated). Window units 20 and 22 can be unlatched from the channels and tilted out for cleaning as shown. Since the top and bottom frame portions 10 and 12 have the same cross-sectional configuration as the side frame portions 14, channels are also present in top and bottom frame portions 10 and 12. An extruded PVC sill 24 is provided with resilient legs which permit sill 24 to be snap-connected to bottom frame portion 12. Balance mechanisms 25 (only one of which is shown) are mounted in the window guidance channels of side frame portions 14 to counterbalance the weight of window units 20 and 22. Window stops 26 (only two of which are shown) are snap-connected to side frame portions 14 to limit the movement of window units 20 and 22 so as to prevent possible damage to hardware mounted on the window units should they be slammed up or down.
FIG. 2 is a cross-sectional view of the bottom portion of the window system shown in FIG. 1, in its installed state. A rectangular opening in a wall 28 is framed by wooden strips 30 and interior trim 32. Nailing fins 34 are lodged into slots in frame portions 10-14 and are nailed into wall 28 to mount the window system in wall 28.
Further information about the construction of the window system can be found in U.S. application Ser. No. 06/929,303, filed Nov. 12th, 1986, the disclosure of which is incorporated herein by reference.
Since the process of extruding a plastic substrate leaves its own characteristically bland appearance to the surfaces of the substrate, in the past facade strips have been laminated to major visible surfaces of prefabricated plastic window systems to provide a wood-grain appearance. Such strips additionally increase the resistance of the window systems to weathering. However prior art techniques for laminating facade strips to extruded plastic substrates have not been entirely satisfactory. Facade strips laminated with prior art methods have had a tendency to become loose at the ends when segments are cut from a substrate and welded together. Furthermore, due to rough handling that may be encountered during transportation, storage, and installation of prefabricated window systems, the lateral edges of facade strips applied by prior art techniques may peel in places. Long years of exposure to the elements after installation, and possibly the prying hands of small children, may lead to further peeling.
In one prior art method for laminating a facade strip to an extruded plastic substrate, the substrate is moved through a cleaning station, where it is cleaned with soap suds. After drying, an adhesive-backed facade strip is pressed onto the member with rollers. |
Tuesday, August 18, 2015
The Real Naqba
Three Weeks ago was Naqba Day – the day on which Palestinians inside and outside Israel commemorate their "catastrophe" - the exodus of more than half of the Palestinian people from the territories occupied by Israel in the 1948 war.
Each side has its own version of this momentous event.
According to the Arab version, the Jews came from nowhere, attacked a peace-loving people and drove them out of their country.
According to the Zionist version, the Jews had accepted the United Nations compromise plan, but the Arabs had rejected it and started a bloody war, during which they were convinced by the Arab states to leave their homes in order to return with the victorious Arab armies.
Both these versions are utter nonsense - a mixture of propaganda, legend and hidden guilt feelings.
During the war I was a member of a mobile commando unit that was active all over the southern front. I was an eye-witness to what happened.
I wrote a book during the war ("In the fields of the Philistines") and another one immediately afterwards ("The Other Side of the Coin"). They appeared in English together under the title “1948: A Soldier's Tale”. I also wrote a chapter about these events in the first half of my autobiography ("Optimistic") that appeared in Hebrew last year. I shall try to describe what really happened.
First of All, we must beware of looking at 1948 through the eyes of 2015. Difficult as it may be, we must try to transport ourselves to the reality of then. Otherwise we shall be unable to understand what actually occurred.
The 1948 war was unique. It was the outcome of historical events which had no parallel anywhere. Without taking into account its historical, psychological, military and political background it is impossible to understand what happened. Neither the extermination of the Native Americans by the white settlers, nor the various colonial genocides resembled it.
The immediate cause was the November 1947 UN resolution to partition Palestine. It was rejected out of hand by the Arabs, who considered the Jews as foreign intruders. The Jewish side did accept it, but David Ben-Gurion later boasted that he had had no intention of being satisfied with the 1947 borders.
When the war started at the end of 1947, there were in British-governed Palestine about 1,250,000 Arabs and 635,000 Jews. They lived in close proximity but in separate neighborhoods in the towns (Jerusalem, Tel-Aviv-Jaffa, Haifa), and next to each other in neighboring villages.
The 1948 war was actually two wars that blended into one. From December 1947 until May 1948 it was a war between the Arab and the Jewish population inside Palestine, from May until the armistices in early 1949 it was a war between the new Israeli army and the armies of the Arab countries – mainly Jordan, Egypt, Syria and Iraq.
In The first and decisive phase, the Palestinian side was clearly superior in numbers. Arab villages dominated almost all highways, Jews could move only in hastily armored buses and with armed guards.
However, the Jewish side had a unified leadership under Ben-Gurion and organized a unified, disciplined military force, while the Palestinians were unable to set up a unified leadership and army. This proved decisive.
On both sides, there was no real difference between fighters and civilians. Arab villagers possessed rifles and pistols and rushed to the scene when a passing Jewish convoy was attacked. Most Jews were organized in the Haganah, the underground armed defense force. The two "terrorist" organizations, the Irgun and the Stern Group, also joined the unified force.
On both sides, everybody knew that this was an existential struggle.
On the Jewish side, the immediate task was to remove the Arab villages along the roads. That was the beginning of the Naqba.
From the start, atrocities cast a sinister shadow. We saw photos of Arabs parading in Jerusalem with the severed heads of our comrades. There were atrocities committed on our side, reaching a climax in the infamous Deir Yassin massacre. Deir Yassin, a neighborhood near Jerusalem, was attacked by an Irgun-Stern force, many of its male inhabitants were massacred, the women were paraded in Jewish Jerusalem. Incidents like these formed part of the atmosphere of existential struggle.
Throughout, this was a total ethnic struggle between two sides, each of which claimed the entire country as its exclusive homeland, denying the claims of the other side. Long before the term "ethnic cleansing" was widely used, it was practiced throughout this war. Only a few Arabs remained in the territory conquered by the Jews, no Jews at all remained in the few areas conquered by the Arabs (the Etzion Bloc, the Old City of Jerusalem.)
With the approach of May and the expectation that the Arab armies would enter the conflict, the Jewish side tried to create a zone from which all non-Jewish inhabitants were removed.
It must be understood that the Arab refugees did not "leave the country". When their village was shot at (generally at night), they took their families and escaped to the next village, which then came under fire, and so on. In the end they found an armistice border between them and their home.
The Palestinian exodus was not a straightforward process. It changed from month to month, from place to place and from situation to situation.
For example: the population of Lod was induced to flee by shooting at them indiscriminately. When Safed was conquered, according to the commander "we did not drive them out, we only opened a corridor for them to flee".
Before Nazareth was occupied, the local leaders signed a surrender document and the townspeople were guaranteed life and property. The Jewish commander, a Canadian officer named Dunkelman, was then verbally ordered to drive them out. He refused and demanded a written order, which never came. Because of that, Nazareth is an Arab town today.
When Jaffa was conquered, most inhabitants fled by sea to Gaza. Those who remained after the surrender were loaded onto trucks and sent on their way to Gaza, too.
While much of the expulsion was dictated by military necessity, there certainly was an unconscious, semi-conscious or conscious wish to get the Arab population out. It was "in the blood" of the Zionist movement. Indeed, long before the founder, Theodor Herzl, even thought about Palestine, when writing the initial draft of his ground-breaking book "Der Judenstaat", he proposed founding his Jewish State in Patagonia (Argentina), and proposed inducing all the native inhabitants to leave.
After the Arab armies entered the war in May, the Egyptians were stopped 22 km from Tel Aviv. A month-long cease-fire was decreed by the UN, and used by the Israeli side to equip itself for the first time with heavy arms (artillery, tanks, air force) sold them by Stalin. In the very heavy fighting in July, the balance shifted and the Israeli side slowly gained the upper hand.
From then on, a political – as distinguished from military – decision was taken to remove the Arab population. Units were ordered to shoot on sight every Arab who tried to return to their village.
The decisive moment came at the end of the war, when it was decided not to allow the refugees to return to their homes. There was no official decision. The idea did not even come up. Masses of Jewish refugees from Europe, survivors of the Holocaust, flooded the country and filled the places left by the Arabs.
The Zionist leadership was certain that within a generation or two the refugees would be forgotten. That did not happen.
It should be remembered that all this happened only a few years after the mass expulsion of the Germans from Poland, Czechoslovakia and the Baltic states, which was accepted as natural.
Like a Greek tragedy, the Naqba was conditioned by the character of all the participants, victimizer and victim.
Any solution of the "problem" must start with an unequivocal apology by Israel for its part in the Naqba.
The practical solution musty include at least a symbolic return of an agreed number of refugees to Israeli territory, a resettlement of the majority in the State of Palestine when it comes into being, and generous compensation to those who choose to stay where they are or emigrate elsewhere.
1
comments
:
Good to have eye-witness accounts. History is usually so much more messy than the masses are comfortable with.
I've been reading today of the events leading up to the UN partition. The British initially proposed partition, but withdrew it and favoured an Arab majority rule, with protection of minority rights - never mind that minority rights were being trampled at that very time in Eastern Europe, and the oppressors appeased.
Even when the gas chambers were in operation, the British were turning back Jewish refugee boats from Palestine.
Anthony McIntyre
Former IRA volunteer and ex-prisoner, spent 18 years in Long Kesh, 4 years on the blanket and no-wash/no work protests which led to the hunger strikes of the 80s. Completed PhD at Queens upon release from prison. Left the Republican Movement at the endorsement of the Good Friday Agreement, and went on to become a journalist. Co-founder of The Blanket, an online magazine that critically analyzed the Irish peace process. |
Q:
Reset Button Issues
I have created a table with a search bar which will allow me to filter the data based on what I type in the search bar. The problem I have is with the reset button I created. What I want is for the search bar to be cleared and the table to return back to its original state showing all the data (NOT the filtered data)
<!DOCTYPE html>
<html lang="en">
<head>
<meta charset=utf-8>
<meta name="viewport" content="width=device-width, initial-scale=1">
<link rel="stylesheet" href="css/RichStyle.css">
<!-- Custom Made CSS -->
<script src="js/jquery-1.11.1.min.js"></script>
<script src="js/bootstrap.min.js"></script>
<script src="js/toggle.js"></script>
<script src="js/jquery%201.4.4.min.js"></script>
<!-- JS -->
<!-- Slide Toggle -->
<script type="text/javascript">
$(document).ready(
function() {
$('td p').slideUp();
$('td h2').click(
function(){
$(this).closest('tr').find('p').slideToggle();
}
);
}
);
</script>
<!-- Search Bar -->
<script language="javascript" type="text/javascript">
$(document).ready(function() {
$('#search').keyup(function() {
searchTable($(this).val());
});
});
function searchTable(inputVal) {
var table = $('#searchTable');
table.find('tr').each(function(index, row) {
var allCells = $(row).find('td');
if (allCells.length > 0) {
var found = false;
allCells.each(function(index, td) {
var regExp = new RegExp(inputVal, 'i');
if (regExp.test($(td).text())) {
found = true;
return false;
}
});
if (found == true)
$(row).show();
else
$(row).hide();
}
});
}
</script>
<title>Report Page</title>
</head>
<body>
<div>
<p>
<form>
<label for="search"></label>
<input type="text" id="search" placeholder="Enter Search Term" />
<input type="reset" value="Reset" />
</form>
</p>
<table id="searchTable">
<thead>
<tr>
<th>User ID</th>
<th>Website</th>
<th>Hours Spent</th>
<th>Data Usage</th>
</tr>
</thead>
<tbody>
<tr>
<td><h2>Luke</h2></td>
<td><h3 id="Luke">
<script>
var website = ["Facebook", "Youtube"];
document.getElementById("Luke").innerHTML = website.length;
</script></h3><p>Facebook</p><p>Youtube</p></td>
<td><h3>2.5h</h3><p>2h</p><p>0.5h</p></td>
<td><h3>1.3gb</h3><p>3mb</p><p>1gb</p></td>
</tr>
<tr>
<td><h2>John</h2></td>
<td><h3 id="John">
<script>
var website = ["Youtube"];
document.getElementById("John").innerHTML = website.length;
</script></h3><p>Youtube</p></td>
<td><h3>3h</h3><p>3h</p></td>
<td><h3>1gb</h3><p>1gb</p></td>
</tr>
<tr>
<td><h2>Peter</h2></td>
<td><h3 id="Peter">
<script>
var website = ["Facebook", "Youtube"];
document.getElementById("Peter").innerHTML = website.length;
</script></h3><p>Facebook</p><p>Youtube</p></td>
<td><h3>1.75h</h3><p>1.5h</p><p>0.25h</p></td>
<td><h3>1.3gb</h3><p>3mb</p><p>1gb</p></td>
</tr>
</tbody>
</table>
</div>
<button onclick="window.print()">Print Report</button>
<!-- Javascript Coding - No Colours Displayed On Print Preview-->
<button>Save Report</button>
<!-- Save Button Does Not Work, Fix Needed-->
</body>
</html>
A:
IF you want to use the <input type="reset" /> tag, the elements you can reset must be editable, i.e. you can type whatever you want into it, so
input type="text"
input type="password"
input type="email"
etc. . .
The above are editable.
So if what you want is that, you have no more data in your table you can use JQuery, here's an example using input reset
$(function(){ // this is === to document.ready
$("#the-table .ins").val("some value here ");
});
<script src="https://ajax.googleapis.com/ajax/libs/jquery/2.1.1/jquery.min.js"></script>
<!-- the tags that you can enter text into can be reset if they resied within the same form that contains the reset button -->
<form action="some url" method="post">
First name: <input type="text" name="firstname" /> <br />
Surname: <input type="text" name="surname" /><br />
<input type="reset" value="Clear form" />
<input type="submit" value="Submit now" />
</form>
<hr />
With a table:
<!-- so when the page loads, we use JQuery to insert so value to your table -->
<form action="some url" method="post">
<table id="the-table">
<tr><td><input class="ins" type="text" /></td>
<td><input class="ins" type="text" /></td>
</tr>
</table>
<input type="reset" value="Clear form" />
</form>
and here's an example using jquery to clear the table data
$(function(){
$('#reset-btn').click(function(){
$('table tr').remove();
});
});
<script src="https://ajax.googleapis.com/ajax/libs/jquery/2.1.1/jquery.min.js"></script>
<h2>click on the table to remove data </h2>
<table style="width:100%">
<tr>
<td>Jill</td>
<td>Smith</td>
<td>50</td>
</tr>
<tr>
<td>Eve</td>
<td>Jackson</td>
<td>94</td>
</tr>
</table>
<button id="reset-btn">reset data</button>
|
---
abstract: 'By networking multiple quantum devices, the Quantum Internet builds up a virtual quantum machine able to provide an exponential scale-up of the quantum computing power. Hence, the Quantum Internet is envisioned as a way to open a new distributed computing era. But the Quantum Internet is governed by the laws of quantum mechanics. Phenomena with no counterpart in classical networks, such as no-cloning, quantum measurement, entanglement and teleporting, impose very challenging constraints for the network design. Specifically, classical network functionalities, ranging from error-control mechanisms to overhead-control strategies, are based on the assumption that classical information can be safely read and copied. But this assumption does not hold in the Quantum Internet. As a consequence, the design of the Quantum Internet requires a major network-paradigm shift to harness the quantum mechanics specificities. The goal of this work is to shed light on the challenges and the open problems of the Quantum Internet design. To this aim, we first introduce some basic knowledge of quantum mechanics, needed to understand the differences between a classical and a quantum network. Then, we introduce quantum teleportation as the key strategy for transmitting quantum information without physically transferring the particle that stores the quantum information or violating the principles of quantum mechanics. Finally, we describe the key research challenges to design quantum communication networks.'
author:
- '\'
- '\'
bibliography:
- 'quantum-04.bib'
title: 'Quantum Internet: Networking Challenges in Distributed Quantum Computing'
---
Quantum Internet, Quantum Network, Entanglement, Teleporting.
Introduction {#sec:1}
============
Nowadays, the development of quantum computers is experiencing a major boost, since tech giants entered the quantum race. In November 2017 IBM built and tested a 50-qubits processor, in March 2018 Google announced a 72-qubits processor, and other big players, like Intel and Alibaba, are actively working on double-digit-qubits proof-of-concepts. Meanwhile, in April 2017 the European Commission launched a ten-years 1 €-billion flagship project to boost European quantum technologies research. And in June 2017 China successfully tested a 1200 km quantum link between satellite Micius and ground stations.
Such a race in building quantum computers is not surprising, given their potential to completely change markets and industries - such as commerce, intelligence, military affairs [@CalCacBia-18; @PirBra-16; @Kim-08]. In fact, a quantum computer can tackle classes of problems that choke conventional machines, such as molecular and chemical reaction simulations, optimization in manufacturing and supply chains, financial modelling, machine learning and enhanced security.
The building block of a quantum computer is the quantum bit (qubit), describing a discrete two-level quantum state as detailed in the next section. By oversimplifying, the computing power of a quantum computer scales exponentially with the number of qubits that can be embedded and interconnected within [@Bou17; @CalCacBia-18; @Kim-08]. The greater is the number of qubits, the harder is the problem that can be solved by a quantum computer. For instance, solving some fundamental chemistry problems is expected to require[^1] “hundreds of thousands or millions of interconnected qubits, in order to correct errors that arise from noise” [@Bou17].
Quantum technologies are still far away from this ambitious goal. In fact, so far, although the quantum chips storing the qubits are quite small, with dimensions comparable to classical chips, they usually require to be confined into specialized laboratories hosting the bulked equipment – such as large near absolute-zero cooling systems – necessary to preserve the coherence of the quantum states. And the challenges for controlling, interconnecting, and preserving the qubits get harder and harder as the number of qubits increases. Currently, the state-of-the-art of the quantum technologies limits this number to double digits (IBM 50-qubits and Google 72-qubits).
Hence, very recently, the *Quantum Internet* has been proposed as the key strategy to significantly scale up the number of qubits [@CalCacBia-18; @Kim-08]. Specifically, by interconnecting multiple quantum devices via a *quantum network*, i.e., through a network able to share quantum information among remote nodes, and by adopting a distributed paradigm, the Quantum Internet builds up a virtual quantum machine constituted by a number of qubits that scales with the number of interconnected remote devices. This, in turn, implies an exponential speed-up of the quantum computing power [@CalCacBia-18].
From an engineering communication perspective, the design of the Quantum Internet constitutes a breakthrough. In fact, a quantum network is governed by the laws of quantum mechanics. Hence, phenomena with no counterpart in classical networks – such as no-cloning, quantum measurement, entanglement and teleporting – imposes terrific constraints for the network design. As instance, classical network functionalities, such as error-control mechanisms (e.g., ARQ) or overhead-control strategies (e.g., caching), are based on the assumption that classical information can be safely read and copied. But this assumption does not hold in a quantum network. As a consequence, the design of a quantum network requires a major paradigm shift to harness the key peculiarities of quantum information transmission, i.e., entanglement and teleportation.
The goal of this paper is to shed light on the challenges in designing a quantum network. Specifically, we first introduce some basic knowledges of quantum mechanics – superposition principle, no-cloning theorem, quantum measurement postulate and entanglement – needed to understand the differences between a classical and a quantum network. Then, we focus on quantum teleportation as *the key strategy* for transmitting quantum information without either the physical transfer of the particle storing the quantum information or the violation of the quantum mechanics principles. Finally, we draw the key research challenges related to the design of a quantum network.
Quantum Mechanics Background {#sec:2}
============================
In this section, we briefly review some quantum mechanics postulates and principles needed to understand the challenges behind the design of a quantum network.
A quantum bit, or *qubit*, describes a discrete[^2] two-level quantum state, which can assume two (orthogonal) basis states: the zero (or ground state) and the one (or excited state), usually denoted[^3] as $\ket{0}$ and $\ket{1}$. As instance, if we represent the state of a photon with a qubit, the two basis states represent the horizontal and the vertical polarization of the photon, respectively.
Superposition Principle {#sec:2.1}
-----------------------
As widely known, a classical bit encodes one of two mutually exclusive states, being in only one state at any time. Conversely, a qubit can be in a superposition of the two basis states, being so simultaneously zero and one at a certain time. As instance, a photon with degrees of polarization is described by a superposed qubit, with an even *amount* of zero and one, being simultaneously horizontally and vertically polarized.
Hence according to the superposition principle, $n$ qubits can simultaneously encode **all** the $2^n$ possible states at once. Differently, $n$ classical bits can encode only **one** of $2^n$ possible states at a certain time.
Quantum Measurement {#sec:2.2}
-------------------
According to one of the quantum mechanics postulates, whenever a measurement can have more than one outcome, as is the case for the two possible states of a qubit, after the measurement the original quantum state collapses in the measured state. Hence, the measurement alters irreversibly the original qubit state [@NieChu-11]. And, the result of such a measurement is probabilistic, since one obtains either the state zero or the state one, with a probability depending on the *amount* of zero and one in the original superposed quantum state. For instance, if the outcome of measuring a superposed qubit is the state zero, the qubit collapses into such a state and any further measurement will give zero as outcome, independently of the original *amount* of one in the superposed state.
As a consequence, although a qubit can *store* more than one classical bit of information thanks to the superposition principle, by measuring a qubit, only one bit of information can be obtained.
The measurement postulate has deep implications on the quantum network design as described in Sec. \[sec:4\]. In fact, we can not share quantum states among remote quantum devices by simply measuring the qubits and transmitting the measurement outcomes. Instead, we must share qubits among remote devices without measuring them by exploiting a fundamental quantum mechanics property: *the entanglement*.
No-cloning theorem {#sec:2.3}
------------------
The no-cloning theorem states that an unknown qubit cannot be cloned. The no-cloning theorem is a direct consequence of the properties of the transformations allowed in quantum mechanics. Specifically, nature does not allow arbitrary transformations of a quantum system. Nature forces these transformations to be unitary. The linearity of unitary transformations alone implies the no-cloning theorem [@NieChu-11].
The *no-cloning theorem* has critical consequences from a communication engineering perspective, since classical communication functionalities are based on the assumption to be able to safely copy the information. This in turn deeply affects the quantum network design, as pointed out in Sec. \[sec:4\].
![Quantum Entanglement: measuring one qubit of an EPR pair instantaneously changes the status of the second qubit, regardless of the distance dividing the two qubits. Specifically, the two particles forming the EPR pair are in superposed state, with an even *amount* of zero and one. Hence, by measuring one of the two qubits, we would obtain either zero or one with even probability. But once the qubit is measured, say particle A with outcome corresponding to state zero, the second qubit instantaneously collapses into state zero as well.[]{data-label="fig:09"}](./Fig-09.png){width=".8\columnwidth"}
Entanglement {#sec:2.4}
------------
The deepest difference between classical and quantum mechanics lays in the concept of *quantum entanglement*, a sort of correlation with no counterpart in the classical word. In a nutshell, the entanglement is a special case of superposition of multiple qubits where the overall quantum state can not be described in terms (or as a tensor product) of the quantum states of the single qubits.
To better understand the entanglement concept, let us consider Figure \[fig:09\], showing a couple of maximally entangled qubits, referred to as *EPR pair*[^4]. The couple of qubits forming the EPR pair are in a superposed state, with an even amount of zero and one. By measuring each of the two qubits independently, one obtains a random distribution of zero and one outcomes with equal probability. However, if the results of the two independent measurements are compared, one observes that every time that the measurement of qubit A yielded zero so did the measurement of qubit B, and the same happened with the outcome 1. Indeed, according to quantum mechanics as soon as one of the two qubits is measured the state of the other is *instantaneously* fixed.
This quantum entanglement behavior induced Einstein and his colleagues to the so-called *EPR paradox*: the measurement of one qubit instantaneously changes the state of the second qubit, regardless of the distance dividing the two qubits. This seems involving information being transmitted faster than light, violating so the Relativity Theory. But the paradox is apparent, since entanglement does not allow to transmit information faster than light, as shown in Sec. \[sec:3.1\].
{width="1\columnwidth"} \[fig:11.a\]
{width="1\columnwidth"} \[fig:11.b\]
Quantum Communications {#sec:3}
======================
{width="1\columnwidth"} \[tab:01\]
So far, quantum communications have been widely restricted to a synonymous of a specific application, e.g. *Quantum Key Distribution* (QKD) [@NieChu-11]. QKD is a cryptographic protocol enabling two parties to produce a shared random secret key by relying on the principles of quantum mechanics, either quantum measurement or entanglement. However, in a QKD system, quantum mechanics plays a role only during the creation of the encryption key: the encrypted information subsequently transmitted is entirely classical. Similarly, *superdense* coding [@NieChu-11] is a communication protocol enabling two parties to enhance the transmission of classical information through a quantum channel, i.e., to exchange two bits of classical information by exchanging a single qubit. Both QKD and superdense coding exploit quantum mechanics to convey classical information.
Differently, as shown in Table \[tab:01\], the Quantum Internet relies on the ability to share quantum states among remote nodes. However, quantum mechanics restricts a qubit from being copied or safely measured. Hence, although a photon can encode a qubit and it can be directly transmitted to a remote node, e.g., via a fiber link, if the traveling photon is lost due to attenuation or corrupted by noise, the quantum information is definitely destroyed. This quantum information can not be recovered via a measuring process and/or a copy of the original information, due to the measuring postulate and the no-cloning theorem. As a consequence, the direct transmissions of qubits via photons is not feasible and quantum teleportation, described in the following, must be employed.
Quantum Teleportation {#sec:3.1}
---------------------
Quantum Teleportation [@BenBraCre-93] provides an invaluable strategy for transmitting qubits without either the physical transfer of the particle storing the qubit or the violation of the quantum mechanics principles. Indeed, with just local operations, referred to as Bell[^5]-State Measurement (BSM)[^6], and an EPR pair shared between source and destination, quantum teleportation allows one to “*transmit*” an unknown quantum state between two remotes quantum devices.
Differently from what the name seems to suggest, quantum teleportation implies the destruction of both the original qubit (encoding the quantum information to be transmitted) and the entanglement-pair member at the source, as a consequence of a measurement operation. Then, the original qubit is *reconstructed* at the destination once the output of the BSM at the source – 2 classical bits – is received through a classical channel.
The teleportation process of a single qubit is summarized in Figure \[fig:11\]. In a nutshell, it requires: i) the generation and the distribution of an EPR pair between the source and destination, ii) a classical communication channel for sending the two classical bits resulting from the BSM measurement. Hence, it is worth noting that the integration of classical and quantum resources is a crucial issue for quantum networks.
Regarding the EPR pair, the measurement at the source side destroys the entanglement. Hence, if another qubit needs to be teleported, a new EPR pair must be created and distributed between the source and the destination.
Quantum Network Design: Challenges and Open Problems {#sec:4}
====================================================
In this Section, we discuss the key research challenges and open problems related to the design of a quantum network, which harnesses quantum phenomena with no-counterpart in the classical reality, such as entanglement and teleportation, to share quantum states among remote quantum devices.
Decoherence and Fidelity {#sec:4.0}
------------------------
Qubits are very fragile: any interaction of a qubit with the environment causes decoherence, i.e., a loss of information from the qubit to the environment as time passes.
Clearly, a perfectly-isolated qubit preserves its quantum state indefinitely. However, isolation is hard to achieve in practice given the current maturity of quantum technologies. Furthermore, perfect isolation is not desirable, since computation and communication require to interact with the qubits, e.g., for reading/writing operations.
Decoherence is generally quantified through *decoherence times*, whose values largely depend on the adopted technology for implementing qubits. For qubits realized with superconducting circuits[^7], the decoherence times exhibit order of magnitude within $10$-$100 \,\mu$s [@BenKel-15; @FilSheMag-15]. Although a gradual decrease of the decoherence times is expected with the progress of the quantum technologies, the design of a quantum network must carefully account for the time constraints imposed by the quantum decoherence.
Decoherence is not the only source of errors. Errors practically arise with any operation on a quantum state due to imperfections and random fluctuations. Here, a fundamental figure of merit is the *quantum fidelity*. The fidelity is a measure of the distinguishability of two quantum states, taking values between 0 and 1. The larger is the imperfection of the physical implementation of an arbitrary quantum operation, the lower is the fidelity. Since teleportation consists of a sequence of operations on quantum states, the imperfection of such operations affects the fidelity of the “transmitted" qubit.
From a communication engineering perspective, the joint modeling of the errors induced by the quantum operations together with those induced by entanglement generation/distribution (as described in the next subsection) is still an open problem.
Furthermore, the no-cloning theorem prevents the adoption in quantum networks of classical error correction techniques, which depend on information cloning, to preserve quantum information against decoherence and imperfect operations. Recently, many quantum error correction techniques have been proposed as in [@Hanzo18]. However, further research is needed. In fact, quantum error correction techniques must handle not only bit-flip errors, but also phase-flip errors, as well as simultaneous bit- and phase-flip errors. Differently, in the classical domain, a single type of error, i.e., the bit-flip error, has to be considered.
From the above, the counteraction of the errors induced by decoherence and imperfect quantum operations in a quantum network is a functionality embracing aspects that traditionally belong to the physical layer of the classical network stack.
Entanglement Distribution {#sec:4.1}
-------------------------
![Entanglement Swapping. Two EPR pairs are generated and distributed: i) between a source (Alice) and an intermediate node (Quantum Repeater), and ii) between the intermediate node and a destination (Bob). By performing a BSM on the entangled particles at the Quantum Repeater, entanglement is eventually generated between Alice and Bob.[]{data-label="fig:12"}](./Fig_3_new_2.png){width="0.9\columnwidth"}
According to the quantum teleportation protocol, as in classical communication networks, the “transmission” of quantum information is limited by the classical bit throughput, necessary to transmit the output of the BSM outcome. But, differently from classical networks, the “transmission” of quantum information is achievable only if an EPR pair can be distributed between remote nodes.
Long-distance entanglement distribution – although deeply investigated by the physics community in the last twenty years – still constitutes a key issue due to the decay of the entanglement distribution rate as a function of the distance.
There is a global consensus toward the use of photons as entanglement carriers, i.e., as candidates to generate entangled pairs among remote devices. Figure \[fig:12\] reports a possible strategy for entanglement distribution and we refer the reader to [@PirEisWee-15] for a survey. Entanglement distribution is achieved through Quantum Repeaters [@VanTou-13], i.e. devices that implement a physical process known as *entanglement swapping* [@VanTou-13]. In practice, two EPR pairs are generated with source (Alice in Figure \[fig:12\]) and destination (Bob in Figure \[fig:12\]) receiving one element of each pair while the other two are sent to an intermediate node (the Quantum Repeater in Figure \[fig:12\]). By performing a BSM on the two entangled particles at the intermediate node, entanglement is created between the elements at the remote nodes. The process does not require the elements of the two pairs to have the same nature, therefore it can be used to distribute entanglement between two remotely located atoms or ions or to enhance the useful distance for the distribution of entangled photons. Hence, instead of distributing the entanglement over a long link, the entanglement is distributed iteratively through smaller links.
Despite these efforts, further research is needed. Specifically, from a communication engineering perspective, the entanglement distribution is a key functionality of a quantum network embracing aspects belonging to different layers of the classical network stack. More in detail:
- *Physical Layer*. The entanglement is a perishable resource. Indeed, due to the inevitable interactions with the external environment, the entanglement among entangled parties is progressively lost over time. Entanglement losses deeply affect the quality of the teleportation process. For the reasons highlighted in the previous subsection, classical error correction techniques cannot be used to counteract these losses. Hence, robust entanglement distribution techniques based on quantum error correction are mandatory for the deployment of a quantum network. This still represents a very hard challenge in this field.
- *Link Layer*: The no-broadcasting theorem, a corollary of the no-cloning theorem, prevents quantum information from being transmitted to more than a single destination. This is a fundamental difference with respect to classical networks, where broadcasting is widely exploited for implementing several layer-2 and -3 functionalities, such as medium access control and route discovery. As a consequence, the link layer must be carefully re-thought and re-designed to distribute the entanglement among multiple quantum devices. Furthermore, effective multiplexing techniques for quantum networks should be designed to allow multiple quantum devices to be connected to a single quantum channel (e.g. a fiber), and the access to the medium could be based on photon-frequency-division for the entanglement distribution.
- *Network Layer*: The entanglement distribution determines the connectivity of a quantum network in term of capability to perform the teleporting among the involved quantum devices. Hence, novel quantum routing metrics are needed for effective entanglement-aware path selection. Furthermore, the teleportation process destroys the entanglement as a consequence of the BSM at the source side. Hence, if another qubit needs to be teleported, a new entangled pair needs to be created and distributed between the source and the destination. This constraint has no-counterpart in classical networks and it must be carefully accounted for an effective design of the network layer.
Matter-Flying Interface {#sec:4.2}
-----------------------
{width="85.00000%"}
As mentioned before, there exists a general consensus about the adoption of photons as substrate for the so-called *flying* qubits, i.e., as entanglement carriers. The rationale for this choice lays in the advantages provided by photons for entanglement distribution: weak interaction with the environment (thus, reduced decoherence), easy control with standard optical components as well as high-speed low-loss transmissions.
The aim of the flying qubits is to “transport” qubits out of the physical quantum devices through the network for conveying quantum information from the sender to the receiver. Hence, as shown in Figure \[fig:07\], a transducer [@Ham10] is needed to convert a *matter* qubit, i.e., a qubit for information processing/storing within a computing device, in a flying qubit, which creates entanglement among remote nodes of the network.
Nowadays, there exist multiple technologies for realizing a matter qubit (quantum dots, trasmon, ion traps, etc) and each technology is characterized by different pros and cons, as surveyed in [@VanDev-16]. As a consequence, a matter-flying interface is required to face with this technology diversity.
Moreover, from a communication engineering perspective, the interface should be compatible also with the peculiarities of the physical channels the flying qubits propagate through. In fact, there exist different physical channels for transmitting flying qubits, ranging from free-space optical channels (either ground or satellite free-space) to optical fiber channels.
In the last ten years, the physics community has been quite active on investigating schemes and technologies enabling such an interface, with an heterogeneity of solutions [@Ham10; @StaRabSor-10] ranging from cavities for atomic qubits to optomechanical transducers for superconducting qubits.
As a consequence, the communication engineering community should join these efforts by designing communication models that account both the technology diversity in fabricating qubits and the propagation diversity in characterizing the different physical channels.
Deployment Challenges {#sec:4.3}
---------------------
Quantum Internet is probably still a concept far from a real world implementation, but it is possible to outline a few deployment challenges for the near future.
- The current technological limits to qubits and quantum processors physical realizations:\
At first, quantum computers will be available in few, highly specialized, data centers capable of providing the challenging equipment needed for quantum computers (ultra-high vacuum systems or ultra low temperature cryostats). Companies and users will be able to access quantum computing power as a service via cloud. Indeed, the quantum cloud market is estimated nearly half of the whole 10 billion quantum computing market by 2024 [@HSRC-18]. IBM is already allowing researchers around the world to practice quantum algorithm design through a classical cloud access to isolated 5-, 16- and 20-qubits quantum devices.
- Existing technological limits to quantum communication and quantum interfaces:\
The first realizations of a Quantum Internet will be small clusters of quantum processors within a data center. Architectures will have to take into account the high cost of data buses (economically and in term of quantum fidelity) limiting both the size of the clusters and the use of connections for processing.
- Hybrid architectures will probably be used for connections faring both the use of cryo-cables (expensive and necessarily limited in length) and of optical fibers or free space photonic links.
- Given the fragile nature of quantum entanglement and the challenges posed by the sharing of quantum resources, a substantial amount of conceptual work will be needed in the development both of novel networking protocols and of quantum and classical algorithms.
In conclusion, the Quantum Internet, though still in its infancy, is a very interesting new concept where a whole new set of novel ideas and tools at the border between quantum physics, computer and telecommunications engineering will be needed for the successful development of the field.
[Angela Sara Cacciapuoti]{} (M’10-SM’16) is a Tenure-Track Assistant Professor at the University of Naples Federico II, Italy. Since July 2018, she held the national habilitation as “Full Professor” in Telecommunications Engineering and, since April 2017, she held the habilitation as “Associate Professor”. Her work appeared in first tier IEEE journals, and she received several awards. Currently, Angela Sara serves as Editor/Associate Editor for: IEEE Trans. on Communications, IEEE Communications Letters, and IEEE Access. In 2016 she was elevated to IEEE Senior Member and she has been an appointed member of the IEEE ComSoc YP Standing Committee. Since 2018, she is the Publicity Chair of the IEEE ComSoc WICE Standing Committee.
[Marcello Caleffi]{} \[M’12, SM’16\] is a tenure-track assistant professor at the University of Naples Federico II. His work has appeared in several premier IEEE transactions and journals, and he has received multiple awards, including best strategy, most downloaded article, and most cited article awards. Currently, he serves as editor for IEEE Communications Magazine, IEEE Access, and IEEE Communications Letters. He serves as Distinguished Lecturer for the IEEE Computer Society and, since 2017, he serves as elected treasurer for IEEE ComSoc/VT Italy Chapter.
[Francesco Tafuri]{} is a Full Professor at the University of Naples Federico II. His scientific interests fall in the field of Superconductivity. He is leading an experimental research group investigating macroscopic quantum phenomena and the Josephson effect in hybrid devices, new routes for the realization of superconducting qubits for hybrid architectures, mesoscopic and nanoscale systems, vortex physics. He is Associate Editor of Journal of Superconductivity, in the Advisory Board of Physica C, and Peer Reviewer of National and European projects. He is author of more than 160 publications on international journals and books, including 13 papers on Science/Nature/Nat.Mat./Nat.Comm./PRL. He has given $>$90 invited talks at international conferences and research institutes. Several manuscripts are the product of international and national collaborations, including IBM T.J. Watson Research Center, USA; Stanford University, USA; Chalmers University of Technology, Goteborg, Sweden; University of Cambridge, UK; Lawrence Berkeley Laboratory, USA; Columbia University, USA.
[Francesco Saverio Cataliotti]{} activity is mainly experimental and is centered on atomic physics and atom-laser interactions. It can be divided in lines that, starting form nonlinear atom-laser interaction, develop into the manipulation of the atomic momentum state, laser-cooling and the realization of Bose-Einstein condensates. The optical manipulation of condensates has allowed him to investigate problems concerning superfluidity and macroscopic quantum states. More recently he has been concerned with coherent micro-manipulation techniques with atoms, molecules and micro-mechanical systems with perspectives ranging from quantum computation to single molecule manipulation. He is author of more than 100 scientific papers on international peer reviewed journals and contributions to conferences and workshops. Overall his works have received more than 2900 citations to date (researcherID K-4772-2015; orcid.org/0000-0003-4458-7977).
[Stefano Gherardini]{} was born in 1989. He received his B.Sc. degree in Electronic and Telecommunications Engineering and his M.Sc. degree in Electrical and Automation Engineering from the University of Florence, Italy, in 2011 and 2014, respectively. He got his Ph.D. degree in Information Engineering, curriculum non-linear dynamics and complex systems, in 2018 with the Automatic Control and QDAB group at the University of Florence. Moreover, he is a visiting student at SISSA, the International School for Advanced Studies, in Trieste, Italy. His main research interests include binary measurements, networked moving-horizon estimation, Kuramoto models, disordered systems, stochastic quantum Zeno effect, open quantum systems theory, and quantum estimation and control.
[Giuseppe Bianchi]{} has been a full professor of networking at the School of Engineering of the University of Roma “Tor Vergata” since 2007. His research activity, documented in more than 200 peer-reviewed international journal/conference papers having received to date more than 14,000 citations, focuses on wireless networks (his WLAN modeling work received the ACM SIGMO-BILE Test Of Time award in 2017), programmable network systems, and privacy and security monitoring in both the wireless and wired domains.
[^1]: Qubit redundancy is mandatory due to the unavoidable presence of an external noisy environment.
[^2]: At nano-scale, physical phenomena is described by discrete quantities, such as the energy levels of electrons in atoms or the horizontal/vertical polarization of a photon. This discrete nature contrasts with classical phenomena, which is commonly described by continuous quantities.
[^3]: The *bra-ket* notation $\ket{\cdot}$, introduced by Dirac, is a standard notion for describing quantum states. In a nutshell, a ket $\ket{\cdot}$ represents a column vector, hence the standard basis $\ket{0},\ket{1}$ is equivalent to a couple of 2-dimensional orthonormal vectors.
[^4]: A couple of maximally entangled qubits is called EPR pair in honor of an article written by Einstein, Podolsky, and Rosen in 1935.
[^5]: So called from John S. Bell who, in 1964, proposed a physical test of the EPR paradox.
[^6]: We emphasize that a solid strategy for a complete BSM with arbitrary photon states is not yet available.
[^7]: Superconducting qubits are based on Josephson tunneling junctions, which allow for a flow of electrons with a practically zero resistance between two superconductor layers. Under certain conditions, current can flow in both the directions, i.e., in a superposition of two states. As the two states are very close energetically, low temperatures (20 mK) are needed to avoid thermal fluctuations [@BenKel-15].
|
927 N.E.2d 343 (2006)
365 Ill. App.3d 1124
IN RE DA.R.
No. 4-05-0766.
Appellate Court of Illinois, Fourth District.
June 13, 2006.
Affirmed.
|
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Danny O'Brien
Danny O'Brien has been an activist for online free speech and privacy for over 15 years. In his home country of the UK, he fought against repressive anti-encryption law, and helped make the UK Parliament more transparent with FaxYourMP. He was EFF's activist from 2005 to 2007, and its international outreach coordinator from 2007-2009. After three years working to protect at-risk online reporters with the Committee to Protect Journalists, he returned to EFF in 2013 to supervise EFF's global strategy. He is also the co-founder of the Open Rights Group, Britain's own digital civil liberties organization.
In a previous life, Danny wrote and performed the only one-man show about Usenet to have a successful run in London's West End. His geek gossip zine, Need To Know, won a special commendation for services to newsgathering at the first Interactive BAFTAs. He also coined the term "life hack"; it has been nearly a decade since he was first commissioned to write a book on combating procrastination.
David Cameron, the British Prime Minister, could have buried almost any bad news on the same day as a royal birth. Instead, the main grievous news he had to offer — his plan for pervasive censorship of the British Internet — was entirely his own making.
Today, EFF announced that it was making a formal objection to including consideration of digital rights management (DRM) in the First Public Working Draft from the HTML working group of the World Wide W
UPDATE 2013-04-12: Apparently as a result of this blog post, social media attention, and questions from the Australian Greens to the Australian Federal Attorney General's Department, the block has been lifted. But there has not yet been any explanation of why these 1,200 sites were blocked in the first place.
In July 2009, South Korea became the first country to introduce a graduated response or "three strikes" law. The statute allows the Minister of Culture or the Korean Copyright Commission to tell ISPs and Korean online service providers to suspend the accounts of repeated infringers and block or delete infringing content online.
What happens when a country's government censors the entirety of its domestic web, with no oversight or transparency? It turns out that politicians aren't the only ones with an interest in repressing free expression — and given a lever of control, a black market of censors quickly emerges. |
I'll probably go to Honeymoon again next time I go, but I doubt if it'll be this weekend due to the holiday crowds. I'd like to get into some trout, and I've been told Honeymoon is a good trout location. I'm not sure what tide condition I shoud try for, or how much it matters. Also not sure about weather or time of day.
Yesterday the tide was running out while I was there, but where I was fishing was largely dictated by wind conditions. Maybe a different spot would've been better with that tide.
I quit fishing about 3:00 when some thunderstorms started moving in. I would have liked to fish longer, but I'm not thrilled with the idea of standing in the ocean waving a rod while lightning is in the area.
My local fly shop recommended a shrimp pattern for trout, but I've also been told to try a topwater popper when the water is murky.
With the shrimp pattern, I've been letting it sink, then retrieving it with short (4" to 6") jerks. I've tried using a steady retrieve with Clousers and Deceivers, as well as short strips with pauses.
Any suggestions you can offer will be appreciated.
Trout = trout do pass honeymoon isl during migrations to the gulf, however right now with water temps in the 80's they start out shallow and by 10am they are set up in 8-15 feet of grass bottom flats. You wont find trout right now wading. I mean you can but not consistently.
Honeymoon Isl is known for their large snook, just Google those two things and you will get all sorts of tips. None of the tips will give you any help with fly fishing because predominantly live bait is used.
If you not over grass or under a dock a shrimp pattern will not do too well.
In Florida when the water gets in the 80's "most" (people will argue) shallow water species, from 10am - 6pm will be in a deep hole, under a dock, under mangroves, or on a deep shady sea wall. Just think where you go when you get hot? That is where they go. The only way to get a good bite going during the heat is to live chum. Which is not a fly type of tactic.
Top water wont get too much action after sun-up in the summer.
So the moral to this story is that from shore, or wading is not very productive when the water is this hot. You can catch fish but nothing on fly that you wont have to work for. (like 1,000 casts)
This is the same for all of your other posts regarding areas like Fort De-Soto, and encompassing areas.
Getting a canoe or kayak is the cheapest way to find some deeper shady areas to fish in the summer for the fly guy's like us.
Now there is a lot more to this area but I get sick of typing so I'm cutting this short.
There are not many anglers on this forum in this area so I will check for your post and try to help the best I can.
__________________
"Snook are like females, you can throw your lure at them all day and they won't bite!" Graham |
#!/usr/bin/env bash
./sbt-dist/bin/sbt "$@" |
Everyone must open arms and welcome the marinated fish tacos served inside corn tortillas with lime pickled vegetables and Mexico City style rice. This rice is as nice as its name. Yes. Yes. Yes. Read more
Private|Social, located in Uptown Dallas, is where the casual and upscale effortlessly mingle, creating an ideal setting for elegance with ease. While menu is chef-driven, it remains accessible to every palette and every wallet |
St. Bartholomew Cathedral
Construction St. Bartholomew began after 1295 and was completed in early 16th century.
Construction St. Bartholomew began after 1295 and was completed in early 16th century. On the main altar you can see the sculpture of the Madonna of Pilsen, 1390, one of the most valuable statues of the "beautiful style," of Czech madonnas (there is a replica on the top of the Plague Column of the 17th century.).The Šternberk chapel with a pendant bracket dates from the 1st half of the 16th century. In 1993, Pope John Paul II. established a bishopric in Pilsen and the church of St. Bartholomew became a cathedral. The cathedral tower with its height of 102.6 metres the highest church tower in Bohemia, is a popular viewing point.
About Portal
The Improvement of the Tourist Portal of the Pilsen Region came into being as an initiative of the Pilsen Region and is being implemented and managed by the staff of the Department of Tourism and the Department of Computer Science. The project is aimed at the promotion of a comprehensive range of services and the development of tourism and tourist services throughout the Pilsen Region.
The Project Improvement of the Tourist Portal of the Pilsen Region is financed by the European Union. |
BRIGHTER, MORE COLORS- Your aquarium is full of spectacular colors, why not bring them all to life. Introducing the new Orbit Marine LED. Now packed with even more ultra-bright LEDs in a far wider color spectrum, it will make your corals and fish look even more spectacular. And it’s now in the LOOP, providing both light and pump control, taking your aquarium experience to a whole new level.
MULTIPLE MODES- With modes that dim periodically to create cloud cover effects, storm modes that will blow you away complete with lightning strikes and innovative evening modes that include lunar and dusk – it’s sure to add excitement and intrigue to any aquarium.
EASY INSTALLATION- Extendable brakets allows this LED light to fit most aquairum fish tanks 18-24 inches wide. The razor thin aluminum profile measures less than ½” thick, and is almost transparent over your aquarium.
REALISTIC EFFECTS- From start-up to sundown, the Orbit Marine gradually mimics the effect of a slow sunrise, bright daylight, dimming sunset and moonlight. Built-in lighting programs create a 24-hour natural biorhythmic lighting cycle, while on-demand dynamic weather effects gently roll clouds across your reef. |
Eto`o in QPR Link!
A subject we`ve touched on before is the apparent future of one Samuel Eto`o.
No longer contracted to Chelsea and free to find another club, we`re interested to see where the Cameroon international striker will end up.
If Harry Redknapp has his way, according to the press, then 34 year-old Eto`o will join Queens Park Rangers and spearhead the Loftus Road attack as wily Harry attempts to keep Queens Park Rangers in the Premier League.
We`re informed by the tabloid press that Claudio Vigorelli, Samuel`s agent, is in London trying to thrash out a deal with Queens Park Rangers.
However, the concerns are that his potential salary could be a stumbling block.
We`re also reliably informed that Eto`o wants to stay in London and in the Premier League, hence the apparent talks with Queens Park Rangers.
Here at Vital Chelsea, all we`ll say is if that is his dream then he`d better only put pen-to-paper on a one year deal because our friends from up the road are not staying in the Premier League much longer than that, you mark my words! |
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<string name="voice_recognition_toast" msgid="1149934534584052842">"ตอนนี้อุปกรณ์บลูทูธที่เชื่อมต่อจะจัดการการจดจำเสียง"</string>
<string name="car_guest" msgid="318393171202663722">"ผู้ใช้ชั่วคราว"</string>
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<string name="user_add_user_message_setup" msgid="1035578846007352323">"เมื่อคุณเพิ่มผู้ใช้ใหม่ ผู้ใช้ดังกล่าวจะต้องตั้งค่าพื้นที่ของตนเอง"</string>
<string name="user_add_user_message_update" msgid="7061671307004867811">"ผู้ใช้ทุกคนจะอัปเดตแอปให้แก่ผู้ใช้คนอื่นๆ ได้"</string>
<string name="car_loading_profile" msgid="4507385037552574474">"กำลังโหลด"</string>
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100 Interesting facts
100 Interesting Facts
The Benedictine Monks ran the first regular ferry from Birkenhead to Liverpool. The Monks would row over to the fishing village in Liverpool on market days, and offer the service to travellers.
The service was granted a Royal Charter by Edward III in 1330.
Edward III also granted the right to the Earl of Chester to run the ferry service from Seacombe to Birkenhead, establishing the Wallasey ferry. These two operations merged as Mersey Ferries in 1968.
Rowing across the Mersey would take 90 minutes in calm weather; however much longer in rough conditions.
In one day, the ferry had over 100 arrivals and departures on the Liverpool – Wirral service.
The ferry used to run all night before the opening of the Mersey tunnels.
The Mersey Ferries were the only way of getting across the river until the opening of the railway tunnel in 1886.
In 1863 when the Channel fleet visited, 55,000 people took the ferry out to see the Navy ships.
Before radar was installed in 1947, ferry captains had to rely on fog bells to give them an audible target to aim for
The Mersey Ferries were the first in the world to install a radar system for safe navigation in fog, in 1947.
In 1357, the charge for a foot passenger was a halfpenny.
The opening of the first underwater railway in 1886 and the lines electrification in 1903 was predicted to be the end of the Mersey Ferries, but in 1919 the Seacombe ferry alone carried 22 million passengers.
After the opening of the road tunnel in 1934, there was a huge decline in numbers using the Mersey Ferries.
The opening of the second Mersey tunnel in 1971 brought the closure of the New Brighton ferry.
The Birkenhead and Wallasey ferry operations merged into Mersey Ferries in 1968.
The Mersey Ferries were under threat in 1977, when a bill was put before Parliament to discontinue ferry services. The bill failed to receive support and the Mersey Ferries survived.
After a decline in commuter traffic using the ferries, in 1990 Merseytravel re-launched the ferries as a heritage and visitor attraction.
Mersey Ferries won Visitor Attraction of the Year award in 1996, by the Merseyside Tourism Board.
The Mersey Ferries took a technological leap in 1815 with the introduction of the first steam ferry, Etna. For the first time, the ferry service could operate a timetable.
Soon after Etna, came Vesuvius, the second paddlesteamer.
By 1840, all ten ferry services across the Mersey were using paddlesteamers.
Crocus, the first coal-fired screw steamer was introduced in 1884, closely followed by Snowdrop the following year.
The first diesel-powered ferry came in 1949
The last steam ferry on the Mersey was the Wallasey, which sailed for the last time at Whitsun in 1963.
The template for the 20th century Mersey Ferries was the Wallasey ship Rose, in 1900.
Cammell Laird built all five ferries that made up the Birkenhead post-war fleet; Upton, Hinderton, Thurstaston, Claughton and Bidston.
Birkenhead and Wallasey followed different trends in naming their ferries; Birkenhead used local place names such as Woodchurch and Claughton, and Wallasey used flowers, such as Daffodil and Primrose.
In 1906 Wallasey took ownership of Iris and Daffodil, which become their most famous ferries.
In 1918, Daffodil and Iris were requisitioned by the Admiralty to take part in a raid on Zeebrugge on St Georges Day, 23 April.
A commemorative service for the Zeebrugge raid is held every year on a Mersey ferry, on the Sunday nearest to St Goerge’s Day.
Eleven Victoria Crosses were awarded for bravery in the Zeebrugge raid.
In recognition of their courageous part in the raid, King George V granted Wallasey Corporation permission to add the ‘Royal’ prefix to both names.
In November 1998, Overchurch went into dry dock in Manchester, where she was refurbished and given new engines. She later emerged as the Mersey Ferries new flagship, Royal Daffodil.
Mountwood went into dry dock for refurbishment in 2001, and was renamed Royal Iris of the Mersey on her return in April 2002.
The current Mersey Ferries fleet consists of two boats: Snowdrop, (previously Woodchurch) and Royal Iris of the Mersey (previously Mountwood). Up to 2013, the Royal Daffodil (previously Overchurch) had been the flagship of the fleet.
The Royal Iris was sold in November 1991, and is now berthed in London, by the Thames Barrier.
Since the name Royal Iris is still in use by the old ferry now berthed in London, we have Royal Iris of the Mersey.
Each ferry holds 35 tons of fuel in its tanks and burns a ton in 12 hours: So, a Mersey ferry could travel across the Atlantic to New York without needing to refuel.
Overchurch was the most recent Mersey ferry to be built, launched at Cammell Laird’s yard in 1962.
The current ferries were built to carry 1,200 passengers; however they are now licensed to carry 650 for their daily service, and 396 for longer cruises.
By 1989 the ferries were running at an annual loss of £2.5 million; a new strategy was then agreed, focusing on tourism and heritage rather than commuter transport.
Between high and low tide, the river Mersey can rise and fall by 30ft.
The ferries all have Fletner system rudders which make them much more manouverable. By using the vessel's twin screws, captains can move the vessels away by using one engine to push the vessel from the stage and the rudders and other engine to point it into the correct direction.
A ferry across the Mersey from Seacombe, the narrowest crossing point of the river, is recorded in the Domesday Book of 1086
During the First World War the steamers Iris and Daffodil were taken out of service from Wallasey to be used as troop ships in the naval raid on Zeebrugge in Belgium
The Manchester Ship Canal is the eighth-longest ship canal in the world. Mersey Ferries offer six hour cruises along the Manchester Ship canal with return bus transport.
Famous people who have travelled on the Mersey Ferries – Queen Elizabeth and Prince Phillip, Princes Anne, Margaret Thatcher, King George V and Queen Mary, The Beatles, Gerry & the Pacemakers.
The Manchester Ship Canal 60ft rise in height from Liverpool to Manchester is resolved by a series of 5 locks
In 1847, the first floating landing stage opened at Liverpool. The landing stage rose and fell with the tide so that the boats could dock at any time.
The first passenger ferry steamer to have a saloon was the Cheshire. It operated from Woodside in 1864.
On 26 November 1878, the ferry Gem, a paddle steamer operated from Seacombe collided with the Bowfell, a wooden sailing ship at anchor on the River Mersey; the collision resulted in the death of five people.
In 1894 trains were carrying 25,000 passengers per day and the ferries 44,000 per day.
The ferry service at Tranmere closed in 1897.
The pier and landing stage at Rock Ferry was built in 1899.
In 1914 King George V and Queen Mary travelled on the ferry S.S. Daffodil from Wallasey to Liverpool.
The ferry service from Seacombe lost two million passengers when the Queensway road tunnel opened in 1934, with people starting to use the tunnel rather than the ferry.
The opening of the road tunnel also had an impact on the luggage boats which were introduced in 1879. Both ferry companies earned a substantial amount from luggage boats, which carried vehicles and goods across the river. The luggage boat services from both Woodside and Seacombe to Liverpool came to a close in the 1940s.
The closure of Eastham in 1929 marked the last use of ferry paddle steamers on the river.
In 1941, mines which had drifted into the River Mersey stopped ferry crossings
In 1950, the ferries carried almost 30 million passengers.
By 1970, the total number had fallen to just 7 million.
In 1956, night boats across the river were cancelled and replaced by bus services through the tunnel.
The future of the Mersey Ferries came under threat in June 1971, with the opening of the Kingsway road tunnel. The Mersey Ferries saw a further decline in passenger numbers; only 4,000-5,000 passengers a day using the service.
1984 was a considerably good year for the Mersey Ferries, and is seen as the beginning of the ferries rise from the slumps of the 1970s.
A special ferry service was provided to Otterspool Promenade throughout the International Garden Festival. This service was usually operated by Overchurch.
In August 1984, sailing ships from the Tall Ships Race visited the river.
The first diesel ferry to enter service was the Royal Iris in 1951.
The most famous Royal is the Royal Iris of 1951. She was by far the best loved of all the Mersey Ferries.
Royal Iris was the first diesel powered vessel of the Wallasey fleet, with four diesel generators connected to two Metrovick marine propulsion units.
Before being sold in the early nineties for use as a floating nightclub, The Royal Iris held hundreds of party cruises, and played host to popular bands such as Gerry & The Pacemakers, The Searchers, and The Beatles.
The Royal Daffodil II was constructed by James Lamont and Co. at Greenock and entered service in 1957.
The Royal Daffodil II is the only Mersey Ferry to ever have docking order telegraphs in the wings.
Leasowe was sold to Greek owners in the mid 1970s; she has been heavily modified and is still cruising around the Greek islands.
The former Royal Daffodil II was converted to a container ship. She sank in November 2007, 20 miles off the coast of Cape Andreas, in heavy seas.
The Mountwood was used in the film "Ferry Cross The Mersey",
The Mountwood was used for the video of the Gerry & The Pacemakers song, “Ferry across the Mersey” It was filmed on two separate journeys across to Liverpool from Birkenhead
In her early years Mountwood was an unreliable ferry, breaking down three times whilst crossing the river and had to anchor. Her passengers had to be rescued by Woodchurch.
In 1981, Woodchurch was withdrawn from operation for almost three years, due to cost cutting measures.
Woodchurch returned to service in 1983, after main engine repairs and a full repaint. Her return freed up Overchurch to work the new Otterspool service, set up for the 1984 International Garden Festival.
In 1996 the Overchurch was given a facelift, enclosing of the promenade deck shelter.
The current fleet has served the river for nearly five decades. The Royal Iris of the Mersey and the Snowdrop celebrated their 50th birthday in 2009.
The new Wartsilla engines fitted on the ferries are much more economical than the previous engines by Crossley Bros of Manchester. They produce much less emissions than the original propulsion units, making them a lot greener.
Each of the Mersey Ferries in the current fleet carries two Kockums Super Tyfon TA 100/150 type fog horns. These are the original horns that were fitted when the ferries were first built. Both Royal Iris of the Mersey and Snowdrop have an E-flat tone, and Royal Daffodil is in F Sharp.
The ferries are known for their ability to operate in very heavy seas.
When berthing the vessel, the captain uses a combination of rudder positions and engine movements.
Although the ferries can cope in heavy seas, berthing the vessel can be extremely hazardous. This can result in the service being suspended.
The ferries all have Fletner system rudders which make them much easier to manoeuver.
The ferries played a big part in Liverpool's European Capital of Culture 2008 celebrations, where they carried a record numbers of passengers
On Monday 21st July 2008, the Royal Daffodil operated a special cruise to witness the parade of sail and departure of the Tall Ships.
The first record of a service from New Ferry to Liverpool was in 1774.
The first steamship to operate on the Mersey was the Elizabeth, a wooden paddle steamer, which was introduced in 1815 to operate between Liverpool and Runcorn.
The Woodside, North Birkenhead and Liverpool Steam Ferry Company was formed in 1835.
The Cheshire, the first passenger ferry steamer to have a saloon, operated from Woodside in 1864.
The iron pier at Eastham was built in 1874.
The pier and landing stage at Rock Ferry was built in 1899.
Wallasey Ferries had a funnel livery of white and black and Birkenhead red and black up until the Second World War, when this was changed to orange when Mountwood and Woodchurch were introduced.
Due to financial losses incurred from a reduction in patronage, Birkenhead Corporation gradually closed its southern terminals; New Ferry on 22nd September 1927, Eastham in 1929 and Rock Ferry on 30 June 1939.
In 1941, The Oxton and Bebington vessels were fitted with cranes to enable them to unload United States aircraft from mid-river and deliver them to the Liverpool landing stage.
The Birkenhead boats Claughton, Bidston, Thurstaston and Upton were seen as the fastest ferries on the river. |
LaRouche Claims No Income of His Own
By Leslie MarshallBy Leslie MarshallOctober 31, 1984
Independent presidential candidate Lyndon H. LaRouche testified in court yesterday that he has had no income and paid no income tax for the past 12 years and does not know who has paid for his food, clothing, travel expenses, lawyers' fees and rent over that period.
"I have not made a purchase of anything greater than a $5 haircut in the last 10 years," said LaRouche, testifying for the second day at the trial of his $150 million libel suit against NBC television in U.S. District Court in Alexandria.
In earlier testimony, LaRouche has said that his concern in the suit is not with financial loss, but with damage to his "prime function" of being a philosopher and an economist and with an increase of personal physical danger that he claims have resulted from two NBC broadcasts that portrayed him as the anti-Semitic leader of a "violence-prone" political cult.
LaRouche, 62, is a three-time presidential candidate who will be on the ballot next Tuesday in 19 states including Virginia.
LaRouche is known as the leader of the right-wing National Democratic Policy Committee, but he testified Monday that he belongs only to a loose association of people around the world.
He was testifying yesterday under cross-examination by NBC attorney Thomas Kavaler, who repeatedly turned to read statements LaRouche made in a deposition this summer that appeared to contradict the answers he gave to the same questions yesterday.
LaRouche disputed statements that were read to him from the deposition.
"That's not the testimony I gave this summer. Your testimony is false," LaRouche declared at one point. LaRouche also criticized Kavaler's questioning as a strategy of "lying by fallacy of composition of the question," taking things out of context and pulling "alligators and washing machines" from a "little bag."
Kavaler also showed the jury a 10-minute excerpt yesterday from a half-hour paid political announcement entitled "Walter Mondale and the Danger of Fascism in West Germany" that LaRouche's campaign committee aired on CBS prime-time television a week ago. In the film clip, LaRouche elaborated on his theory that presidential candidate Mondale is an "agent of influence" of the Soviet Union's secret intelligence service.
In testimony yesterday, LaRouche said that as of next Monday, his campaign will have produced a total of 15 such nation-wide televised political spots in the past year, at an average cost of about $250,000 per spot. Three are scheduled to appear on prime-time television next Monday, he said.
During his testimony, LaRouche acknowledged that he had once referred to one of the defendants in his suit, Pat Lynch, a producer for one of the the NBC broadcasts in question, as "Fat" Lynch, and described another defendant, NBC correspondent Brian Ross, as "a scoundrel" who has committed "virtual treason as well as thuggery."
In reference to a document he circulated critical of former secretary of state Henry Kissinger, LaRouche testified that "I wanted Kissinger to sue me because I have evidence." Kissinger, LaRouche said, has been "attacking me, killing my friends and wrecking governments" for years.
Later in the day, LaRouche was cross-examined by an attorney for the Anti-Defamation League of B'nai B'rith, which is also a defendant in the case because of an ADL official's statements in the broadcasts that LaRouche is a "small-time Hitler."
"The IRS ought to look into the ADL," LaRouche testified at one point, as he explained his belief that the ADL should be registered as a political action committee instead of a nonprofit organization. |
Studies are in progress on the membrane properties and contractile responses of intrafusal muscle fibers in isolated cat muscle spindles. Intracellular micropipettes are used for recording and current passage. Contractile responses are recorded through an inverted microscope with differential interference contrast, using high speed cinematography. Spontaneous miniature endplate potentials are also being recorded from intrafusal muscle fibers. These studies aim towards an understanding of how nuclear bag and nuclear chain fibers respond to their motor innervation and modify the response on sensory endings to muscle stretch. |
(function () {
'use strict';
angular.module('CounterApp', [])
.controller('CounterController', CounterController);
CounterController.$inject = ['$scope'];
function CounterController($scope) {
$scope.counter = 0;
$scope.upCounter = function () {
$scope.counter++;
};
}
})();
|
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Traps are a great way of catching exceptions and handling errors manually but this does not seem to work all of the time. This example catches the error: Trap { 'Something terrible happened.' ; Continue } 1 / $null However, this example does not...
Traps are a great way of handling errors but you may want to control where PowerShell continues once an error occurs. There is a simple rule: a trap that uses the Continue keyword continues execution in the next line in the scope of the current trap....
Traps are exception handlers that help you catch errors and handle them according to your needs. A Trap statement anywhere in your script: Trap { 'Something awful happened' } 1 / $null Whenever an error occurs and an exception is raised, PowerShell... |
Tag Archives: Gun Control
One of my favorite commentators on Youtube is Mr. Colion Noir. Most of his stuff rings true with me. I’m also extremely jealous of the weapons he gets to shoot. I was excited when he became a commentator for NRA news. The first video of his that I watched was this one. I remember thinking, “wow this guy has his shit together.” He has a litany of videos dispelling gun myths and hysteria in a way that is both objective and non partisan. He’s also apparently got a ton of hats. Seriously dude who are you a fan of? I count at least seven different baseball team hats. You can’t like both the Red Sox and the Yankees. Anyways, for me his latest video hit the closest to home:
I have one issue with the video in that I don’t really think people are pro-gun. I’m actually quite anti-gun. I mean, I wish they didn’t have to exist. I’m pro self defense. I’m pro liberty. I’m pro freedom. It just so happens that the gun is the tool that protects. The tool that defeats tyranny and ensures freedom. I don’t think that people are against those things; we just have different ideas on how to accomplish it.
It always disturbs me when people try to put me in a box because of my defense of the Second Amendment. I’ve said before that I’m fairly moderate and not religious. However, when I rebuff people’s arguments for gun control all of a sudden I’m a privileged, racist white male. I’m a religious right wing fanatic. Oh, you didn’t think that when when I was arguing on your side in support of gay marriage. Now I’ve come out against the gun control statists and I’m bad guy. I get it. The strawman argument.
I hate being placed in a political box. I don’t think either side has it entirely right. I think both sides happen to get a couple of things right and a lot of things wrong. I’m all for freedom. As one of my friends on the radio always says, “I don’t care if you think you’re a liberal. I don’t care if you think you’re a conservative. All I care about is that you think.” Think for yourselves people. Don’t let any ultra-anything tell you how you should believe. Get out. Do the research. Especially if someone is advocating for the loss of rights for a hundred million people.
Like this:
I find myself repeatedly correcting my fellow Second Amendment advocates. “It’s a magazine not a clip.” “It’s not an assault rifle.” “The AR-15 is not a “high powered rifle.” Usually they just shine me on and laugh. Some use the terms on purpose around me to elicit a response. It’s important for us as 2A defenders to use the correct terms and here’s why; how can you defend something you don’t know anything about? I mean, I know you like your guns and you make statements that you’ll die before you let someone take them, but you sound like an idiot. You’re doing more harm by using the wrong terms and making stupid statements like that.
Worse than not knowing what a clip or what an assault rifle is is using the anti-gun terminology. If you’ve ever said “gun show loophole” or “assault weapon” you’re letting the anti-gunners control the conversation. There is no such thing as “the gun show loophole.” It’s a device used by anti-gunners to make people think that some trick is being played that lets people get away with buying a firearm without a background check. It’s called a private party sale, and is legal in most states without a background check. When you use terms like this you are in fact supporting their argument. I know that’s not what you’re trying to do.
The last reason why the jargon is important is; that’s how I win arguments against anti-gunners. When they use the wrong terminology I point out that they don’t know what they’re talking about. How could someone advocate for the loss of rights for almost 100 million people without knowing the subject? It’s irresponsible at best and willful ignorance at worst. The flip side of this is true as well. How can you defend your rights, which people see as something bad, without truly knowing the subject? What’s your basis?
Let’s take back the conversation. We do this by using the right jargon, the correct terminology. Don’t laugh at people when they correct you and try your best to not undermine the work that is being done to protect your rights. Above all, stop saying clip when you mean a magazine. Seriously just stop it. If you’re trying to shorten it to sound cool say mag. Please. |
Nokia could launch a new smartwatch soon, according to its board of directors’ proposed amendments to the company’s constitution. The changes will add “consumer wearables” and “Internet of Things” devices to its list of Nokia’s |
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