text stringlengths 8 5.77M |
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Medication use in the treatment of migraine during pregnancy and lactation.
Migraine is very common in women of reproductive age. With peak prevalence of migraine occurring during childbearing years, many women with migraine may knowingly or unknowingly use medication during pregnancy. Although migraine tends to improve during pregnancy, many women may still experience moderate to severe disabling headache and need pharmacologic treatment for the pain, nausea, and vomiting. This article explores the physiologic changes occurring during pregnancy that can affect pharmacokinetic properties of drugs and their metabolism. Acute and preventive treatment of migraine during pregnancy and lactation is discussed, with an emphasis on safety to the fetus and nursing infant. Safety and recommended use of medication during pregnancy may be different when use is considered during breastfeeding. A goal of treatment is to balance potential risk of treatment to the fetus and nursing infant with significant relief and return to normal function of the mother. |
'use strict';
const loadRule = require('../utils/load-rule');
const ContextBuilder = require('../utils/contextBuilder');
const UserBuilder = require('../utils/userBuilder');
const ruleName = 'aspnet-webapi';
describe(ruleName, () => {
let rule;
let context;
let user;
let globals;
let request;
const expectedCustomId = 'testId';
beforeEach(() => {
globals = {
auth0: {
users: {
updateAppMetadata: jest.fn()
}
},
configuration: {
YOURWEBSITE_SECRET_TOKEN: ';ojhsajk;h;Kh:Jh'
}
};
request = {
post: jest
.fn()
.mockImplementation((url, cb) => {
cb(null, null, { customId: expectedCustomId });
})
};
user = new UserBuilder()
.build();
context = new ContextBuilder()
.build();
rule = loadRule(ruleName, globals, { request });
});
describe('when aspnet post request is successful', () => {
it('should update the user metadata and set idToken', (done) => {
const updateAppMetadataMock = globals.auth0.users.updateAppMetadata;
updateAppMetadataMock.mockReturnValue(Promise.resolve());
rule(user, context, (e, u, c) => {
const call = updateAppMetadataMock.mock.calls[0];
expect(call[0]).toBe(user.user_id);
expect(call[1].customId).toBe(expectedCustomId);
expect(context.idToken['https://example.com/custom_id']).toBe(expectedCustomId);
expect(user.app_metadata.customId).toBe(expectedCustomId);
done();
});
});
});
});
|
Polycystic kidneys as the presenting feature of tuberous sclerosis.
Tuberous sclerosis is an inherited neurocutaneous disorder characterized by seizures, mental retardation, cutaneous lesions and visceral hamartomas. We describe a 17-year-old boy in whom polycystic kidneys of the adult type were fortuitously detected on routine check-up. The patient enjoyed good health and had no evidence of renal dysfunction. Closer scrutiny of his past history and his physical and laboratory findings disclosed that he had tuberous sclerosis. Our case adds to the scant reported experience with the association of tuberous sclerosis and adult-type polycystic kidneys, and suggests that a search of additional manifestations of tuberous sclerosis is warranted in children in whom adult-type polycystic renal disease is detected. |
Role of microangiopathy in diabetic cardiomyopathy.
Although heart disease due to diabetes is mainly associated with complications of the large vessels, microvascular abnormalities are also considered to be involved in altering cardiac structure and function. Three major defects, such as endothelial dysfunction, alteration in the production/release of hormones, and shift in metabolism of smooth muscle cells, have been suggested to produce damage to the small arteries and capillaries (microangiopathy) due to hyperglycemia, and promote the development of diabetic cardiomyopathy. These factors may either act alone or in combination to produce oxidative stress as well as changes in cellular signaling and gene transcription, which in turn cause vasoconstriction and structural remodeling of the coronary vessels. Such alterations in microvasculature produce hypoperfusion of the myocardium and thereby lower the energy status resulting in changes in Ca(2+)-handling, apoptosis, and decreased cardiac contractile force. This article discusses diabetes-induced mechanisms of microvascular damage leading to cardiac dysfunction that is characterized by myocardial dilatation, cardiac hypertrophy as well as early diastolic and late systolic defects. Metabolic defects and changes in neurohumoral system due to diabetes, which promote disturbances in vascular homeostasis, are highlighted. In addition, increase in the vulnerability of the diabetic heart to the development of heart failure and the signaling pathways integrating nuclear factor κB and protein kinase C in diabetic cardiomyopathy are also described for comparison. |
Docker Finds Open Source Success
PaaS firm formerly known as dotCloud changes its name to Docker after developers embrace open source Docker.org project.
The San Francisco-based company behind the popular open source code project Docker.io is changing its name to Docker, effective on Tuesday. The firm was formerly known as dotCloud.
DotCloud was founded in 2010 to provide a multi-language platform-as-a-service for developers. In doing so, the company came up with the idea of containerized virtual machines, in which sets of virtual machines run in a container under one operating system instead of each virtual machine having its own operating system. This approach saves server memory and speeds up virtual machine operations.
In Java programming, a container is a logical construct that defines a set of resources for use by the virtual machines launched inside it. Sun Microsystems, for example, used "zones" as virtual machines running under one copy of Solaris on a host server. Docker implements a similar approach under Linux, explained Ben Golub, CEO of Docker.
Launched six months ago, the Docker.io project was created from dotCloud's early work. Since then, it has grown popular with developers, reminiscent of Linux, the Apache Web server and MySQL in their early days. Docker has been downloaded 100,000 times, and 200 outside developers have contributed to the Docker project.
The firm has established a registry of applications that have been placed in Docker containers and that are available for public use. Applications and their dependencies, such as application servers, Web servers or connections to databases, all fit into one container file, which can be downloaded or moved from one cloud location to another. The registry shows 20,000 applications.
At least 13,000 developers have competed online Docker training, and 50 developer meet-ups have taken place in 30 cities on use of Docker, Golub said in an interview.
A driver allowing Docker to be used from inside an OpenStack cloud was included in the recent Havana release of the cloud software, potentially easing the task of migrating from one OpenStack cloud to another. The Docker driver is also integrated in Red Hat's OpenShift platform-as-a-service and Red Hat's distribution of OpenStack in Red Hat Enterprise Linux.
"It was a great validation to be accepted into OpenStack," said Golub, adding that Docker representatives will attend the upcoming OpenStack Summit in Hong Kong, where they will demonstrate how to create Docker containers and move applications between clouds.
Docker also plans to provide professional consulting on implementing Docker, as well as technical support, in 2014.
Containers as discrete holders of multiple virtual machines are a good idea, but it took a little thinking outside of the large virtualization vendor box to come up with it. Docker is that most valuable of projects, a simple idea, propagated through open source, whose time has come.
There's no doubt Google has made headway into businesses: Just 28 percent discourage or ban use of its productivity products, and 69 percent cite Google Apps' good or excellent mobility. But progress could still stall: 59 percent of nonusers distrust the security of Google's cloud. Its data privacy is an open question, and 37 percent worry about integration. |
Personal History:
Joseph L. Bryant, DVM, directs the IHV's Animal Model Division and Animal Core Facility. A board certified Laboratory Animal Veterinarian with a background in Comparative Medicine, Dr. Bryant's current research program is an extension of work begun at the National Institutes of Health, where he played a major role in developing a transgenic and gene Knockout Program.
His research team at the IHV has created the first transgenic rats whose DNA has been manipulated to incorporate genes of HIV-1. The rat model opens new areas of human HIV/AIDS study for scientists; its potential is enhanced by similarities in the structure of the rat genome with humans. Ongoing studies include the use of the transgenic rat as a model for HIV/drug abuse, HIV-associated skin diseases and HIV-associated neurological diseases.
Other research efforts focus on the mechanisms of human diseases caused by bacteria and viruses and on details of virus replication to develop better therapies and improved or new diagnostics. The current focus of this work is on the continual development of animal models for AIDS and AIDS-associated malignancies, such as Kaposi's sarcoma and B-cell lymphoma, as well as animal models for breast and prostate cancer. The most recent studies in the division involve the development of animal models of b-cell lymphoma as it relates to infectious causes i.e. mycoplasma fermentans, HIV-1 transgenic mouse model of b-cell lymphoma, and the use of natural plant extracts as anti-cancer agents. Currently, we are also working closely with groups from Jamaica and Nigeria.
Research Interests:
The use of animals as models for human disease has been indispensable in understanding the causes, biology and prevention of disease; the Animal Models Division at the Institute of Human Virology has developed the following models for studying AIDS and AIDS-associated cancers:
The SIV Non-human primate models for studying HIV-1 pathogenesis and for the development of HIV-1 vaccines
All of these models have and are playing a crucial role in understanding the pathogenesis of AIDS and various cancers with the ultimate intent of providing models for pre-clinical testing of new anti-HIV and anti-cancer drugs and treatment. The goal of the continuing development of the HIV transgenic rat model is to develop a small animal model that can be used for testing vaccines.
Clinical Speciality:
Board Certified in Laboratory Animal Medicine.
Lab Techniques and Equipment:
The Animal Core Facility is fully equipped to house and care for animals used in basic bio-medical research. We have over 20,000 square feet of space, and we have 3 laboratories for basic research.
Grants and Contracts:
09/01/2010-08/31/2013
NIH/NINDS
A Model of Stem Cell-Based Treatment of HIV-Related Neurological Disease
The goal of this project is to evaluate the efficacy of brain derived neurotrophic factor (BDNF) in suppressing nervous system abnormalities that can be observed with HIV infection.
Role: Co- PI
04/26/2011-04/30/2013
OPP1017606 (Bryant)
Gates Foundation
Phase I Clinical Trial of a Noval HIV Protein Construct that presents CD4 Induced Epitopes (Obj. 4)
The main goal of this project is to support a Phase I clinical trial of a novel HIV protein construct that presents CD4 induced epitopes. This research will allow for clinical testing of FLSC evaluating immune response and safety in humans, and optimization of the prime-boost vaccination strategy.
Role: PI |
Q:
Laptop is treating speakers as headphones
Up until recently, whenever I plugged in my headphones, the sound would switch over from my laptop's speakers to my headphones. Now, plugging in headphones only results in the speakers becoming lower, which makes me think the speakers are being treated as headphones.
Any solutions? I have not changed any drivers recently, or software for that matter.
A:
I found the solution (rather simple, really). Somehow, I had set Independent Dual Headphones as my default playback device. Going to the Sound control panel, I just had to set Speakers and Dual Headphones as the default device, and everything is working again.
|
2 New Orleans officers charged in beating death
NEW ORLEANS
Two New Orleans police officers were indicted Thursday on federal charges in the beating death of a 48-year-old man, part of a sprawling Justice Department probe that has led to charges against 18 of the city's officers.
Melvin Williams, one of the two officers charged, is accused of kicking Raymond Robair and beating him with a police baton, causing his fatal injuries. Williams and a fellow officer, Matthew Dean Moore, encountered Robair while patrolling a New Orleans street on July 30, 2005.
Both officers dropped him off at a hospital, where he later died of a ruptured spleen. The officers didn't tell anyone at the hospital that Williams struck Robair, the indictment said. Both men are still on the force.
A police report submitted by Williams and Moore described the encounter as a "medical incident" and included a false description of how Robair was injured, according to the indictment.
Williams is charged with deprivation of rights under color of law in Robair's death. Both officers are charged with obstructing a federal investigation. Moore is charged with lying to the FBI when he said Williams never struck Robair.
The case is one of at least eight investigations of the New Orleans Police by the Justice Department's Civil Rights Division. So far, Robair's death is the only incident being investigated that happened before Hurricane Katrina in August 2005.
Williams' attorney, Frank DeSalvo, said his client has been given several citations over the years for being a "really good cop."
"I am surprised by this indictment," he said. "I just don't think they have a case. I don't think they did anything wrong."
Moore's lawyer, Eric Hessler, said the officers didn't know how Robair was injured.
"It was a very quick encounter," Hessler said. "It certainly wasn't a homicide, and it wasn't caused by any use of force by these officers."
Mary Howell, a lawyer for Robair's family, said they are grateful for the Justice Department's intervention.
"The family is hopeful that there will be justice and that those responsible for Raymond's death and the cover-up of the circumstances of his death will ultimately be held accountable for their actions," Howell said.
Sixteen other current or former officers are charged in an unrelated pair of post-Katrina police shootings.
Five former officers already have pleaded guilty to helping cover up a deadly shooting of unarmed civilians on a bridge less than a week after the storm's landfall. Six others are charged in that case.
Five current and former officers are charged in the death of 31-year-old Henry Glover, whose charred remains turned up in a car that had been torched and abandoned on a river levee after Katrina.
If convicted, Williams faces a maximum possible sentence of life in prison or the death penalty if prosecutors seek the latter. U.S. Attorney Jim Letten said the Justice Department has not decided whether to seek the death penalty.
The charges against Moore carry a maximum sentence of 25 years in prison. A date for their initial court appearances wasn't immediately set. |
all: mono
mono: swf-combobox-ownerdraw.cs
mcs swf-combobox-ownerdraw.cs /r:System.Windows.Forms.dll /r:System.Drawing.dll
dotnet: swf-combobox-ownerdraw.cs
csc swf-combobox-ownerdraw.cs /r:System.Windows.Forms.dll /r:System.Drawing.dll
clean:
rm swf-combobox-ownerdraw.exe -r -f
|
430 F.3d 1305
UNITED STATES of America, Plaintiff-Appellee,v.Emiliano W. ALVARADO a/k/a Alvarado-Guerra, Defendant-Appellant.
No. 05-4064.
United States Court of Appeals, Tenth Circuit.
December 13, 2005.
Stephen R. McCaughey, Salt Lake City, UT, for Defendant-Appellant.
Diana Hagen, Assistant United States Attorney, Paul M. Warner, United States Attorney, District of Utah, Salt Lake City, UT, for Plaintiff-Appellee.
Before BRISCOE, ANDERSON, and O'BRIEN, Circuit Judges.
ANDERSON, Circuit Judge.
1
Emiliano Alvarado entered a conditional plea of guilty to one count of possessing cocaine with intent to distribute, in violation of 21 U.S.C. § 841(a)(1), reserving his right to appeal the district court's denial of his motion to suppress. He now appeals that ruling, arguing that the district court erred in holding that the police stop of his vehicle was reasonable based on a single instance of crossing over the right fog line of the highway, where the relevant Utah statute requires driving "as nearly as practical entirely within a single lane." Utah Code Ann. § 41-6-61(1).1 We affirm.
BACKGROUND
2
At approximately 3 p.m. on February 19, 2004, Utah Highway Patrol Trooper Nick Bowles was driving eastbound on Interstate 70 and observed a Jeep Cherokee, which Alvarado was driving, cross "about a foot" over the right fog line of the highway, continue traveling over the line "for a few seconds," and then cross back to the righthand lane. Mot. to Suppress Hr'g, R. Vol. II at 5. According to Trooper Bowles' testimony and the findings of the district court, "[i]t was a clear and sunny day with no wind or other adverse weather conditions." Mem. Decision & Order at 2, R. Vol. I. The highway at that location "was straight and flat" and "was dry at the time," with "no pot holes, debris, or other obstructions in the roadway." Id. Based on these "ideal driving conditions," id., Trooper Bowles testified that he "felt there was no reason" for the Jeep "to be crossing the line" and was therefore "concerned that [the driver] might be impaired or fatigued." Mot. to Suppress Hr'g, R. Vol. II at 5. According to Trooper Bowles, the majority of accidents that he had handled on Interstate 70 involved "single vehicle rollovers, most of which are caused by vehicles crossing the fog line, either they never correct themselves and go off or else they over-correct and come back on and roll." Id. at 20. Trooper Bowles was also aware that failing to maintain one's vehicle in a single lane was a traffic infraction under Utah state law.
3
Thus, after observing the Jeep cross the fog line, Trooper Bowles "turned on his emergency lights and pulled over [Alvarado]." Mem. Decision & Order at 2, R. Vol. I. Trooper Bowles asked Alvarado to sit in the front passenger seat of the patrol car, where the trooper asked him about his travel plans while dispatch ran a check on Alvarado's license, registration, and criminal history. Trooper Bowles then gave Alvarado a written warning for crossing the fog line, returned Alvarado's documents, and told him "you're free to leave, drive safely." Id. at 4.
4
As Alvarado was returning to the Jeep, Trooper Bowles asked him if he could talk with him for another minute. Alvarado agreed. Subsequently, the trooper asked Alvarado for permission to search the vehicle, and Alvarado consented. During the search, Trooper Bowles discovered illegal narcotics hidden in the rear of the Jeep and consequently placed Alvarado under arrest.
5
Alvarado was charged with one count of possession with intent to distribute 500 grams or more of cocaine, in violation of 21 U.S.C. § 841(a)(1). He moved to suppress the evidence of drugs, arguing that his initial stop was illegal, that he had not given valid consent to search the vehicle, and that the trooper did not have probable cause to search or detain the vehicle. In denying this motion, the district court reasoned that Trooper Bowles was justified in stopping the Jeep because Utah Code Ann. § 41-6-61(1) required remaining in a single lane "as nearly as practical," and "the record in this case does not contain evidence of other conditions or circumstances that would make it impractical for [Alvarado] to drive the vehicle within a single lane." Mem. Decision & Order at 10, R. Vol. I.
6
The court therefore held that the initial stop was reasonable under the Fourth Amendment because it was supported by a reasonable articulable suspicion that Alvarado was in violation of the Utah statute. The court further held that the traffic stop became a consensual encounter after Alvarado's documents were returned and he was told he was free to leave, and that Alvarado voluntarily consented to the search of the Jeep. Alvarado then entered a plea of guilty to the single count in the indictment but reserved his right to appeal the district court's order denying his motion to suppress. He filed a timely notice of appeal of that order, raising only the issue of the reasonableness of the initial stop.
DISCUSSION
7
In reviewing a district court's denial of a motion to suppress evidence, "we accept the factual findings of the district court, and its determination of witness credibility, unless they are clearly erroneous." United States v. Cline, 349 F.3d 1276, 1286 (10th Cir.2003) (internal quotation omitted). "In conducting our review, we consider the evidence in the light most favorable to the district court's ruling." United States v. Zabalza, 346 F.3d 1255, 1258 (10th Cir.2003). However, "[w]e review de novo the `ultimate determination of reasonableness under the Fourth Amendment.'" Cline, 349 F.3d at 1286 (quoting United States v. Cervine, 347 F.3d 865, 868 (10th Cir.2003)).
8
Alvarado's sole argument on appeal relates to the reasonableness of Trooper Bowles' initial stop of his vehicle. We have held that, in order to satisfy the Fourth Amendment's reasonableness requirement, a law enforcement officer "`must have an objectively reasonable articulable suspicion that a traffic violation has occurred or is occurring before stopping [an] automobile.'" Cervine, 347 F.3d at 869 (quoting United States v. Soto, 988 F.2d 1548, 1554 (10th Cir.1993)). Thus, "[w]hen evaluating the reasonableness of the initial stop [of a vehicle], `[o]ur sole inquiry is whether this particular officer had reasonable suspicion that this particular motorist violated any one of the multitude of applicable traffic and equipment regulations of the jurisdiction.'" Zabalza, 346 F.3d at 1258 (quoting United States v. Botero-Ospina, 71 F.3d 783, 787 (10th Cir.1995) (en banc)) (further quotation omitted).
9
Here, the relevant Utah statute in effect at the time of the stop provided that "[a] vehicle shall be operated as nearly as practical entirely within a single lane." Utah Code Ann. § 41-6-61(1). We have previously addressed the validity of traffic stops in relation to this statute and a similar Kansas statute, Kan. Stat. Ann. § 8-1522 (requiring vehicles to be driven "as nearly as practicable entirely within a single lane"), in a number of cases. In United States v. Gregory, 79 F.3d 973 (10th Cir.1996), the officer had stopped a truck after it "briefly crossed into the right shoulder emergency lane" where "[t]he road was winding, the terrain mountainous and the weather condition was windy." Id. at 978. We held that the stop was unreasonable in light of the Utah statute's qualification that vehicles remain in a single lane only "as nearly as practical," reasoning that under the particular weather and road conditions present on that occasion, "any vehicle could be subject to an isolated incident of moving into the right shoulder of the roadway, without giving rise to a suspicion of criminal activity." Id.; see Cervine, 347 F.3d at 869 (interpreting Gregory); see also State v. Bello, 871 P.2d 584, 587 (Utah Ct.App.1994) (holding that an officer improperly stopped a truck for briefly moving out of its lane because Utah Code Ann. § 41-6-61(1) was not violated by a single instance of weaving where it was "extremely windy" and the truck had a camper shell "that caused it to catch the wind more easily than other vehicles").
10
Subsequent to our decision in Gregory, we have emphasized that that case does not "stand[ ] for the proposition that a single instance of drifting onto the shoulder can never be a violation of a traffic statute like section [41-6-61(1)]." Cline, 349 F.3d at 1287. Rather, a court must "analyze objectively all the surrounding facts and circumstances to determine whether" the officer had reasonable suspicion that a violation of the statute had occurred. United States v. Ozbirn, 189 F.3d 1194, 1198 (10th Cir.1999). Thus, in Ozbirn, we held the traffic stop was reasonable where no "adverse physical conditions existed" and the driver of a motor home passed over onto the shoulder "twice within a quarter mile." Id. We reached the same conclusion in Zabalza where the officer observed the vehicle cross the center line twice, 346 F.3d at 1258, and in Cline where the officer observed the truck "swerve" onto the shoulder of the road, nearly hitting a bridge abutment, 349 F.3d at 1287.
11
Here, as indicated above, the district court found that, similar to the situation in Ozbirn, Zabalza, and Cline, there were no adverse weather or road conditions that might have made it impractical for Alvarado to prevent his vehicle from drifting out of the righthand lane and over the fog line. Based on our review of the record, this finding is not clearly erroneous. Alvarado attempts to distinguish the circumstances of this stop from those present in the above-mentioned cases in that here, unlike in Cline, there were no "roadside structures that would have alerted him of the need to stay clear of the shoulder of the road," and, unlike in Ozbirn, "Alvarado's vehicle crossed the fog line only once." Appellant's Br. at 9.
12
We are unpersuaded that these distinctions warrant a different conclusion in this case. Alvarado has failed to point to any objective factor that might have made it impractical for him to remain in a single lane. Rather, his argument rests solely on the proposition that "[a] reasonable driver operating a motor vehicle at or near interstate speed limits has a difficult task of operating the vehicle entirely within a single lane for the entirety of his trip" and that "[v]ehicles traveling at interstate speeds, even with optimal road and weather conditions, do not typically stay in the exact center of the lane." Id. at 11. Essentially, Alvarado asks us to hold that an officer must observe something more than a single lane crossing in order to reasonably suspect a violation of Utah Code Ann. § 41-6-61(1) has occurred because any driver on the highway might inadvertently cross out of his lane once, regardless of the conditions that are present. However, as explained above, we have already rejected the argument that the "as nearly as practical" qualification in § 41-6-61(1) requires the conclusion, as a matter of law, that a single instance of crossing over the fog line can never violate the statute. Rather, as previously discussed, we understand it to require a fact-specific inquiry into the particular circumstances present during the incident in question in order to determine whether the driver could reasonably be expected to maintain a straight course at that time in that vehicle on that roadway.
13
Under the particular facts and circumstances of this case, where there is an utter absence of any weather conditions, road features, or other circumstances that could have interfered with Alvarado's ability to keep his vehicle in a single lane, we hold that Trooper Bowles had a reasonable articulable suspicion that Alvarado, by crossing one foot over the fog line, had violated § 41-6-61(1). The initial stop of Alvarado's Jeep was therefore reasonable under the Fourth Amendment. Because Alvarado raises no other objection on appeal, we therefore affirm the district court's order.
CONCLUSION
14
For the foregoing reasons, the district court's denial of Alvarado's motion to suppress is AFFIRMED.
Notes:
1
Although it has no relevance to our analysis in this case, we note that in 2005, Utah Code Ann. § 41-6-61 was renumbered and amended by changing its wording to active voiceSee Utah Code Ann. § 41-6a-710(1)(a). We refer herein to the provision as it existed at the time of the vehicle stop in question.
|
Q:
Compiler for C++ 17
Is there any online place where I can run a C++17 code, as I want to learn the new features of C++ 17. I tried running it on inbuilt Coliru compiler GCC 6.1 C++ 17. But it is unfortunately not compiling.
Is the compiler for C++17 not yet out, I searched it everywhere on Internet?
A:
wandbox supports g++ 7.0 SVN and clang++ 4.0 SVN versions, which have (experimental) C++17 feature support.
gcc C++1z support
clang C++1z support
A:
For most of the compilers c++17 isn't fully implemented yet. You can try using clang with flag -std=c++1z.
List of all availible functionalities can be found at http://clang.llvm.org/cxx_status.html.
|
Walmart sells its UK Asda business to hone its focus on competing with Amazon - Eurongreyjoy
https://techcrunch.com/2018/04/30/walmart-retreats-from-its-uk-asda-business-to-hone-its-focus-on-competing-with-amazon/
======
edf13
Not read the full article above - but I'm aware that Walmart is keeping circa
40% ownership in the final merged group... that's if the competition authority
in the UK gives it the go-ahead (Which is a big hurdle).
|
Q:
Ember RESTAdapter with custom JSON format
I'm currently enjoying diving into Ember and learning a complete Front End MVC solution.
Currently, I'm a little stumped on getting data from our API as it doesn't follow the standard required by Ember's Adapter. Currently, our team are unable to change the structure of the API / JSON response due to the dependencies of third party applications.
RESTAdapter is looking for JSON like this:
{
'episodes' : [
{
id : '1',
title : 'my first title'
}
]
}
Unfortunately all we get back is an array with each episode as an object, i.e. it cannot be a key with a value of array of objects.
[
{
id : 1,
title : 'my first title'
},
{
id : 2,
title : 'my second title'
}
]
Can anyone provide assistance on how to extend DS.RESTAdapter to follow this format?
Again, our server devs cannot change the API so we have to take the JSON response as is.
Thanks
A:
Override extractArray in your serializer. You can then modify the payload to match what ember data is looking for.
App.ApplicationSerializer = DS.RESTSerializer.extend({
extractArray: function(store, type, payload){
var plural = Ember.String.pluralize(type.typeKey),
fixed = {};
fixed[plural] = payload;
return this._super(store, type, fixed);
}
});
Example: http://emberjs.jsbin.com/OxIDiVU/953/edit
|
Q:
Byte Stream Unit Test
I'm utilizing the Reporting Services web service to generate a report and allow the user to download it. This is occuring via PDF, Excel, XML, etc. and working just fine. I'm trying to create some seperation between my reporting class and the implementation, but am struggling with how I can can do this in a manor that is still testable.
Since my custom Reports Object/Class is calling the web service directly, should I seperate this out even further with the use of interfaces? Any reccomendations on this and how it would still be unit testable regardless of the byte source would be much appreciated.
A:
Russell, I think your answer lies in learning about Dependency Injection/Inversion of Control. You might start here...
http://codebetter.com/blogs/jeremy.miller/archive/2005/10/06/132825.aspx
http://misko.hevery.com/2009/01/14/when-to-use-dependency-injection/
http://iridescence.no/post/Using-Unit-Tests-to-Uncover-Design-Flaws.aspx
http://en.wikipedia.org/wiki/Dependency_injection
http://martinfowler.com/articles/injection.html
|
Terpenes are a large and structurally diverse family of natural products that range from simple hydrocarbons associated with flavors and fragrances, to complex, highly oxidized polycyclic molecules such as the anti-malarial drug artemisinin, and the anticancer compounds ingenol and taxol. Although terpenes are isolated from natural sources, it can be challenging to translate their biological activity into a practical application. In some cases, the hurdle is low natural abundance; other times, it is the difficulty encountered by chemists seeking to precisely edit a terpene's molecular structure in order to improve its drug-like properties or interrogate its role in modulating disease pathways. The development of concise chemical syntheses of terpenes can transform the ability to use these molecules and their synthetic derivatives as biological probes or as lead compounds for the development of new medicines. Furthermore, these scientific efforts often innovate chemical reactivity or synthetic design concepts.
The natural product ryanodine (1) and its hydrolysis product ryanodol (2) are among the most highly oxidized and synthetically challenging diterpenoids reported to date.
Isolated from the tropical shrub Ryania speciosa Vahl in connection with its insecticidal properties, ryanodine is the namesake ligand of the ryanodine receptors (RyRs), an important family of ion channels that regulate intracellular Ca2+ release and play a critical role in signal transduction. In mammalian cells, these receptors exist in multiple isoforms (RyR1, RyR2, and RyR3) that serve to mediate both movement and cognitive function. Mutations of RyRs are associated with genetic diseases such as malignant hyperthermia and central core disease, while altered expression of RyR2 and RyR3 has been associated with the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. Ryanodine binds with high affinity to the conducting state of RyRs, exerting concentration dependent modulation of Ca2+ release: at nanomolar concentrations, ryanodine locks RyRs in an open, subconductance state, whereas at higher concentrations, ryanodine causes closure of the channels. The deacylated compound ryanodol binds with lower affinity than 1 to mammalian RyRs; however, it still induces a subconductance state and has been reported as a reversible probe of RyR-mediated Ca2+ release in cells.
Since the initial reports describing the isolation of ryanodine from Ryania, a number of congeners (known as ryanoids) that vary in oxidation pattern have been isolated. Whereas ryanodol—the compound obtained by hydrolysis of ryanodine—has not yet been isolated directly from a natural source, the closely related compound C3-epi-ryanodol (4) was isolated by Gonzaléz-Coloma from Persea indica.
Indeed, due to their structural similarities, C3-epi-ryanodol (4) was initially erroneously reported to be ryanodol (2); however, the structure of the Coloma-Gonzaléz isolate was recently reassigned through the synthetic efforts of Inoue. (M. Koshimizu et al, Angew Chem. Int. Ed. Engl. 55, 2493-2497 (2016)). These subtle differences in structure exert a pronounced effect on RyR-binding: C3-epi-ryanodine (5), prepared from 4, binds 100 fold more weakly to RyRs than 1. (W. Welch et al. Biochemistry 36, 2939-2950 (1997)).
Given the biological importance of the RyRs, the ryanoids have been the focus of both total synthesis and derivatization efforts. (A. Belanger et al. Can. J. Chem. 57, 3348-3354 (1979); P. Deslongchamps et al. Can. J. Chem. 68, 115-126 (1990); P. Deslongchamps et al. Can. J. Chem. 68, 127-152 (1990); P. Deslongchamps et al. Can. J. Chem. 68, 153-185 (1990); P. Deslongchamps et al. Can. J. Chem. 68, 186-192 (1990); M. Nagatomo et al. J. Am. Chem. Soc. 136, 5916-5919 (2014); M. Nagatomo et al. Chemistry 22, 222-229 (2016); K. Masuda et al. Chemistry 22, 230-236 (2016); A. L. Waterhouse, et al. J. Med. Chem. 30, 710-716 (1987); W. Welch et al. Biochemistry 33, 6074-6085 (1994); J. L. Stuko et al. Pharmacol. Rev. 49, 53-98 (1997)). These synthetic efforts, however, include up to 41 steps.
Alternative routes of preparing (+)-ryanodol and ryanodol, as well as derivatives thereof, are needed |
Now Available With Steam Workshop - Scribblenauts Unlimited |
Q:
Can "à moins que" be used as a subtle, indirect way to ask for something?
I’m not sure how the following sentences sound, compared to straightforwardly asking someone to do something with "can you, will you, please" etc.
Je vais aller à la gare à pied.
À moins que vous ne me conduisiez là ?
À moins que vous n'ayez l'amabilité de me conduire là ?
À moins que vous ne me conduisiez là...
À moins que vous n'ayez l'amabilité de me conduire là...
A:
First, ne me conduisiez là is not idiomatic, that should be ne m'y conduisiez.
With à moins que vous me conduisiez là, you are somewhat rude because it is very clear you are asking but you are missing a formule de politesse. I wouldn't say that unless I'm familiar with the listener.
You are more polite with adding vous n'ayez l'amabilité but depending on the context, it might be "too formal".
This is how I would ask using à moins que :
Je vais aller à la gare à pied à moins que vous puissiez m'y conduire. (formal)
Je vais aller à la gare à pied à moins que ça ne vous/te dérange pas de m'y conduire. (informal)
|
Q:
Javafx: Reusable collections using FXML
I'd like to bind a single collection to multiple ChoiceBox's in FXML.
However the only way I know how is using:
<ChoiceBox fx:id="cb00" prefWidth="150.0" GridPane.rowIndex="0" GridPane.columnIndex="0">
<items>
<FXCollections fx:id="test" fx:factory="observableArrayList">
<String fx:value="1" />
<String fx:value="2" />
<String fx:value="3" />
<String fx:value="4" />
<String fx:value="5" />
<String fx:value="6" />
<String fx:value="7" />
<String fx:value="8" />
<String fx:value="9" />
</FXCollections>
</items>
</ChoiceBox>
Is it possible to declare the collection in the controller and refer to it in FXML instead of copying the collection for each ChoiceBox?
A:
You can define the items in the controller:
public class Controller {
private ListProperty<String> choiceBoxItems = new SimpleListProperty(FXCollections.observableArrayList());
public Controller() {
IntStream.range(1,10).mapToObj(i -> Integer.toString(i))
.forEach(choiceBoxItems::add);
}
public ListProperty<String> choiceBoxItemsProperty() {
return choiceBoxItems ;
}
public ObservableList<String> getChoiceBoxItems() {
return choiceBoxItemsProperty().get() ;
}
public void setComboBoxItems(ObservableList<String> choiceBoxItems) {
choiceBoxItemsProperty().set(choiceBoxItems) ;
}
// ...
}
and then (this is not tested, but I think it will work):
<ChoiceBox fx:id="cb00" items="${controller.choiceBoxItems}" prefWidth="150.0" GridPane.rowIndex="0" GridPane.columnIndex="0">
See expression binding in the FXML documentation. (It's not actually documented that the controller is available in the FXML namespace with key controller, but I think it is safe to use this.)
You can also just define the list in the FXML using fx:define:
<fx:define>
<FXCollections fx:id="choiceBoxItems" fx:factory="observableArrayList">
<String fx:value="1"/>
<String fx:value="2"/>
<String fx:value="3"/>
<!-- ... -->
</FXCollections>
</fx:define>
and then refer to it in each choice box:
<ChoiceBox fx:id="cb00" items="${choiceBoxItems}" prefWidth="150.0" GridPane.rowIndex="0" GridPane.columnIndex="0">
|
Ventilatory compensation for continuous inspiratory resistive and elastic loads during halothane anesthesia in humans.
Inspiratory mechanical loads were applied to the airway continuously for 5 min in healthy young adult volunteers maintained in a near steady-state of halothane anesthesia 1.1 MAC. The loads, both flow resistive and elastic in nature, had been selected to reduce the first loaded tidal volume approximately 10, 30 or 50%--these being designated "small," "medium," and "large" loads, respectively. The actual magnitudes of resistive load were 8 +/- 1, 21 +/- 3, and 48 +/- 6 cmH2O X l-1 X s, and of elastic load 6 +/- 1, 18 +/- 1, and 41 +/- 5 cmH2O X l-1 (mean +/- SEM). All loads caused an immediate reduction of ventilation proportional to the size of the load. This was followed by a gradual recovery of ventilation toward control values over approximately 2 min and then nearly stable ventilation for the rest of the loading period. Respiratory frequency was unchanged throughout. At 5 min of loading, ventilation and PaCO2 had been nearly steady for 3 min and O2 uptake and CO2 output at the airway were unchanged from control, suggesting the establishment of a near steady respiratory state. With the small and medium loads of both types, ventilation and PaCO2 in this near steady-state were not detectably different from control. With the large loads, however, ventilation was significantly reduced and PaCO2 slightly increased. The end-expiratory position of the chest wall and the relative contributions of the rib cage and abdomen-diaphragm to ventilation, as estimated by anteroposterior chest wall magnetometers, were not consistently altered by any load.(ABSTRACT TRUNCATED AT 250 WORDS) |
<?xml version="1.0" encoding="utf-8"?>
<Project ToolsVersion="12.0" DefaultTargets="Build" xmlns="http://schemas.microsoft.com/developer/msbuild/2003">
<Import Project="$(MSBuildExtensionsPath)\$(MSBuildToolsVersion)\Microsoft.Common.props" Condition="Exists('$(MSBuildExtensionsPath)\$(MSBuildToolsVersion)\Microsoft.Common.props')" />
<PropertyGroup>
<Configuration Condition=" '$(Configuration)' == '' ">Debug</Configuration>
<Platform Condition=" '$(Platform)' == '' ">AnyCPU</Platform>
<ProjectGuid>{F831A8E1-4494-41DA-B219-6D9A64E67DF2}</ProjectGuid>
<OutputType>Exe</OutputType>
<AppDesignerFolder>Properties</AppDesignerFolder>
<RootNamespace>RobustGuy</RootNamespace>
<AssemblyName>RobustGuy</AssemblyName>
<TargetFrameworkVersion>v4.5</TargetFrameworkVersion>
<FileAlignment>512</FileAlignment>
</PropertyGroup>
<PropertyGroup Condition=" '$(Configuration)|$(Platform)' == 'Debug|AnyCPU' ">
<PlatformTarget>AnyCPU</PlatformTarget>
<DebugSymbols>true</DebugSymbols>
<DebugType>full</DebugType>
<Optimize>false</Optimize>
<OutputPath>bin\Debug\</OutputPath>
<DefineConstants>DEBUG;TRACE</DefineConstants>
<ErrorReport>prompt</ErrorReport>
<WarningLevel>4</WarningLevel>
</PropertyGroup>
<PropertyGroup Condition=" '$(Configuration)|$(Platform)' == 'Release|AnyCPU' ">
<PlatformTarget>AnyCPU</PlatformTarget>
<DebugType>pdbonly</DebugType>
<Optimize>true</Optimize>
<OutputPath>bin\Release\</OutputPath>
<DefineConstants>TRACE</DefineConstants>
<ErrorReport>prompt</ErrorReport>
<WarningLevel>4</WarningLevel>
</PropertyGroup>
<ItemGroup>
<Reference Include="System" />
<Reference Include="System.Core" />
<Reference Include="System.Xml.Linq" />
<Reference Include="System.Data.DataSetExtensions" />
<Reference Include="Microsoft.CSharp" />
<Reference Include="System.Data" />
<Reference Include="System.Xml" />
</ItemGroup>
<ItemGroup>
<Compile Include="Program.cs" />
<Compile Include="Properties\AssemblyInfo.cs" />
<Compile Include="RobustGuy.cs" />
</ItemGroup>
<ItemGroup>
<None Include="App.config" />
</ItemGroup>
<Import Project="$(MSBuildToolsPath)\Microsoft.CSharp.targets" />
<!-- To modify your build process, add your task inside one of the targets below and uncomment it.
Other similar extension points exist, see Microsoft.Common.targets.
<Target Name="BeforeBuild">
</Target>
<Target Name="AfterBuild">
</Target>
-->
</Project> |
Q:
Cannot assign value of type ‘ViewController‘ to type ‘GADInterstitialDelegate?‘
I have looked around the answers but I cannot seem to find one for GADInterstitialDelegate
I am following the tutorial: https://developers.google.com/admob/ios/interstitial but all I get is this error:
Cannot assign value of type ‘ViewController‘ to type ‘GADInterstitialDelegate?‘
I have deleted the project and started again. Can anyone help?
A:
Taken from: Swift AdMob Tutorial
Reason
This means that your ViewController does not conform to the type GADInterstitialDelegate
Solution
Change your ViewController Class declaration from :
class ViewController: UIViewController {
To this:
class ViewController: UIViewController, GADInterstitialDelegate
|
Minimalism & The Pursuit of Happiness
When my wife and I first started decluttering our house, it felt incredible.
With each unneeded item we donated, recycled, or took out of our home, we felt lighter and experienced new space opening around us. Over the next several months, a few thousand items went out the door. The contrast was stark: surfaces were clear and our home felt more welcoming than ever.
Coming home after a long day at work became something we looked forward to—a peaceful sight rather than reminders of things to be picked up. Cleaning up before having guests over, what used to take us a couple hours, took us only ten minutes, which subsequently increased the frequency of invitations and time spent with friends.
However, a few months after making the bulk of the changes, the glaringly positive effects began to fade.
Our lives were still significantly easier due to the streamlining, but the happiness and satisfaction we felt as a result of the minimizing process seemed to return back to normal.
At first, we wondered if we didn’t fully complete the appropriate amount of downsizing. Did we need to continue donating, selling, and recycling to get that happiness boost again? It might solve the problem, at least temporarily, but even that process would meet an eventual end once we ran out of things we didn’t want. We’d have to face the “Now what?” sooner or later.
We were experiencing “hedonic adaptation,” the observed tendency of humans to quickly return to a relatively stable level of happiness despite major positive or negative changes. We had grown accustomed to less time spent on cleaning and organizing; other activities began to fill in the gaps—some productive and some not so much.
After reading extensively about the joy that minimalism brings, we found this frustrating. We had seemingly arrived at our desired destination but didn’t feel that our journey was complete. There was a missing piece to the puzzle.
What we found was that other distractions gradually crept into our lives as we freed up time from other tasks. No matter how much time we saved, it wouldn’t be helpful unless we used that time efficiently. Hours spent on a smartphone throughout the day did not add to our happiness. But working on a project, simply being still, reading for pleasure, or learning about something new did continually make our lives better.
It became clear the pursuit of satisfaction in life is a daily practice that never ends, and that’s for the best.
The purging process eventually reaches a point of winding down, but the gratitude and contentment that comes from appreciating all that we have should never stop. The calming of our desire for what we don’t need is an enduring task that continues to this day.
Getting rid of the clutter in our lives created more opportunity for us to pursue the things we’re passionate about. But it was still up to us to make the most of the opportunity.
For us, that means traveling, spending time with people we care about, dedicating time to our passion projects, creating more, and consuming less. What brings the greatest satisfaction in life will differ with each person. But the important part is we continually pursue them. It is with this intentional, consistent pursuit that happiness will follow.
***
Anthony Ongaro blogs and vlogs at Break the Twitch where he helps others explore ways to live a more intentional life. I also recommend following him on Twitter.
About Joshua Becker
Writer. Inspiring others to live more by owning less.WSJ Bestselling author of The More of Less.
Comments
“Getting rid of all the clutter in our lives created more opportunity for us to pursue the things we’re passionate about. But it was still up to us to make the most of the opportunity.”
That is both literally and metaphorically the meaning of pursuing happiness: make the time and space for the things you want and need, and leave all else aside. I wrote about struggling to do just that, as a new mum of two, and how inevitably some things are left aside, including ironing (!) and spending time with people who don’t ‘add’ to my life. http://goo.gl/qHzlCh
Within the last couple of weeks I’ve gotten rid of 5 large bags of things that no longer brought me joy or use. It’s felt great…and I am now on a roll. My summer project is to whip this house into shape. It looks so much better already :) I’ve had to replace a few things (old curtains and bedding) — I hate being a consumer, but certain things do need to be replaced from time to time. I’ve never gotten to the point Anthony, where I say…”OK, I’m done. What now”? I doubt that I ever will. For me it seems to be a constant pursuit. I love the journey. Just love it!!! :)
I live in a 500 square foot condo, which is a great way for me to work on minimalism. As I refuse to get a storage locker, I have to fit everything I own in this condo. I can’t bring in items that are redundant or useless. Everything has to have a space for it, and it has to be used within 3 months or donated. Everyone I know tells me that eventually I will have to move into a bigger space so I can fit in the stuff that I will eventually get.
They don’t understand that I deliberately choose less everyday, so I can not only have a more fulfilling financial life, but more time, more energy, and less hassle of taking care of my stuff.
You have the perfect life! I live with my bf in a 2 bedroom apt & may have to downsize to 1 bedroom & although I am a minimalist, I’m afraid if I downsize some more & get rid of the extra yet loved peices, I won’t ever want a bigger place! It’s good to have that problem though, isn’t it?! So good of you not to get a storage unit & to continue & always have less! Keep doing what you’re doing, you have the perfect life!
Good for you for not indulging in a storage locker/room! I had friends who paid $100/mo. for one for 30 years! You do the math! When it was finally decided upon to clean out all the family treasures, they were broken, rotted, etc. = Worthless to anyone. And these were not people who could afford to waste that amount of money.
I so agree! We went overseas for two years and stored out things and it’s taken 1.5 years since we have been back to get rid of the lock up and we said it would only be 3 months :/. The upside is that not living with the extra ‘stuff’ for 3.5 years means we are now less attached to it (and can”t remember half of it!). We’ve finally let go of the lock up and that’s $150 per month saved but we’ve brought 7 boxes back with us. I need to look more positively at what I have got rid off but sometimes all I can see is ‘stuff’ and, now, we are down to the curly stuff. Mementoes, photos, ‘precious things’, letters and it’s like wading through mud – it takes ages and I just seem to move it around rather than OUT. The wardrobes and books were easier than this!
Thank you so much for this post! I’ve been a ‘middle class’ minimalist (What I call it) for years now & every time I think I’m happy, I keep trying to find things to get rid of! Why? Because I need that high! This post made me realize that I’m not not happy because I have more to get rid of, I’m not happy because I want that continued high of getting rid of items. And this post has also shown me that instead of looking for items, I should fill my time with more hobbys I enjoy & to just sit sometime & take it all in.
This post couldn’t have been more timely for me, Anthony. After getting rid of 65% of my belongings, you’d think I’d feel satisfied with my progress – and I did for a while – but then I found myself looking around and wondering if I should get rid of more. That wonderment was definitely coming from a place of want: wanting to feel, once again, how good it felt to get rid of so much stuff in the first place; wanting to remember how good it felt to realize I could live with less – and even be happier for it. Eventually, I came to a similar conclusion: that I need to stop chasing that feeling and instead just appreciate how good life is these days. Now I’m even more excited to chat tomorrow. :)
This is EXACTLY what happened with me. After downsizing and decluttering to a wonderful level, I found myself with a lot more time and energy, but didn’t really know what to do with it! What a great problem to have, but it was one which took some intentional thought in order to move out of that stage. It still amazes me just how much the minimalist mindset can open up for a person and a family by removing the unnecessary.
Great post! I feel like my heart and mind are constantly telling me to simplifiy but I struggle to actually do it. I think I start believing I already have little but I know I can have much less…and we would be more than okay.http://www.sweetlytattered.com
This reminds of me of stories you hear of people who retire and then don’t know what to do with themselves. They think that not working will bring happiness, and initially it does. Then the novelty wears off. This is something that I have been thinking about as we simplify. If acquiring all this stuff didn’t make me happy, it is unlikely that getting rid of it will bring long term happiness either. Getting rid of my stuff will eliminate all the onerous tasks involved in maintaining my stuff, which will be awesome, but that is not a substitution of long term happiness or life purpose. So hopefully I will find those answers in the open spaces left behind.
My wife and I are retired 63 year old global nomads. We allow ourselves one suitcase and one backpack each. If it doesn’t fit into one suitcase then we probably don’t need it. We travel the world staying one year each in a different country. Two more weeks in Turkey and then we are off to Belize. Living life the way it was meant to be lived.
Thank you, Anthony, for saying something that I’ve thought about for a long time but haven’t seen addressed in minimalist blogs: that the buzz that comes from letting things go quickly fades. I do experience an ongoing satisfaction from living with less, but it’s more a quiet contentment now. The truth is, clutter can be a barrier to happiness, but its removal does not necessarily guarantee it. Thanks for voicing this important idea.
I loved this! I’ve been spending way too much time on my smart phone and laptop lately and actually find a direct correlation with how much time I spend on it with how happy I feel at any given time. It can be easy to passively take part in those activities, but it definitely doesn’t leave us feeling good, productive, or happy!
I agree i have been getting rid of things for years and yes it does feel great in the beginning and i felt it consumed me to keep on and on with it, trying different numbers ie 10 items this week etc, but i soon realised that life holds much more than that, but i also know that even though i know a lot of truths about possessions we need things and as long as they don,t control us then things are fine, i just like neat and tidy but the unpredictable is always lurking isn,t it
love Jacqueline
Love this post. I have found a lot of joy and peace in getting rid of the unnecessary junk in my life. One of the biggest things that brings me joy in my life is decluttering the amount of virtual and digital clutter that I have.
Too true! The cycle of consumerism trains us to keep seeking instant gratification and it takes self awareness and a commitment to break the cycle and seek sustainable satisfaction to overcome it. It was a struggle for me as well until I realized that I didn’t have purge and reorganize everything at once. Some items like clothing I did in a day or two, other things like kitchen items and cosmetics I did over weeks/months. In the meantime I kept a list of things I would finally be able to do as I cleared stuff out and didn’t have to tend to it anymore. It helped me stay focused and appreciate how far I had come.
Oh so that’s how it works! I still haven’t gotten to the part where the house is streamlined. Actually, it is on the surface, but please don’t open a closet! Thank you Anthony for the inspiration and sage advise.
We’re the opposite, Sunny. We have a little less that’s visible, but we were pretty neat before, so it’s not a big change. What is enormously different for us is that, now, when you open a closet or a cupboard or look a a shelf, most are much emptier than before.
This post really caught my attention, thank you Anthony. Two aspects in particular struck me:
*I love that with less cleaning up to do, you found that you increased the frequency of invitations to friends – that’s such a great and unexpected bonus of de-cluttering.
* You’ve described the slight roller coaster of emotions you went through – I’m intrigued as to what the time frame was for your ‘clear space, clear brain euphoria to level out?
I “minimalized” and redecorated my guest room a couple of years ago. For a while I found myself going in there for a mini vacation from clutter in the rest of the house. Now I don’t do that anymore.
Though I purged a couple hundred items of clothing from my closet recently, the momentum hasn’t carried me to the rest of the house. Maybe just the commitment to cease acquiring and the occasional giveaway will bring more contentment, if not a real “high.”
I loved this post. It’s important to be aware of that transition between decluttering the stuff and creating meaning out of the space and time left behind. I copied (and linked) a big chunk of this on my own blog post for today:) Thank you!
Plateau reached – what do I do now? Every spare hour, or 1/2 day for the last 8 months has been spent on decluttering – the thrill of throwing something away is addictive. Now my gaps feel large – not the physical gaps, but the gap of time – what does my heart desire to do? I’m quite scared of finding something to do – and having to buy the materials to do it with … And then ending up back where I came from.
I am so totally here as well. Still looking around in our house after more things to get rid of, but seldomly finding any. The feeling of “now what” is definitely there. And some days indeed, spending way too much time distracting myself on my smartphone and not so useful things. Trying to organize myself though to spend time on things that are more meaningful.
Thanks for letting me know I´m far from being alone in this feeling :-)
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The history of cardiopulmonary resuscitation.
The development of modern cardiopulmonary resuscitation (CPR) is an exciting and surprising history to modern health professionals who rarely are aware of how new CPR really is. Artificial respiration began in the 16th century with Vesalius's work on living animals; progressed with the rise and fall of mouth-to-mouth, manual, and positive pressure ventilation methods of the 18th and 19th centuries; and culminated in 1958 with demonstration of the superiority of the mouth-to-mouth technique. Cardiac massage began in 1874, with the open chest method gaining ascendancy until the 1960 demonstration of the equality and greater ease of closed chest cardiac massage. Electrical defibrillation may have begun in 1775, but was not proven successful in animals internally until 1899. The technique was applied to man internally in 1947 and externally in 1956. The simultaneous use of all these modern CPR methods dates back only 20 years. |
257 Cal.App.2d 117 (1967)
CAROLYN H. SCHOENFELD et al., Plaintiffs and Respondents,
v.
DONALD M. PRITZKER et al., Defendants and Appellants.
Civ. No. 792.
California Court of Appeals. Fifth Dist.
Dec. 18, 1967.
Mack, Bianco & Means and H. C. Mack, Jr., for Defendants and Appellants.
Siemon & Patterson and Bennett Siemon for Plaintiffs and Respondents.
CONLEY, P. J.
The plaintiffs, Carolyn Schoenfeld, Rosalie J. Levy and Clarisse Haberfelde Main, are the owners of the northwest quarter of section 17, township 30 south, range 30 east, M. D. B. & M., while the defendants, Donald M. Pritzker and Janet Wyman own adjoining land, namely, the northwest quarter of the northeast quarter of the same section, township and range. The plaintiffs were successful in their suit to quiet their title as against the defendants and the *118 latter appeal. The strip of land in question contains roughly six acres and is now planted to orange trees. The essential elements here involved consist of the fact that for many years without specifically claiming the intervening strip of land adversely to the plaintiffs the defendants and their predecessors used the parcel to raise annual crops without objection by the plaintiffs. However, the defendants never paid taxes on the land and there was never an agreement express or implied between the owners that there should be erected a division fence, or that a pipeline or farm road installed unilaterally by the plaintiffs' predecessors on their own land should constitute a borderline as between the two parcels.
We are bound, of course, by the findings of fact made by the trial judge which actually had ample substantial evidence in their support, and which include the following: (1) "At some time during the year 1939, plaintiffs caused a pipeline to be constructed from the North line of said section running in a southerly direction" which pipeline "was located approximately 200 feet westerly of the North-South center line of said section"; (2) "After the construction of such pipeline defendants occupied the land lying 30 feet easterly of such pipeline by planting and harvesting yearly crops thereon"; (3) "Defendants did not attempt to construct any permanent improvements upon such strip of land until the summer of 1963, at which time they prepared the land for the planting of orange trees"; (4) "Plaintiffs, on June 11, 1963, immediately upon being advised of such acts, gave defendants notice in writing that they were encroaching upon plaintiffs' land and demanded that defendants desist from such action"; (5) "Defendants did not pay any taxes levied or assessed upon the land, or any portion thereof, lying easterly of the pipeline and westerly of the North-South center line of said section"; (6) "Defendants occupied said strip under the mistaken belief that the pipeline was located on the boundary and defendants did not intend to claim title to any property they did not own"; (7) "Neither plaintiffs nor defendants were uncertain as to the location of said boundary line ..."; (8) "... plaintiffs and defendants did not enter into an agreement fixing the boundary line at the pipeline or at any other point"; (9) "The use of the land belonging to plaintiffs by defendants was under a mistake on the part of the defendants that the pipeline had been constructed by plaintiffs on the boundary between the two parcels"; (10) "Plaintiffs did not at any time make any false statements and did not conceal *119 any material fact from defendants as to the location of the boundary line ..."; (11) "... plaintiffs notified defendants of the encroachment upon learning of defendants' actions in commencing to place permanent improvements consisting of orange trees upon plaintiffs' property; that such notification was given promptly and before defendants had suffered substantial damage ..."; (12) "... that the true boundary was ascertainable and that defendants' act in planting such trees upon said strip of land was done and performed with knowledge of plaintiffs' claims"; (13) "Defendants' occupation of said strip of land was under and subordinate to plaintiffs' legal title."
As conclusions of law the court held that the plaintiffs were owners of their quarter section of land and that the boundary line between the parcels of land owned by the plaintiffs and defendants is the north-south center line of said section, being a straight line between the north quarter corner and the south quarter corner; that defendants' occupancy of the easterly 200 feet of the northwest quarter of section 17 was without title or right; that plaintiffs' action is not barred by the provisions of section 318 of the Code of Civil Procedure or any other statute of limitations, and that plaintiffs are not estopped or barred by laches from asserting title to the whole of their quarter section of land; that no boundary other than the true boundary was established between the plaintiffs and defendants under the doctrine of agreed boundaries or otherwise, and that the defendants have not acquired title to any portion of the northwest quarter of section 17, township 30 south, range 30 east, M.D.B. & M. by adverse possession or otherwise. Consequently, the title of plaintiffs to the whole of their quarter section of land was quieted by the decree.
[1] The principal defense urged by the defendants is that respondents are barred by the provisions of section 318 of the Code of Civil Procedure which hold that: "No action for the recovery of real property, or for the recovery of the possession thereof, can be maintained, unless it appear that the plaintiff, his ancestor, predecessor, or grantor, was seized or possessed of the property in question, within five years before the commencement of the action."
Defendants cite Townsend v. Driver, 5 Cal.App. 581 [90 P. 1071], and Haney v. Kinevan, 73 Cal.App.2d 343 [166 P.2d 361], to the alleged effect that plaintiffs' action is an action to quiet title, and, consequently, that it is for the recovery of real property within the meaning of section 318 of the Code *120 of Civil Procedure, and that under the holdings of Cocking v. Fulwider, 95 Cal.App. 745 [273 P. 142], Sibbett v. Babcock, 124 Cal.App.2d 567 [269 P.2d 42], and Ernie v. Trinity Lutheran Church, 51 Cal.2d 702 [336 P.2d 525], because defendants were in open possession of the disputed strip from 1939 and that because defendants refused to assent to what Mr. Haberfelde called the "true surveyed line," they were in hostile possession of the strip of land for more than five years and the statute of limitations should apply to bar the plaintiffs' action.
The Haney case, supra, placed the burden of proof on plaintiff in a quiet title action, and the Cocking case, supra, denied a legal owner a decree quieting title when the defendant proved actual possession for 30 years, even though defendant was unable to establish title by adverse possession because of his failure to pay taxes. The Cocking case sounds impressive; however, it was criticized in 17 California Law Review 390, 393-394, where it is stated: "In the recent case of Cocking v. Fulwider, however, the California District Court of Appeal considered, among other grounds for refusing to quiet title in the plaintiff, the effect of Section 318 of the Code of Civil Procedure, and held that, since the language of the section required 'seisin or possession' by the plaintiff within five years before the commencement of the action, the plaintiff could not recover, although he was, and he and his predecessors for thirty years had been, in actual possession of all the property except the disputed boundary strip, and although he had legal title to the entire tract. It was admitted that the defendant had acquired no title to the disputed strip, because neither he nor his predecessors had ever paid taxes on it as required by Section 325 of the Code of Civil Procedure. The decision appears to rest upon a very narrow interpretation of the expression 'seised or possessed' as used in the Code. It seems that the court here requires actual possession or occupation of the property by the plaintiff within five years preceding the bringing of the action in order that he be allowed to bring it at all. When, however, this case is considered in the light of other decided cases, both in California and in other states having similar statutes, the question arises whether the expression 'seised or possessed' as used in the Code does not include the theoretical possession of a legal owner whose property is not in the adverse possession of another. Although the legal owner may not be in the actual occupation of his land, is he not still to be regarded as 'seised or possessed' so *121 long as no one else is actually holding adversely to him?" (Italics added.)
Witkin states that the correct rule is that "seisin" or the right of possession is in the legal owner, even while an adverse claimant is actually occupying the land, and a showing of legal title is enough to satisfy section 318 of the Code of Civil Procedure. That work cites People's Water Co. v. Boromeo, 31 Cal.App. 270 [160 P. 574]; Westphal v. Arnoux, 51 Cal.App. 532 [197 P. 395]; Philp v. Hobart, 103 Cal.App.2d 446, 448 [229 P.2d 783]; and Clark v. Stotts, 127 Cal.App.2d 589 [274 P.2d 172], in support of the rule as announced. (2 Witkin, Summary of Cal. Law (1960) Real Property, 24, p. 880.)
In Balestrieri v. Sullivan, 142 Cal.App.2d 332, 340-341 [298 P.2d 688], it is said: "It is urged that in any event, because of appellants' long continued possession of the strip, respondents cannot quiet title, for they must show that they were seized or possessed of the property within five years preceding the action. (Code Civ. Proc., 318.) Recovery was denied in Cocking v. Fulwider, 95 Cal.App. 745 [273 P. 142], where defendant proved actual possession for 30 years, although defendant was unable to get title by adverse possession because of failure to pay taxes. In that case there was evidence that in the year 1888 the adjoining owners had agreed upon the common boundary, that this boundary had been acquiesced in for more than 30 years by the parties and their predecessors in title. In 1917 a new survey was made under which appellant claimed title to a strip of land beyond the old boundary."
"In People's Water Co. v. Boromeo, 31 Cal.App. 270 [160 P. 574], an ejectment action, it was held that findings that defendants were in open, notorious possession of the land for a period of five years preceding the bringing of the action, and that plaintiffs were seized of said land within the last five years as required by section 318, Code of Civil Procedure, where the defendants had not paid the taxes for the requisite years, were not inconsistent. It was said that seisin of plaintiff in the premises was the right of possession which could only be destroyed by defendants' compliance with the requirements for establishing title by adverse possession, and that section 318 is to be read in connection with sections 322, 323 and 325 of the same code which set forth the legal prerequisites for such title by adverse possession as would defeat plaintiff's seisin in law. In Westphal v. Arnoux, 51 Cal.App. *122 532, 534 [197 P. 395] an ejectment action, it was said that the requirement of section 318, Code of Civil Procedure, was satisfied when it was shown that plaintiff had legal title, and that the burden was then placed on defendant to show that he had held the property adversely for five years. In the recent case of Madden v. Alpha Hardware & Supply Co., 128 Cal.App.2d 72, 75 [274 P.2d 705], it is said that plaintiffs having shown legal title in themselves 'it is presumed that they were possessed of the property within the time required by law and that the occupation thereof by defendant is deemed to have been in subordination of plaintiff's title, unless it be shown that defendant's possession was adverse for the statutory period.' (And see Wilkerson v. Thomas, 121 Cal.App.2d 479, 486 [263 P.2d 678].)" (Italics added.)
Plaintiffs correctly argue that the authorities hold that section 318 must be read in connection with those sections which set forth legal prerequisites for obtaining title by adverse possession, otherwise, the record owner would be unable to recover possession, and a possessor would be unable to establish title and the rule that the right of possession is an incident of title would be destroyed. In Labory v. Los Angeles Orphan Asylum, 97 Cal. 270, 273 [32 P. 231], the court comments that " 'The rule is well settled that title draws to it the possession, and it remains with the owner of the legal title until he is divested of it by an actual adverse possession; ...' " The finding that appellants did not pay any taxes upon the strip, has not been attacked on the appeal; consequently, one of the essential elements of title by adverse possession is lacking, and the exception contained in Code of Civil Procedure, section 321, does not apply: "In every action for the recovery of real property, or the possession thereof, the person establishing a legal title to the property is presumed to have been possessed thereof within the time required by law, and the occupation of the property by any other person is deemed to have been under and in subordination to the legal title, unless it appear that the property has been held and possessed adversely to such legal title, for five years before the commencement of the action. (Enacted 1872.)" (Italics added.)
(See also Wood v. Henley, 88 Cal.App. 441, 460 [263 P. 870]; Nutting v. Herman Timber Co., 214 Cal.App.2d 650, 658 [29 Cal.Rptr. 754]; Clark v. Stotts, supra, 127 Cal.App.2d 589, 591; McKelvey v. Rodriquez, 57 Cal.App.2d 214, 223 [134 P.2d 870]; Ward Redwood Co., Inc. v. Fortain, 16 *123 Cal.2d 34, 42 [104 P.2d 813]; People's Water Co. v. Lewis, 19 Cal.App. 622, 625 [127 P. 506]; Sharp v. Daugney, 33 Cal. 505, 511; Borden v. Boyvin, 55 Cal.App.2d 432, 436 [130 P.2d 718]; Comstock v. Finn, 13 Cal.App.2d 151, 158 [56 P.2d 957].)
In its criticism of the Cocking case, supra, 17 California Law Review, pages 394-395 further states: "In Section 325 the acts and circumstances which constitute adverse possession are set forth; and it seems clear that unless these requirements are fully met by the one in actual occupation, he is not an adverse possessor within the meaning of the statute, and the legal owner should be considered as still 'seized or possessed' notwithstanding the actual occupation, not adverse, of the other. There is, at present, no single section in either the Civil Code or the Code of Civil Procedure which, by virtue of its separate provisions alone bars both the right and the remedy. In the Code of Civil Procedure, the remedy of the legal owner is barred by Section 318, which is the statute of limitation; Section 321, which should be read with Section 318, establishes the presumption of possession in the legal owner, unless such presumption is rebutted by the actual adverse possession, as defined in Section 325, of another; and in the Civil Code, Section 1007 confers title upon one who has occupied for the period which bars the legal owner of his remedy, subject, however, to the qualification that such occupation is not adverse unless it fulfills the statutory requirements."
These authorities support the trial court's holding that plaintiffs' action was not barred by the statute of limitations; appellants' possession was subordinate to respondents' title.
[2] The defendants next maintain that the court erred in not finding that there was an "agreed boundary." However, a review of the record makes it clear that the necessary elements to authorize an agreed boundary were totally lacking. To prove an agreed boundary there must be an uncertainty and an agreement shown. (See 2 Witkin, Summary of Cal. Law (1960) Real Property, 46, pp. 900-901, 47, pp. 901-902.)
Clapp v. Churchill, 164 Cal. 741, 746 [130 P. 1061], holds: "In the case under consideration plaintiff's evidence completely breaks down in its failure to show an uncertainty touching the boundary line which would support such an agreement for, as is said in Lewis v. Ogram [149 Cal. 505 (87 P. 60, 117 Am.St.Rep. 151, 10 L.R.A. N.S. 610)]: 'such an *124 agreement necessarily is not valid for any other purpose than that of settling an uncertainty in regard to common boundary.'plaintiff shows that he did not know where the true boundary line was until he caused his land to be surveyed when it was easily determined. But his uncertainty was not the defendants' uncertainty, and there is not the slightest evidence that they considered that their northern boundary line was uncertain in its location. Therefore, the acquiescence of the defendants in the acts of the plaintiffs in their exercise of dominion over the strip might make against them for their failure to assert their right, if title were claimed by adverse possession, a claim which has heretofore been said could not, in this instance, be sustained. But it is without meaning or potency under the contention of an agreed boundary line because, as has been said and shown, an agreement fixing a common boundary line can only have efficacy where the true boundary is either uncertain in fact or is believed by the contracting parties to be uncertain."
Mrs. Main, one of the present owners of the quarter section, testified that until 1963 all the crops on defendants' property had been annual, and there were no permanent improvements on the strip of land in question; she testified that plaintiffs never gave permission to appellants to use plaintiffs' land, that plaintiffs regularly paid all of the taxes on the quarter section, that annual crops as they were being produced on the strip were of little consequence and use of this small strip of land seemed unimportant until appellants began planting a permanent crop, and at that time plaintiffs instructed their attorneys to make a demand upon appellants to remove themselves from the premises.
In 8 California Jurisprudence, Second Edition, Boundaries, section 38, pages 761-763, it is said: "One of the requisites to the valid establishment of a boundary line by oral agreement between adjoining owners, in the absence of adverse possession or estoppel, is uncertainty or dispute as to the location of the true boundary. The requirement is the same when it is sought to establish a line on the basis of an implied agreement based on recognition and acquiescence for the requisite period of time. Accordingly, an agreement fixing a boundary is not valid for any other purpose than that of settling an uncertainty in regard to a common boundary. If adjoining owners agree orally on a division line, knowing that it is not the true line, and with a purpose of thereby transferring from one of them to the other a body of land which they know his true *125 line does not bound, the agreement will not be enforced. Land cannot be conveyed by the device of moving fences or changing the marks or monuments which define its boundaries. To allow parties whose common boundary line is not uncertain or in dispute to give title in such a manner would involve recognition of a mode of transferring title not countenanced by law."
"Uncertainty sufficient to satisfy the rule as to agreed boundaries exists where at the time of the agreement there is a lack of knowledge by both parties of where the line is or should be drawn. An actual dispute in the sense of a quarrel or ill feeling is not essential. A doubt as to the location of a common boundary such as will sustain a finding of an agreed boundary may arise from a believed uncertainty which may be proved by direct evidence or inferred from the circumstances existing at the time of the asserted agreement. The fact that an actual survey is possible is not conclusive of the question whether a doubt existed as to the location of a common boundary so as to sustain a finding of an agreed boundary."
"Uncertainty need not appear from the deed or from an attempt to make a survey. Nor is it essential that the true location of the boundary be absolutely unascertainable, mere uncertainty being sufficient. But a boundary line is not unknown or uncertain within the rule where the fact that the agreed line is not the true line is known to any party, where an accurate description of the true line may be ascertained from fixed existing government monuments and field notes; or where a boundary fence is built on what the parties suppose to be the true line, and there is no dispute." (Italics added.)
There is no evidence that the parties "agreed" upon a dividing line. The dirt road on plaintiffs' land was a convenient way for vehicles to enter upon the property; it is common for dirt roads to spring into existence next to power lines or pipelines or any obvious physical mark in large areas of farming. As is obvious from the testimony, the dry farming of annual crops would lend itself easily to just such a situation as developed here around the pipeline, power line, and road, without any agreement that these physical marks on plaintiffs' land should designate a boundary on their land.
The plaintiffs did not have to take affirmative action in the circumstances while the occupation was " 'not of a nature to ripen into a valid adverse title.' " (Wareham v. Randolph, 184 Cal.App.2d 218, 227 [7 Cal.Rptr. 483]; Farmers' Loan & *126 Trust Co. v. Denver L. & G. R.R. Co., 126 F. 46, 53 [60 C.C.A. 588]; Hays v. Marsh, 123 Iowa 81 [98 N.W. 604].)
The orange trees were planted in 1963; appellants were immediately told they were trespassing; they would not acknowledge the surveyed boundary; plaintiffs brought suit in 1965; the judge found no laches on the part of plaintiffs and the evidence presents no showing of an abuse of discretion in the finding of the court. (Wilkerson v. Thomas, 121 Cal.App.2d 479, 490 [263 P.2d 678].) Appellants put their permanent improvements upon plaintiffs' strip of land with full knowledge and warning. The evidence does not indicate any error in the judgment.
The judgment is affirmed.
Gargano, J., concurred.
Stone, J., deeming himself disqualified, did not participate.
|
Frankies 570 Spuntino, the Manhattan annex of Frank Castronovo and Frank Falcinelli’s Brooklyn restaurant empire, will close in about two weeks.
But the corner restaurant in Greenwich Village shouldn’t be empty for long. Come November, the Franks, as they are known, plan to open a new restaurant. They are partnering with the chef Nick Anderer, formerly of the Union Square Hospitality Group.
“I hate to use the word ‘close,’” Mr. Castronovo said. “It’s going to go into a transition, and then it’s going to be reborn.”
Enter Anton’s. The restaurant, named after Mr. Anderer’s great-great grandfather, who immigrated from Germany, will be “an updated, more modern version of a nostalgic New York cafe,” Mr. Anderer said. Still-life paintings will hang on the walls, and there will be some antique furniture. Think old-fashioned without being quaint. |
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Space Age Looks Now!
Jul 11, 2012 5:02:14 PM
The Tulip chair was designed by Eero Saarinen in 1955 and 1956 for the Knoll company of New York City. It was designed primarily as a chair to match the complementary dining table. The chair has the smooth lines of modernism and was experimental with materials for its time. The chair is considered a classic of industrial design.
The chair is often considered "space age" for its futuristic use of curves and artificial materials. The design was popularized by its use on the original Star Trek television series.
In the late 1960's the Tulip chair played a highly visible role on the TV show "Star Trek", where they appeared on the bridge set of the "U.S.S. Enterprise", as well as throughout the rest of the ship. The bridge chairs were slightly modified Tulip chairs that featured plastic appliques attached to their back panels. After the show was cancelled most of the set furnishing were discarded into trash dumpsters. Fortunately, several of the show's bridge chairs were salvaged, and have become highly prized collectibles. An original bridge Tulip chair was offered during the Profiles in History Hollywood Auction #17, selling for $18,000.
Create your own futuristic look from prices from the past! We offer a stylish replica of this infamous side chair for $175 and the armchair for $189. We also carry a replica of the Tulip table in 35 or 47 inches starting at $495. The shipping and tax are free*! |
Alex Rodriguez could not be more serious about his war with Major League Baseball.
The Yankees third baseman has enlisted the help of the same private-investigation firm Dominique Strass-Kahn did to successfully contest a 2011 rape allegation, according to New York Magazine. Earlier this summer, Rodriguez hired Guidepost Solutions, a New York firm run by former federal prosecutor Bart Schwartz, a source told the publication, and the sleuths there have been working closely with A-Rod’s lawyers in recent weeks.
On Monday, Rodriguez was suspended by MLB for 211 games for his ties with anti-aging clinic Biogenesis, which has allegedly distributed performance-enhancing drugs to professional athletes in multiple sports. While 12 other players accepted 50-game suspensions, Rodriguez could not reach a deal and MLB and commissioner Bud Selig hit him with a suspension that would go through the 2014 season.
A-Rod has decided to appeal the penalty and he’ll be able to play with the Yankees until that process is over – likely not until November or December when an arbitrator finishes with the case. Rodriguez will make his 2013 Yankee Stadium debut Friday night against the Tigers after playing three road games against the White Sox earlier this week.
Both Guidepost and A-Rod’s lawyers declined to comment on a relationship between the two. Some have questioned the means in which MLB acquired the evidence against the players.
Two years ago, Guidepost was able to dig up damaging information about Strauss-Kahn’s accuser, Nafissatou Diallou. Prosecutors dropped all charges against the former International Monetary Fund chief quickly after.
Schwartz said “old-fashioned detective work” was the key to the DSK case in an interview with New York Magazine earlier this year. Surveillance footage from the Sofitel hotel where Strauss-Kahn was staying spurred suspicions, he said.
“It was evidence that something was wrong with the story being told,” he said.
DSK lead lawyer Ben Brafman said Guidepost’s “breathtaking speed and vast resources they were able to bring to such an assignment was nothing short of extraordinary.” |
Christian Living with Chronic Illness
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Tag Archive | Identity in Christ
Last Thursday, I shared an important lesson God reminded me of as I was reading the book I Still Believe, an autobiography written by Contemporary Christian singer and song writer Jeremy Camp. Before I put this book aside, I wanted to share another truth that I learned while reading this book. It has to do with the purpose of trials in our lives.
“The Word of God never promises that we won’t go through trials. Actually, it’s pretty much a sure thing that we will go through them. In fact, James exhorts us to ‘consider it pure joy, my brethren, whenever you face trials of many kinds.’ We aren’t guaranteed a perfect life. We’re going to struggle and endure hardships.”
But that’s not the end of the message. Jeremy continues:
“And yet God does promise that in our trials, He will stand right next to us and be there every moment. He will be faithful to lead us and guide us, to breathe life into us and heal our hearts.”
But honestly, the statement that touched the deepest place in my heart from this book was the following one. Frankly, it opened my eyes to a truth I hadn’t seen before, the difference between being refined by our trials and being defined by the difficult circumstances God allows to touch our lives. One is a part of the plan of God in allowing suffering in our lives on this earth, the other was never meant to be.
“What I have walked through has refined me. It hasn’t defined me – this is not who I am, ‘the guy whose wife passed away and who has a powerful testimony because if that’- but it has refined me and deepened my dependence on the Rock of my salvation.”
As many of you know, we lost our thirty-four year old special needs son David last November. And after his death, one of my biggest struggles was feeling like I’d lost a major part of who I was. Suddenly, I was no longer the mother of a child with special needs. For thirty-four years, my life (and my husband’s as well) had centered around meeting David’s extensive medical needs. When that was no longer my responsibility, I felt lost.
Until I read the above quote, I really didn’t understand I had been allowing the suffering in my life to define me, to determine how I saw myself. Trials that don’t just come for a short time and then go away can do that if we aren’t careful. But I was not primarily the mother of a child with special needs. My identity is found in Christ and my relationship with Him.
Yes, trials are a part of life on this earth. Yes, they refine us, changing us from within. But, no, the difficult circumstances we walk through are not meant to define who we are. Unfortunately, when trials drag on and on and on, they have the potential of doing just that. What we are walking through becomes so much a part of who we are that it can become how we see outselves, our identity.
Ladies who are reading this on our GLG page, remember your chronic illnesses do not define who you are. You are a child of God, an heir of God and joint heir with Christ (Romans 8:16-17), who happens to have one or more chronic illness. If you are reading this on my personal blog, perhaps your prolonged trial is of a different kind, but the same lesson applies. Life on this earth and trials go together but the suffering we go through does not determine who we are.
On this Teach Me Tuesday, let’s remember our trials do refine us, but they don’t define us. We are God’s beloved children who will one day be whole, when we see Him face-to-face. Allow your trials to remind you of this truth, and look forward to that day when we will leave behind these broken bodies and live in the future God has promised us, when “He will wipe away every tear from their eyes, and death shall be no more, neither shall there be mourning, nor crying, nor pain anymore, for the former things have passed away.” (Revelation 21:4 ESV)
Today I am starting a new series that examines who we are in Christ. These will be short posts, partly because that’s all I can handle as I continue on my recovery process from a major surgery, and partly because they will be most useful if we take the time to meditate on the truths included. Today I’m examining: I am a child of God.
“So also, when we were underage, we were in slavery under the elemental spiritual forces of the world. But when the set time had fully come, God sent his Son, born of a woman, born under the law, to redeem those under the law, that we might receive adoption to sonship. Because you are his sons, God sent the Spirit of his Son into our hearts, the Spirit who calls out, “Abba, Father.” So you are no longer a slave, but God’s child; and since you are his child, God has made you also an heir.” (Galatians 4:3-7 NIV)
There is one sure proof that you are a child of God – the Spirit of the Son is in your heart! When we surrender our lives to the Lordship of Jesus Christ, the Holy Spirit comes to dwell within us, to guide us into all truth and to enable us to live lives pleasing to God. Where before, we were slavess to such sins as fear, selfishness, and any other fruit of living in the fallen flesh, now the Holy Spirit is at work within us to develop the fruit of the Spirit: “… love, joy, peace, forbearance, kindness, goodness, faithfulness, gentleness and self-control. Against such things there is no law.” (Galatians 5:22-23 NIV)
God is Creator – we were each formed by Him in our mother’s womb – but this alone does not make us children of God. Being a child of God has nothing to do with our age or nationality. Only one thing matters: “But you are not in the flesh but in the Spirit, if indeed the Spirit of God dwells in you. Now if anyone does not have the Spirit of Christ, he is not His.” (Romans 8:9 NKJV) Do you know that the Spirit of God lives within you? If so, you can be confident that you are a CHILD OF GOD!
When I was growing up, I remember my mother collecting S&H Green Stamps, which she received at grocery stores. These were redeemable for household items, personal items, and even toys.
Mother and I would sit at the dining room table, licking the stamps and applying them to the pages of the S&H Stamps redemption books. Occasionally, I would go with her the next day to the S&H Stamps Redemption Store, and she would give me one or two books of my own to “buy” something for helping her.
This was my first exposure to the idea of something (or someone) being redeemed, which simply meant that the stamps she had collected were exchanged for money or goods. But this falls far short of the biblical meaning of being redeemed.
So what do I mean when I say “we are redeemed?” According to Baker’s Evangelical Dictionary, redemption is “deliverance from captivity by means of a ransom price paid.” Before we became Christians, we were in bondage to sin and Satan. Jesus paid the “ransom price” by shedding His blood on the cross He paid what we owed and could not pay. Baker goes on to say, “The central theme of redemption is that God has taken the initiative to act compassionately on behalf of those who are powerless to help themselves.”
What was the main effect of our redemption? It was a change from one kingdom to another, from darkness to light. Colossians 1:13-14 explains what happens.
“For he has rescued us from the dominion of darkness and brought us into the kingdom of the Son he loves, in whom we have redemption, the forgiveness of sins.”
What should be our response to Jesus’ gift of redemption? 1 Peter 1: 17-19 gives us the answer.
“Since you call on a Father who judges each person’s work impartially, live out your time as foreigners here in reverent fear. For you know that it was not with perishable things such as silver or gold that you were redeemed from the empty way of life handed down to you from your ancestors, but with the precious blood of Christ, a lamb without blemish or defect.” |
The father of the gunman behind the deadliest shooting in modern U.S. history robbed a string of banks in Arizona, escaped prison in Texas and tried to start a new life as the manager of a bingo parlor in Oregon, according to historical newspaper articles.
Eric, the brother of Las Vegas shooter Stephen Paddock, who killed at least 59 people from his hotel room at the Mandalay Bay Resort and Casino late Sunday, told the Orlando Sentinel that their father was Benjamin Hoskins Paddock.
The elder Paddock, born in Wisconsin in 1926, had a host of other fake names and nicknames, including “Big Daddy” and “Old Baldy," and was on the FBI’s Top Ten Most Wanted list from 1969 to 1977.
Paddock was indicted in 1960 on three counts of robbing Phoenix branches of Valley National Bank, the Arizona Republic reported on Oct. 6 that year. He was accused of stealing close to $25,000 and was caught in the summer of 1960 by FBI agents in Las Vegas.
"The FBI said Paddock tried to resist arrest and attempted to run down an agent with his car," read a July 28, 1960, report from the Tuscon Daily Citizen.
The 6-foot-4, 245-pound Paddock was convicted and slapped with a 20-year prison sentence, but the lengthy jail term was cut short when he busted out of a federal prison in Texas in 1969, the Eugene Register-Guard reported.
STEPHEN PADDOCK: WHAT WE KNOW ABOUT LAS VEGAS SHOOTING SUSPECT
An escaped federal prisoner poster issued by the FBI at the time said Paddock was “diagnosed as psychopathic” and “reportedly has suicidal tendencies and should be considered armed and dangerous.”
About six months after the escape, Paddock was involved in an armed robbery at a bank in San Francisco and in September 1978, he was awaiting trial related to charges from that incident, according to the Oregon newspaper.
“My view is let him go… and good riddance.” — Circuit Judge George Woodrich on Benjamin Hoskins Paddock's legal saga
Paddock, described by the FBI as being an “avid bridge player,” had managed to live a secret life centered on another game --bingo -- as a parlor manager in Springfield, Ore.
The FBI said Paddock lived for years in the Eugene-Springfield area under the alias of Bruce Werner Ericksen and managed to stay one step ahead of law enforcement by constantly changing his appearance and avoiding contact with police, which may have resulted in fingerprinting, the Eugene Register-Guard reported.
The man dubbed by the newspaper as “Bingo Bruce” appeared to have run out of luck in 1978 when he was arrested, but the feds paroled him and he was back in the number-calling game just a year later.
“He was a nice guy, and helped a lot of people financially and did one hell of a lot for the kids,” former Junction City Mayor Chuck Ivey, who was on the parole board, told the newspaper.
“All that stuff is old news,” Ivey said when he asked about Paddock’s past.
LAS VEGAS SHOOTING: TRUMP CALLS MASSACRE 'PURE ACT OF EVIL'
In 1987, the gig finally ended when the Oregon Attorney General’s Office filed seven racketeering charges against Paddock related to his bingo operation. On top of that, he was charged with rolling back car odometers.
Paddock settled the racketeering charges for $623,000 and pleaded no contest to the odometer case, while simultaneously claiming he had cancer.
Among his other life claims: being an auto crew racing chief, Chicago Bears football player and survivor of a World War II mine sweeper sinking, according to the Eugene Register-Guard.
When the final verdict in Paddock’s legal saga came in, and Circuit Judge George Woodrich decided to let him off with a $100,000 fine and no jail time.
“He could be conning everybody, but this is an economic crime and he’s an old man,” the newspaper quoted Woodrich as saying. “My view is let him go… and good riddance.”
Paddock then went back to the Lone Star state and lived there until his death in 1998. Laurel Paulson, a woman he met while living in Oregon, told the Eugene Register-Guard that he got by on a VA pension and helped her run a machine shop.
“He was a man that people either loved or hated,” she said. “He always said he was a dinosaur.” |
2009 Luxembourg general election
General elections were held in Luxembourg on 7 June 2009, together with the 2009 election to the European Parliament. All sixty members of the Chamber of Deputies were elected for five years. The polls were topped by the Christian Social People's Party, which built upon its already high number of seats to achieve a commanding victory, with the highest vote share and number of seats of any party since 1954. Incumbent Prime Minister Jean-Claude Juncker, who is longest serving head of government in the European Union, renewed the coalition agreement with Deputy Prime Minister and Luxembourg Socialist Workers' Party leader Jean Asselborn and formed the Juncker-Asselborn Ministry II, which was sworn-in on 23 July 2009.
Parties
Seven parties ran candidates in all four circonscriptions, of which, five were already represented in the Chamber of Deputies: the Christian Social People's Party (CSV), the Luxembourg Socialist Workers' Party (LSAP), the Democratic Party (DP), the Greens, and the Alternative Democratic Reform Party (ADR). Two parties that were not then represented also ran: The Left and the Communist Party (KPL). In addition, the Citizens' List, which was headed by current independent deputy Aly Jaerling, ran in two constituencies.
Results
By locality
As in 2004, the CSV won pluralities in each of Luxembourg's four circonscriptions, and pluralities in nearly all of Luxembourg's communes. Only four communes didn't register pluralities for the CSV (down from seven in 2004). Wiltz in the north and Dudelange, Kayl, and Rumelange in the southern Red Lands voted for the LSAP.
The CSV's performance improved most markedly in Centre, where it increased its vote from 35.5% to 38.6%. In Centre, the CSV received almost twice as many votes as the Democratic Party in, only ten years after the DP won a plurality by over 2%. It gained one extra seat in Centre, and another in Est.
Aftermath
The CSV's large margin of victory guaranteed that it would form the government once again, with Jean-Claude Juncker appointed as formateur and likely to remain as Prime Minister. Before the election, Juncker, Europe's longest-serving head of government, had told his party that he intended to step down as Minister for Finances, to be replaced by Luc Frieden. This brought into question his chairmanship of the Europe-wide Eurogroup, which he had chaired since 2005. However, he has since stated that he would remain in charge of monetary policy and relations with the European Central Bank.
The CSV was in a strong enough position to form a coalition with any one of three parties: LSAP (partner in the Juncker-Asselborn Ministry I), the DP (partner in the Juncker-Polfer Ministry), and the Greens (who had never previously entered the government). However, the DP and Greens had both ruled out the possibility of a coalition with the CSV, leaving only the previous coalition partners, LSAP, in the running. The CSV and LSAP formed a coalition agreement, with Juncker as Prime Minister and Jean Asselborn as Deputy Prime Minister, with the new government forming on 23 July.
References
Category:2009 elections in Europe
Category:2009 in Luxembourg
2009
Category:June 2009 events in Europe |
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921 S.W.2d 417 (1996)
Martha GARCIA, Individually and on Behalf of the Estate of Juan L. Tijerina, Jr., Appellant,
v.
CAREMARK, INC. and Baxter Healthcare Corp., Miles Inc., and Alpha Therapeutics Corporation, Appellees.
No. 13-95-128-CV.
Court of Appeals of Texas, Corpus Christi.
April 4, 1996.
Rehearing Overruled May 16, 1996.
*419 James B. Ragan, Corpus Christi, for Appellant.
Ferriel C. Hamby, Jr., Adams & Graham, Harlingen, David Bell, Daphne B. Subar, Knapp, Petersen & Clarke, Glendale, CA, Roger W. Hughes, Adams & Graham, Harlingen, Ricardo A. Garcia, McAllen, Lisa D. Powell, Stephen C. Haynes, William M. Mills, Atlas & Hall, McAllen, Terry O. Tottenham, Fulbright & Jaworski, Austin, Brynley James, III, W. Wendell Hall, Renee A. Forinash, Fulbright & Jaworski, San Antonio, for Appellees.
Before DORSEY, FEDERICO G. HINOJOSA, Jr., and RODRIGUEZ, JJ.
OPINION ON MOTION FOR REHEARING
DORSEY, Justice.
Our opinion of February 29th, 1996 is withdrawn and the following substituted in its place. Martha Garcia, individually and on behalf of the estate of Juan Tijerina, Jr., appeals from a take-nothing summary judgment granted in favor of Caremark, Inc., and numerous other appellees,[1] on the ground that the statute of limitations barred Garcia's survival claims asserted on behalf of her deceased minor son. Garcia raises two points of error complaining that the limitations period had not expired at the time she filed the present lawsuit. We affirm in part and reverse and remand in part.
Garcia's minor son, Juan L. Tijerina, Jr., died intestate on November 1, 1989, from the AIDS virus, which Garcia asserts was negligently transmitted through blood products supplied by Caremark. On October 28, 1992, more than two years after her son's death, Garcia filed the present lawsuit asserting wrongful death and survival claims based on products liability in connection with the production and marketing of the blood products that killed her son.
Caremark answered and moved for summary judgment on the ground that Garcia's claims were barred by the two-year limitations period for wrongful death and survival claims. TEX.CIV.PRAC. & REM.CODE ANN. § 16.003(b) (Vernon 1986). Garcia concedes that her wrongful death claim is barred by the two-year limitations period, but asserts that the time for filing the survival claim asserted on behalf of her son's estate was tolled for one year following his death. The trial court granted summary judgment against Garcia on all claims.
As an initial issue, appellee Alpha Therapeutics Corp. claims that this court lacks jurisdiction to hear this case because appellant failed to timely file an adequate appeal bond. Appellee notes that the first appeal bond filed by appellant was a copy, rather than an original. Some time later, appellant filed a second bond, which was an original. Appellee claims that the second bond was filed out of time, and therefore did not perfect the appeal. We disagree.
The Texas Supreme Court has determined that courts of appeals have jurisdiction over "any appeal where the appellant files an instrument that `was filed in a bona fide attempt to invoke appellate court jurisdiction.'" Grand Prairie Indep. Sch. Dist. v. Southern Parts Imports, Inc., 813 S.W.2d 499, 500 (Tex.1991) (per curiam) (quoting Walker v. Blue Water Garden Apartments, 776 S.W.2d 578, 581 (Tex.1989)). In this case, although the second bond was filed after the time for perfection of the appeal had passed, we hold that the first bond filed was a bona fide attempt to invoke the jurisdiction of this court, and therefore that the second bond adequately corrected any alleged defect in the first bond.
Appellee also argues that the appeal bond was defective because it lacked an affidavit of solvency for the surety. Appellee claims that the lack of such affidavit made the bond defective under Rule 46(a) of the Texas Rules of Appellate Procedure. Rule 46(a) states, in relevant part:
*420 Unless excused by law, the appellant shall execute a bond payable to the appellee in the sum of $1000.... The bond on appeal shall have sufficient surety and shall be conditioned that appellant shall prosecute his appeal or writ of error with effect and shall pay all costs which have accrued in the trial court and the cost of the statement of facts and transcript. Each surety shall give his post office address.
TEX.R.APP.P. 46(a). Nothing in Rule 46(a) requires that appellant file an affidavit of solvency with the appeal bond. See Smith v. Valdez, 737 S.W.2d 141, 142 (Tex.App.San Antonio 1987, no writ). The only requirement in the rule is that "[t]he bond on appeal shall have sufficient surety...." In any case, appellant did file an affidavit of solvency with the second appeal bond. We hold that under these circumstances, appellant successfully invoked this court's jurisdiction. Appellee's cross-point on appeal is overruled.
By two points of error, Garcia complains that the two-year statute of limitations on her survival cause of action was tolled by the provisions of section 16.062 of the Texas Civil Practice and Remedies Code:
(a) The death of a person against whom or in whose favor there may be a cause of action suspends the running of an applicable statute of limitations for 12 months after the death.
(b) If an executor or administrator of a decedent's estate qualifies before the expiration of the period provided by this section, the statute of limitations begins to run at the time of the qualification.
TEX.CIV.PRAC. & REM.CODE ANN. § 16.062 (Vernon 1986). Resolution of the present summary judgment and appeal depend upon whether a personal representative of Garcia's son's estate "qualified" within a year after his death and thus commenced the running of limitations against the survival claim.
The Texas Probate Code provides the procedure for the application and appointment of personal representatives. See TEX.PROB. CODE ANN. §§ 72 et seq., 178 et seq. (Vernon 1980). Specifically, section 181 provides authority for the probate court to order the issuance of letters of administration appointing a personal representative after that person has qualified according to law. TEX. PROB.CODE ANN. § 181(e) (Vernon 1980). The Probate Code provides for the qualification of a personal representative as follows:
A personal representative shall be deemed to have duly qualified when he shall have taken and filed his oath and made the required bond, had the same approved by the judge, and filed it with the clerk. In case of an executor who is not required to make bond, he shall be deemed to have duly qualified when he shall have taken and filed his oath required by law.
TEX.PROB.CODE ANN. § 189 (Vernon Supp. 1996).
In the present case, it is undisputed that neither Garcia, nor anyone else, formally applied for letters of administration or "qualified" as personal representative of her son's estate during the period in question under the terms of the Probate Code.
However, Caremark asserts that Garcia informally qualified and became the de facto administrator of her son's estate by the actions that she took and the authority she assumed over his possessions after his death. Caremark points to the fact that, within a short time after her son's death, Garcia collected and stored his toys, distributed his clothes to family members, collected $2000.00 as beneficiary of his insurance policy which she used along with donations to pay for his funeral and final medical bills, and began seeking representation in regard to the current claim.
Caremark points to Scofield v. Douglass, 30 S.W. 817, 820 (Tex.Civ.App.1895, no writ) (on rehearing), as authority for the recognition that such a de facto administration will cause the statute of limitations to begin running again. However, Scofield was decided on the distinction between an action against the estate, which is subject to the tolling provisions, and an action regarding the lands of the decedent which according to law pass immediately to the beneficiaries. Moreover, we have found no subsequent cases that have otherwise recognized this form of de facto qualification as a means of defeating the statute of limitations extension provided by section 16.062.
*421 Formal qualification provides a definite date upon which limitations begins to run again after death and avoids the problem of having to find some nebulous and uncertain point at which it could be said that a de facto administration begins. We do not believe that this one-year respite from the running of limitations provides the potential plaintiff with such an opportunity for abuse as to justify straying from the plain meaning of the language used in the statute. Accordingly, we hold that the "qualification" contemplated by section 16.062 requires formal compliance with those provisions of the Probate Code specifying the manner of applying for, and qualifying as, personal representative of a decedent's estate.
In the present case, Garcia proved her entitlement to the extension and has properly defeated the limitations defense asserted by Caremark. We sustain Garcia's points of error.
Finally, we note that Caremark has independently challenged Garcia's standing to bring the present survival cause of action absent formal qualification as a personal representative of her son's estate.
Under the Texas Survival Statute, a personal injury action survives to and in favor of the heirs, legal representatives, and estate of the injured person. TEX.CIV.PRAC. & REM.CODE ANN. § 71.021(b) (Vernon 1986). However, for an heir to have standing to bring a survival action within the period allowed for administration of an estate, the heir must generally plead and prove that no administration of the decedent's estate is pending and that none is necessary. Frazier v. Wynn, 472 S.W.2d 750, 752 (Tex.1971); City of San Antonio v. Rodriguez, 856 S.W.2d 552, 564 (Tex.App.San Antonio 1993, writ denied); Johnson v. Holly Farms of Texas, Inc., 731 S.W.2d 641, 647 (Tex. App.Amarillo 1987, no writ).
Standing is a jurisdictional question and can be raised for the first time on appeal. Texas Ass'n of Bus. v. Texas Air Control Bd., 852 S.W.2d 440, 443 (Tex.1993); Rodriguez, 856 S.W.2d at 563. However, when standing is reviewed for the first time on appeal, the appellate court "must construe the petition in favor of the party, and if necessary, review the entire record to determine if any evidence supports standing." Texas Ass'n of Bus., 852 S.W.2d at 446; Rodriguez, 856 S.W.2d at 564.
In the present case, Garcia sued as parent on behalf of her son's estate for survival claims, though she did not in her petition allege that no administration was pending or necessary. When an heir's standing is challenged for the first time on appeal, we construe the petition liberally in support of standing. Accordingly, when an heir sues in a representative capacity on behalf of the estate, the evidence shows that no administration is pending and there is no indication that administration is necessary, we assume on appeal that the heir had standing to assert the survival claims on behalf of the estate. See Rodriguez, 856 S.W.2d at 564. We assume in the present case that Garcia has standing, although Caremark is free to further substantiate its challenge to Garcia's standing in the trial court upon remand.
The trial court's summary judgment against Garcia on her wrongful death claim is AFFIRMED. The trial court's summary judgment on Garcia's survival claim is REVERSED AND REMANDED for trial.
NOTES
[1] Caremark, Inc., Cutter Biological, Inc., A Division of Miles, Inc., Miles Laboratories, Inc., Miles Pharmaceutical Biological Products, Baxter Healthcare Corp., Travenol Laboratories, Inc., and Alpha Therapeutics Corp.
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Cardiologist explains disease’s symptoms
By BRENDA SCHORY - bschory@shawmedia.com
Feb. 14, 2013
GENEVA – Heart disease has been identified as the No. 1 cause of death in the United States, and Dr. Michelle Montpetit, a cardiologist at Delnor Hospital in Geneva, educates patients about avoiding heart disease and when to call 911.
“A heart attack is when the coronary artery of the heart is blocked and that causes lack of blood flow, and the heart muscle can die from that,” Montpetit said. “The symptoms that are most commonly seen on television is that the man grabs his chest … and has heavy chest pressure and just collapses. That is really not how most heart attacks happen in anyone, and in women, it is even more nonspecific.”
Classic symptoms of a heart attack are chest pressure going up to the jaw and down the left arm and sweating, she said.
“Heart attacks are a very common killer of women,” Montpetit said. “And in women, sometimes the chest pressure is not there. It can be an uneasy feeling, it can be some pain in the neck and jaw – almost always on left side – sometimes on both sides, not usually just on the right.”
Women’s heart attack symptoms can begin with back pain that starts in the middle and radiates to the middle of the chest. A woman’s heart attack can begin with pain that starts in middle of the body and radiates more to the back than to the chest, she said.
“Also breaking out in a sweat, which women at menopausal age tend to do,” Montpetit said. “It would be something distinctly different than a hot flash.”
The incidence of heart disease among women is nearly 50 percent, she said.
Heart disease, which can include coronary artery disease, rhythm problems or heart failure, affects almost one in two to three women, she said.
“Some diseases that are more common in women is heart failure that occurs in older women,” Montpetit said. “Diastolic heart failure ... is when the heart is stiff. And a loss of estrogen contributes to that stiffening occurring after menopause. [It is] more common with high blood pressure, and it isn’t that the heart isn’t pumping, but it’s not relaxing well because it’s stiff and then fluid builds up and you get shortness of breath.”
Many heart problems can be attributed not directly to obesity but to the things obesity causes or is associated with, Montpetit said.
“Obesity leads to diabetes, which is becoming extremely prevalent … more than 23 million Americans have diabetes, and 57 million have pre-diabetes,” Montpetit said. “ I do find that women do not like to admit that they have diabetes.”
Among the things people can address to reduce the incidence of heart attack is maintain a healthy weight, don’t smoke, watch blood pressure and cholesterol, and get exercise.
A moderate amount of exercise, 2.5 hours a week, best done in 25- to 35-minute increments that raise the heart rate for 30 minutes at a time, is the best goal, she said.
“Walking at a slow pace is good, but you should walk at a brisk pace so your heart rate gets up,” she said. “If you have bad knees, swimming is good.” |
Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic articular inflammation and pain with progressive joint destruction[@b1]. Low-dose administration of methotrexate (MTX) is widely used as a disease-modifying antirheumatic drugs (DMARDs) for RA patients with the best efficacy in relation to its toxicity[@b2]. Although originally developed as an antimetabolite for the treatment of cancer, the anti-inflammatory mechanism of low-dose of MTX in RA is mainly attributed to its capacity to increase extracellular adenosine concentrations[@b3]. However, in 30--40% of early RA patients, MTX monotherapy does not suppress inflammation and reduce disease activity satisfactorily, requiring combinations of other non-biological DMARDs or biologic agents[@b4][@b5].
Adenosine is purine nucleoside that in the extracellular compartment can activate four different G protein--coupled receptors, denoted A1R, A2aR, A2bR, and A3R. Among them, the A2aR subtype is mainly involved in anti-inflammatory and immunosuppressive effects[@b6][@b7]. Degradation of extracellular ATP by sequential activities of two ectonucleotidases, mainly ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1, also known as CD39) and ecto-5′-nucleotidase (E5NT, also known as CD73), has been considered as the main pathway for extracellular adenosine production[@b8][@b9][@b10]. CD39 converts extracellular ATP (or ADP) to AMP, whereas CD73 converts AMP to adenosine[@b9].
Fructose 1,6-bisphosphate (FBP) is an endogenous intermediate of the glycolytic pathway that is produced by the phosphofructokinase-1 activity through phosphorylation of fructose 6-phosphate[@b11]. There are evidence that, when administered exogenously, FBP provides anti-inflammatory effects[@b12][@b13][@b14]. Interestingly, as described for MTX, it was proposed that extracellular adenosine also mediates the anti-inflammatory effects of FBP, since its effects were abolished by simultaneous treatment with adenosine deaminase, an enzyme that converts adenosine into its inactive metabolite[@b15]. In the present study, using two different mouse models of experimental arthritis, we addressed the role of the CD39/CD73 adenosinergic pathway and the contribution of the A2aR to the anti-inflammatory effects of exogenous treatment with FBP.
Results
=======
FBP promotes anti-inflammatory effect in two models of acute experimental arthritis
-----------------------------------------------------------------------------------
To evaluate the anti-inflammatory effect of FBP, we employed two different experimental models of arthritis. Firstly, we used zymosan-induced arthritis (ZIA), an acute model of arthritis that involves mainly innate immune response[@b16][@b17]. The intra-articular injection of zymosan induced a marked infiltration of neutrophil in the knee joint 6 h after challenge, as evidenced in cytospin preparations of joint synovial lavage fluid stained with May-Grünwald-Giemsa ([Fig. 1A](#f1){ref-type="fig"}). Notably, mice treated with different doses of FBP (10, 30 and 100 mg.kg^−1^, i.p.), given 24 h and 30 min before intra-articular injection zymosan (30 μg/knee joint), showed significant reduction neutrophil infiltration into the joint ([Fig. 1A,B](#f1){ref-type="fig"}). We also employed mice expressing eGFP under the control of the endogenous lysozyme-M promoter (LysM-eGFP). Lysozyme-M (LysM) is a marker of myelocytic cells, which is mainly expressed in neutrophils[@b18]. As observed with cytospin preparations ([Fig. 1A](#f1){ref-type="fig"}), we found that mice treated with FBP (100 mg.kg^−1^, i.p.) showed reduction of *in vivo* fluorescence localized in the knee joint when compared to vehicle-treated mice 6 h after zymosan challenge ([Fig. 1C,D](#f1){ref-type="fig"}). Moreover, we assessed *in vivo* imaging of myeloperoxidase (MPO) activity of activated neutrophils in mice after injection of zymosan using a chemiluminescent substrate. In accordance with neutrophil counts, FBP (100 mg.kg^−1^) significantly reduced joint MPO activity, determined by reduction of bioluminescence emission from the zymosan-administrated joints ([Fig. 1E,F](#f1){ref-type="fig"}). Furthermore, FBP treatment reduced articular hyperalgesia in a dose-dependent manner when compared to control mice (Veh) ([Fig. 1G](#f1){ref-type="fig"}). Mice treated with FBP (100 mg.kg^−1^) also showed marked reduction of joint swelling, being significantly evident 1 h after zymosan injection ([Fig. 1H](#f1){ref-type="fig"}, *P* \< 0.01). Finally, we assessed the concentrations of pro-inflammatory, TNF-α and IL-6, and anti-inflammatory, IL-10, cytokines in arthritic joint tissue. As expected, intra-articular injection of zymosan induced production of all cytokines. Interestingly, treatment with FBP reduced the concentrations of TNF-α and IL-6 and enhanced the release of IL-10 in the joint tissue ([Fig. 1I--K](#f1){ref-type="fig"}). Collectively, these data indicate that FBP display marked anti-inflammatory effect in experimental arthritis.
To corroborate these findings, we next investigated the anti-inflammatory effects of FBP in antigen-induced-arthritis (AIA) model, which involves adaptive immunity for the generation of the acute inflammatory response[@b17][@b19]. To this end, mice were immunized with methylated bovine serum albumin (mBSA) and, on day 21, they were intra-articularly injected with mBSA into the knee joint. Mice were treated with FBP given 24 h and 30 min before intra-articular challenge with mBSA (30 μg/knee joint). The serum levels of anti-mBSA specific total IgG antibody were significantly higher in immunized mice than in naive control mice (Ctrl), but it was not affected by the acute treatment with FBP ([Fig. 2A](#f2){ref-type="fig"}). However, consistent with the findings in ZIA model, mice treated with FBP at different doses (10, 30 and 100 mg.kg^−1^, i.p.) showed reduction of clinical symptoms of early arthritis (articular hyperalgesia and swelling) induced by challenge with mBSA ([Fig. 2B,C](#f2){ref-type="fig"}). Moreover, FBP reduced articular MPO activity and neutrophil infiltration into the joints induced by mBSA ([Fig. 2D--F](#f2){ref-type="fig"}). In line, FBP also reduced the concentration of TNF-α and IL-6, while increased IL-10 production in the articular tissue after challenge with mBSA ([Fig. 2G--I](#f2){ref-type="fig"}). Taken together, these data show a strong anti-inflammatory effect of the FBP in two models of arthritis.
Anti-arthritic effect of FBP requires adenosine receptor A2a signalling
-----------------------------------------------------------------------
It was previously reported that the anti-inflammatory effects of FBP are mediated by extracellular adenosine[@b15][@b20]. To further explore the role of extracellular adenosine in the anti-inflammatory effect of FBP, mice were treated with a selective A2aR antagonist (8,3-CSC, 1 mg.kg^−1^, i.p.) 1 h before treatments with FBP (100 mg.kg^−1^, i.p.). Inhibition of neutrophil infiltration into the joint and articular hyperalgesia induced by FBP (100 mg.kg^−1^, i.p.) was completely abolished by concomitant treatment with 8,3-CSC in both models of acute arthritis ([Fig. 3A--D](#f3){ref-type="fig"}). It is noteworthy that the treatment with 8,3-CSC alone had no effect on inflammatory parameters of acute arthritis ([Fig. 3A--D](#f3){ref-type="fig"}). Similarly, 8,3-CSC prevented the inhibitory effects of FBP on joint swelling induced by zymosan injection ([Fig. 3E](#f3){ref-type="fig"}). Moreover, FBP failed to inhibit the production of pro-inflammatory cytokines TNF-α and IL-6 and enhance the release of IL-10 in articular tissue in the presence of A2aR antagonist in ZIA or AIA models ([Fig. 3F--H](#f3){ref-type="fig"} and [Supplementary Fig. S1](#S1){ref-type="supplementary-material"} online). Altogether, these results suggest that FBP promotes anti-inflammatory responses through activation of A2aR.
Extracellular accumulation of adenosine by FBP requires ectonucleotidase activity
---------------------------------------------------------------------------------
Systemic administration of FBP promotes the accumulation of extracellular adenosine by an unknown mechanism[@b15][@b20]. Degradation of extracellular ATP by sequential phosphohydrolysis activity of two ectonucleotidases, CD39 and CD73, has been considered as the main pathway for extracellular adenosine production[@b7][@b10]. Notably, mice showed an increase of serum ATP levels when compared to control naive mice 6 h after FBP injection ([Fig. 4A](#f4){ref-type="fig"}). We then hypothesized that ectonucleotidases could be directly involved in the extracellular increase of adenosine by FBP. To test this hypothesis, we first performed kinetic studies to measure the concentration of adenosine in the mice blood after FBP treatment, using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. [Figure 4B,C](#f4){ref-type="fig"} show that the basal serum concentration of adenosine is in the low micromolar range in naive mice, which is consistent with previous reports[@b15]. Interestingly, a single injection of FBP (100 mg.kg^−1^, i.p.) markedly increased the serum adenosine levels, in a time-dependent manner, with a peak at 24 h after administration. Consistent, FBP given 30 min before intra-articular injection zymosan failed to reduce neutrophil recruitment into the joint ([Fig. 4D](#f4){ref-type="fig"}). Notably, pre-treatment of mice with ARL67156 (2 mg.kg^−1^, i.p.), a selective inhibitor of CD39 (CD39i), or adenosine 5′-(α,β-methylene) diphosphate (4 mg.kg^−1^, i.p.), a selective inhibitor of CD73 (CD73i), completely prevented the increase of serum adenosine concentration induced by FBP ([Fig. 4E](#f4){ref-type="fig"}).
Inhibition of CD39 or CD73 abrogates anti-arthritic effect of FBP
-----------------------------------------------------------------
Finally, we addressed whether CD39/CD73 adenosinergic pathway plays a role in the anti-inflammatory effect of FBP. Flow cytometric analysis shows that acute treatment with FBP did not alter the expression of CD39 or CD73 on blood CD11b^+^ leukocytes or splenic CD11c^+^ dendritic cells (CD11c^+^ cells) or regulatory T cells (CD4^+^Foxp3^+^ cells) ([Fig. 5A,B](#f5){ref-type="fig"}; [Supplementary Fig. S2](#S1){ref-type="supplementary-material"} online). Next, mice were pre-treated with selective inhibitors for CD39 (ARL67156--2 mg.kg^−1^, i.p.) or CD73 \[CD73i, adenosine 5′-(α, β-methylene) diphosphate---4 mg.kg^−1^, i.p.\] 1 h before treatment with FBP (100 mg.kg^−1^, i.p.). The inhibition of CD39 or CD73 alone had no effect on inflammatory parameters of acute arthritis. However, the blockade of CD39 and CD73 activities completely abolished the anti-inflammatory effects of FBP, as can be observed by the neutrophil infiltration, hyperalgesia and oedema of the joint ([Fig. 5C--H](#f5){ref-type="fig"}). In addition, the inhibition of CD39 and CD73 also prevented the reduction of TNF-α and IL-6 and the increase of IL-10 in the articular tissue induced by FBP ([Fig. 5I--K](#f5){ref-type="fig"}), providing further evidence of the importance of the CD39/CD73 adenosinergic pathway on the anti-inflammatory effect of FBP *in vivo*.
Discussion
==========
Although there is multiples evidence showing that FBP, a high-energy intermediate of glycolysis, has anti-inflammatory properties[@b12][@b13][@b14], it is less clear how FBP promotes its effects. Here we demonstrated that exogenous treatment with FBP markedly attenuates arthritis, reducing joint swelling, neutrophil infiltration, articular hyperalgesia and pro-inflammatory cytokine production, while boosting IL-10 production in two experimental models of arthritis. Our mechanistic studies showed that FBP reduces joint inflammation through the generation of extracellular adenosine and subsequent activation of adenosine receptor A2a. Moreover, we showed that the activity of the ectonucleotidase CD39 is required for the generation of extracellular adenosine induced by FBP, implicating the CD39/CD73 adenosinergic pathway in the anti-inflammatory mechanism of FBP.
Adenosine is a purine nucleoside that, in the extracellular compartment, represents an important endogenous mechanism for regulating inflammatory and immune responses[@b7]. Extracellular adenosine exerts its effects by binding to surface receptors, of which the A2aR is predominantly involved with the anti-inflammatory and immunosuppressive activities[@b10][@b21][@b22]. Indeed, the selective activation of A2aR suppresses joint inflammation and reduces progression of experimental rheumatoid arthritis[@b23][@b24]. Moreover, there is now unequivocal evidence showing that MTX, one of the most effective DMARDs used to treat RA, mediates its immunoregulatory effects via generation of adenosine[@b3][@b25][@b26]. Consistent with these findings, our study demonstrate that the blockade of A2aR completely abrogated the inhibition of joint inflammation induced by FBP, suggesting that A2aR plays a crucial role in the anti-arthritic effects of FBP.
Under physiological condition, the extracellular concentration of adenosine is relatively constant, but it can rise dramatically as a result of ATP catabolism[@b27]. The hydrolysis of extracellular ATP to adenosine is orchestrated by ectonucleotidases, especially CD39 and CD73, which are expressed by a broad range of cells, including myeloid, endothelial and regulatory T cells[@b8][@b9][@b10]. Although there is a conventional view that charged molecules, such as phosphorylated sugars, cannot easily cross the cell membrane, there are evidences demonstrating that FBP can cross biological membranes and acts as a high-energy glycolytic substrate, bypassing the two prior ATP-consuming phosphorylation steps and providing accumulation of intracellular ATP[@b28][@b29][@b30][@b31]. Indeed, exogenous administration of FBP has also been shown to exert protective effects in a variety of ischemic injury models, which are attributed to its ability to sustain glycolysis and increase ATP production in a low oxygen environment[@b30][@b32][@b33][@b34][@b35]. In accordance, we found that a single injection of FBP increased serum concentration of ATP. Moreover, Sola *et al.*[@b15] reported that FBP attenuates intestinal ischemia/reperfusion injury by inducing accumulation of extracellular adenosine into the intestinal tissue. These observations raise the possibility that the hydrolysis of extracellular ATP by ectonucleotidases might play a central role in the generation of extracellular adenosine induced by FBP. Supporting this hypothesis, we found that a single injection of FBP markedly increases serum concentration of adenosine, which was completely abrogated by inhibition of CD39 or CD73. Moreover, inhibition of CD39 or CD73 prevented the reduction of joint inflammation induced by FBP. Therefore, our results implicate ectonucleotidases, CD39 and CD73, on the mechanistically accumulation of extracellular adenosine and subsequent anti-inflammatory properties induced by FBP. In line, we have recently reported that pharmacological inhibition of CD39 suppressed the anti-inflammatory effects of MTX[@b25]. Moreover, chronic inhibition of CD39 or genetic deficiency of CD73 aggravates experimental arthritis[@b25][@b36].
FBP has also been demonstrated to be the endogenous activator of Pyruvate Kinase M2 (PKM2), an enzyme that catalyses the last step of glycolysis[@b37][@b38]. Evidence are now emerging indicating that activation of PKM2 inhibits LPS-induced IL-1β while enhances IL-10 production by macrophages[@b39][@b40]. In our study, we also found that FBP reduced TNF-α and IL-6 while strongly increased the release of IL-10 in the joint tissue. Notably, it was described that the production of IL-10 through activation of A2aR is crucially required for the anti-inflammatory properties of adenosine[@b21]. In fact, FBP failed to enhance the release of IL-10 in articular tissue in the presence of A2aR antagonist. However, whether exogenous FBP can activate PKM2 and how this might account for the generation of extracellular adenosine are important questions to be addressed in future studies.
In summary, our findings provide a new insight into the molecular mechanism underlying the anti-inflammatory effect of FBP. Moreover, our study provides further evidence that FBP effectively attenuates experimental arthritis. Importantly, preclinical studies showed that there is a relatively wide margin of safety between toxic and therapeutic doses of FBP[@b41][@b42]. Therefore, FBP may represent a new therapeutic strategy for RA treatment, mainly as adjuvant therapy for RA patients refractory to MTX monotherapy or associated with others DMARDs.
Methods
=======
Mice
----
Male C57BL/6 mice (6--8 weeks old) were bred and housed in the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo. LysM-eGFP mice were generated as previous described[@b43] and were kindly provided as a gift from Prof. Gustavo Batista Menezes (Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brazil). All mice received water and food *ad libitum*. All protocols were conducted in accordance with ethical guidelines and approved by the Animal Welfare Committee of FMRP (Protocol: 53/2013).
Experimental models of arthritis
--------------------------------
ZIA was induced as described previously[@b17]. In brief, 30 μg of zymosan from *Saccharomyces cerevisiae* (Sigma-Aldrich, St. Louis, MO, USA), diluted in PBS, was injected into the femur--tibial joint of mice. Control mice were injected with vehicle (PBS). For AIA, mice were immunized with mBSA (methylated bovine serum albumin, Sigma-Aldrich, St. Louis, MO, USA) as described previously[@b19]. Briefly, mice were immunized with subcutaneous injections of an emulsion containing mBSA (500 μg, Sigma-Aldrich, St. Louis, MO, USA) and Freund's complete adjuvant (CFA, 2 mg.ml^−1^ of inactivated *Mycobacterium tuberculosis*, Sigma-Aldrich, St. Louis, MO, USA). Booster injections of mBSA dissolved in Freund's incomplete adjuvant (IFA) were given at 7 and 14 days after the first immunisation. Sham-immunised mice received similar injections but without mBSA. On day 21 after the first immunization, mice were challenged with an intra-articular injection of 30 μg of mBSA in PBS.
Pharmacological protocol
------------------------
Fructose 1,6-bisphosphate (FBP, Sigma-Aldrich, St. Louis, MO, USA) was given intraperitoneally twice at 24 h and 30 min before intra-articular challenges with zymosan or mBSA (10, 30 and 100 mg.kg^−1^). In some experiments, FBP was also given orally (100 mg.kg^−1^). 8-(3-chloro-styryl)-caffeine (1 mg.kg^−1^), a selective antagonist of the A2aR; ARL 67156 (2 mg.kg^−1^;), an inhibitor of CD39; and adenosine 5′-(α,β-methylene) diphosphate (4 mg.kg^−1^), an inhibitor of CD73 (all from Sigma-Aldrich, St. Louis, MO, USA), were given intraperitoneally 1 h before treatment with FBP.
Determination of joint neutrophil infiltration
----------------------------------------------
Neutrophil infiltration into the joints was assessed 6 h after intra-articular challenges with zymosan or mBSA by counting the number of cells harvested from in articular cavities as previously described[@b44]. Briefly, articular infiltration of neutrophils was assessed by washing femur-tibial joint three times with 3.3 μl of PBS + EDTA (0.2 M) for subsequent counting in a Neubauer chamber. For differentials counts, cells harvested from articular lavage fluid were stained with May-Grünwald-Giemsa in cytospin preparations and analysed their morphological features in an optic microscope (Carl Zeiss, Oberkochen, Germany). The results were expressed as the number of neutrophil ×10^4^ (mean ± SEM)/joint.
*In vivo* bioluminescence imaging
---------------------------------
To determine myeloperoxidase activity *in vivo*, mice were anesthetized with isoflurane and injected intraperitoneally with XenoLight Rediject Inflammation Probe (100 mg.kg^−1^, Caliper Life Sciences) 6 h after intra-articular challenges with zymosan or mBSA. Luminescence image acquisitions were performed using an IVIS Spectrum System (Caliper Life Sciences) at 10 min post injection of the probe. Images were captured and analyzed with Living Image Software (Caliper Life Sciences). The results were expressed as the intensity of radiance (p/sec/cm^2^/sr).
Assessment of articular hyperalgesia
------------------------------------
Articular mechanical hyperalgesia was assessed 6 h after intra-articular challenges with zymosan or mBSA using an electronic pressure meter (model 1601C, Life Science Instruments California, USA) as previously described[@b19]. The results were expressed as the flexion-elicited withdrawal threshold in grams (g).
Determination of knee joint swelling
------------------------------------
Knee joint swelling was assessed 6 h after intra-articular challenges with zymosan or mBSA using a digital caliper (Digmatic Caliper, Mitutoyo Corp., Kanagawa, Japan). The results were expressed as the difference (Δ) between the transverse diameters of ipsilateral (inflamed) and contralateral (control) knee joints measured after induction of articular inflammation in millimeters (mm).
Quantification of cytokines
---------------------------
The levels of TNF-α, IL-6 and IL-10 in joint tissue were determined 6 h after intra-articular challenges with zymosan or mBSA using ELISA kits (R&D Systems, Minneapolis, MN, USA). The results were expressed as pg of cytokine/joint.
Blood collection and adenosine quantification
---------------------------------------------
Blood was withdrawn from mice by cardiac punction at different times after FBP-treatment in tubes containing 10 μM of 5′-deoxycoformycin (adenosine deaminase inhibitor, Sigma-Aldrich, St. Louis, MO, USA)[@b45]. In some experiments, mice were pre-treated with ARL 67156 (2 mg.kg^−1^;) or adenosine 5′-(α,β-methylene) diphosphate (4 mg.kg^−1^) intraperitoneally 1 h before treatment with FBP and blood was withdrawn from by cardiac punction 24 h later. Blood samples were centrifuged at 10,000 × g for 10 min at 4 °C, and serum samples were stored at −70 °C until the analyses. For adenosine quantification, 300 μl of serum was added in 1 ml of acetonitrile and theophylline (internal standard). The tubes were vortexed for 2 min, centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatants were lyophilized by vacuum concentrator system---CentriVap (Labcongo Corporation, Missouri, USA). The lyophilized pellets were resuspended in 100 μl of mobile phase (water with 0.1% formic acid). Samples (100 μl) were analyzed by LC-MS/MS (Liquid chromatography--mass spectrometry).
LC-MS/MS equipment and conditions
---------------------------------
The Shimadzu (Kyoto, Japan) LC-MS/MS equipment consisted of an LC-10ADVP binary solvent delivery pumps, SLC-10AVP system controller, SIL-20A Prominence auto sampler and CTO-10ASVP column oven set at 25 °C. The separations was performed using a 100 × 3.9 mm XTerra MS C~18~ column with a particle size of 3.5 μm (Waters, Milford, MA, USA) and a 20 × 3.9 mm XTerra MS C~18~ guard column with a particle size of 5 μm (Waters, Milford, MA, USA). The mobile phase was composed of (A) water with 0.1% formic acid and (B) acetonitrile. Each mobile phase component was filtered through a 0.22 μm membrane and degassed ultrasonically before use. The binary gradient elution (A:B proportion, v/v), at a flow rate of 0.3 ml.min^−1^, was composed by 96:4 from 0 to 5 min; switching to 50:50 from 5 to 7 min; maintained by 11 min; switching back to the initial condition from 11 to 13 min; and maintaining on this proportion till 16 min. The tandem mass spectrometry (MS/MS) system employed for quantitative analyses was a Quattro LC triple-quadrupole (Micromass, Manchester, UK) fitted with a Z-electrospray (ESI) interface operating with positive ion modes. The temperatures of the source block and desolvation gas were set at 100 °C and 350 °C, respectively. Nitrogen was used as both desolvation (nearly 360 l.h^−1^) and nebulizer (nearly 40 l.h^−1^) gas while argon was used as collision gas. The voltages employed in the ESI source during the analyses were 20 V for the cone, 3 kV for the capillary and 3 V for the extractor. The ions detection were carried out in the multiple reaction monitoring (MRM) mode, employing collision energy of 15 eV, monitoring the transitions of the m/z 268 precursor ion to the m/z 136 product ion for adenosine (268 \> 136) and 181 \> 124 for theophylline (internal standard). The analytical data were processed by MassLynx software (Micromass, Manchester, UK).
Anti-mBSA antibody titer measurement
------------------------------------
The titers of serum anti-mBSA antibody were measured by ELISA. Briefly, 96-well plates were coated with mBSA overnight at 4 °C. After blocking with 2% casein in PBS at room temperature for 1 h, serially diluted serum samples were added and incubated overnight at 4 °C. For detection of anti-mBSA, biotin-conjugated rabbit anti-mouse-IgG antibody was incubated at room temperature for 2 h. Finally, avidin-HRP was added for 30 min, and plates were washed and ortho-phenylenediamine was added for 15 min. The reaction was stopped with 1 M H~2~SO~4~, and the OD read at 490 nm. The data are expressed as optical density values.
ATP quantification
------------------
Blood was withdrawn from mice by cardiac punction 6 h after FBP treatment. The levels of serum ATP were measured using ATPlite Luminescence ATP detection assay system (PerkinElmer Inc., Waltham, MA, USA) according to the manufacturer's instructions.
Flow cytometry
--------------
Cell suspensions (1 × 10^6^ cells) from blood or spleen were stained with fluorochrome-conjugated antibodies for CD11b (M1/70), CD11c (HL3), CD4 (H129.19), CD39 (eBioA1) CD73 (eBioTY/11.8), or FoxP3 (FJK-16s) from BD Biosciences (San Diego, CA, USA) or eBioscience (San Diego, CA, USA). Intracellular FoxP3 staining was carried out according to the manufacturer's instructions (BD Biosciences, San Diego, CA, USA). Stained cells were acquired on a FACSVerse (BD Biosciences, San Diego, CA, USA). Data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
Statistical analysis
--------------------
Statistical analyses were performed using analysis of variance one-way nonparametric (ANOVA) followed by Bonferroni's t test (for three or more groups) comparing all pairs of columns, or two-tailed Student's t-test (for two groups). P \< 0.05, P \< 0.01 and P \< 0.001 were considered statistically significant. Statistical analysis was performed with GraphPad Prism (GraphPad Software, San Diego, CA, USA).
Additional Information
======================
**How to cite this article**: Veras, F. P. *et al.* Fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, attenuates experimental arthritis by activating anti-inflammatory adenosinergic pathway. *Sci. Rep.* **5**, 15171; doi: 10.1038/srep15171 (2015).
Supplementary Material {#S1}
======================
###### Supplementary Information
We thank Roberta Rosales for help with IVIS Spectrum analysis, Giuliana Bertozi for help with ELISA assay and Prof. Gustavo Batista Menezes (Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brazil) for providing LysM-eGFP mice. Funding: The research leading to these results received funding from the European Union Seventh Framework Programme \[FP7-2007-2013\] under grant agreement n° HEALTH-F4-2011-281608 (TIMER), from the São Paulo Research Foundation (FAPESP) under grant agreements n° 2009/54014-7, 2011/19670-0 (Projeto Temático) and 2013/08216-2 (Center for Research in Inflammatory Diseases) and from the University of São Paulo NAP-DIN under grant agreement n° 11.1.21625.01.0.
**Author Contributions** F.P.V., R.S.P. and J.C.A.--F., designed the study. F.P.V. and J.C.A.-F. planned experiments and analyzed data. F.P.V., R.S.P., A.L.L.S. and L.G.P., performed the experiments. P.L.-J. and J.A.R.P. provided essential materials. F.P.V., R.S.P., F.Q.C., T.M.C. and J.C.A-F. wrote the manuscript. J.C.A.-F. supervised the study.
{#f1}
{#f2}
{#f3}
![FBP promotes systemic extracellular adenosine generation.\
(**A**) ATP concentration in the serum of C57BL/6 mice treated with FBP (100 mg.kg^−1^) or vehicle (Veh) collected 6 h after treatment. (B-D) C57BL/6 mice were previously treated with CD39 (ARL67156, 2 mg.kg^−1^, CD39i) or CD73 \[adenosine 5′-(α,β-methylene) diphosphate, 4 mg.kg^−1^, CD73i\] inhibitors 1 h before administration of FBP (100 mg.kg^−1^). Serum from mice was collected at several times after FBP treatment to measure adenosine concentration by LC-MS/MS analyses. (**B**) Representative spectrograms of adenosine levels on serum from naive C57BL/6 mice 6, 12 and 24 h after FBP treatment. (**C**) Adenosine concentrations on serum from C57BL/6 naive mice treated with FBP or saline (Ctrl). mBSA-immunized C57BL/6 mice were treated once (30 min) or twice (24 h and 30 min) before arthritis induction. (**D**) Intra-articular neutrophils migration 6 h after mBSA challenge. (**E**) Adenosine levels on serum from C57BL/6 naive mice 24 h after treatment with FBP plus CD39i or CD73i administration. Data represent mean ± s.e.m., n = 5 mice per group. \*\*\*P \< 0.001 compared with control group and ^\#\#\#^P \< 0.001 compared with FBP group; \*\*P \< 0.01 compared with vehicle group.](srep15171-f4){#f4}
{#f5}
|
Enhanced transmission of electromagnetic waves through 1D plasmonic crystals.
Transmission of electromagnetic waves through thick perfect conducting slabs perforated by one-dimensional arrays of rectangular holes was studied experimentally in the microwave frequency range. The observed thickness-dependent transmission clearly exhibits the evanescent and propagating nature of the involved electromagnetic excitations on the considered structures, which are effective surface plasmons and localized waveguide resonances, respectively. The 1D crystals showing transmission based on localized resonances further manifests the frequency-dependent effective refractive index depending on the filling ratio of the holes and accompanies resonant guided wave propagation. |
Q:
How should i guide a program to perform correct things?
I want to make a small model of A.I. which can learn itself. I am inspired by 1000+ monkey theorem which states that if 1000+ monkey bangs a keyboard for enough long, then they will eventually produce a Shakespeare's play. So, if you give a banana to one monkey when he produce a correct word, then he would eventually learn to do correct things. I think it is related to neural network.
So, practically i want to start with basic alphabets and digits and then my program would permutate and combine those to form words. Now, if the words they form matches with those in English Dictionary, i want to reward the program. However i couldn't think of any possible approach to this. How could this be implemented?
A:
The "Infinite Monkey Theorem" doesn't say that monkeys will learn. On the contrary: it says that if monkeys keep typing random stuff long enough (i.e. not using any sort of learning), the works of Shakespeare will eventually come out purely by chance. It is the Law of Truly Large Numbers.
So you're right: you need to build feedback into the system. The program should change its behavior based on your feedback. This is known as reinforcement learning and it is used a lot in AI. An overarching term for this, I believe, is machine learning.
|
Q:
What's the correct way to print a matrix with labels in a Django template?
I want to do something very simple in Django: Print a matrix as an HTML table, and add labels for the rows and columns. These are the variables in my view:
matrix = np.array([
[101, 102, 103],
[201, 202, 203],
])
colnames = ['Foo', 'Bar', 'Barf']
rownames = ['Spam', 'Eggs']
I want a to get a table that looks like this:
Foo Bar Barf
Spam 101 102 103
Eggs 201 202 203
My template code looks like this:
<table>
<tr>
<th></th>
{% for colname in colnames %}
<th>{{ colname }}</th>
{% endfor %}
</tr>
{% for rowname in rownames %}{% with forloop.counter0 as rowindex %}
<tr>
<th>{{ rowname }}</th>
{% for colname in colnames %}{% with forloop.counter0 as colindex %}
<td>TABLECELL</td>
{% endwith %}{% endfor %}
</tr>
{% endwith %}{% endfor %}
</table>
Output for different values of TABLECELL:
{{ rowindex }}, {{ colindex }} --> table with the indices :)
{{ matrix.0.0 }} --> table full of 101s :)
{{ matrix.rowindex.colindex }} --> table with empty cells :(
As the first two things work, it doesn't seem crazy to assume that the last one would give the intended result. My only explanation is that rowindex and colindex might be strings — and of course int() is among the many things that are forbidden in Django templates.
Does anyone know how I can make this work? Or ideally:
Does anyone know how this is intended to be done in Django?
EDIT 1:
It seems I have to pass the enumerated lists to the template. I provide them as enum_colnames and enum_rownames, but now I can't even execute the nested for loop:
<table>
<tr>
<th></th>
{% for unused_colindex, colname in enum_colnames %}
<th>{{ colname }}</th>
{% endfor %}
</tr>
{% for rowindex, rowname in enum_rownames %}
<tr>
<th>{{ rowname }}</th>
{% for doesnt_work_anyway in enum_colnames %}
<td>You don't see me.</td>
{% endfor %}
</tr>
{% endfor %}
</table>
This gives a table with all the <th>s filled with the correct labels, but no <td>s at all.
EDIT 2:
I found an insanely ugly "solution", which I publish here as an example of something that "works", but is clearly not an answer to my question — how this should be done in Django. Here it comes:
derp = ['', 'Foo', 'Bar', 'Barf', 'Spam', 101, 102, 103, 'Eggs', 201, 202, 203]
iderp = enumerate(derp)
<table>
{% for i, d in iderp %}
{% if i < 4 %} <!-- if top row: th -->
{% cycle '<tr><th>' '<th>' '<th>' '<th>' %}
{% else %} <!-- else: td -->
{% cycle '<tr><th>' '<td>' '<td>' '<td>' %}
{% endif %}
{{ d }}
{% if i < 4 %} <!-- if top row: th -->
{% cycle '</th>' '</th>' '</th>' '</th></tr>' %}
{% else %} <!-- else: td -->
{% cycle '</th>' '</th>' '</td>' '</td></tr>' %}
{% endif %}
{% endfor %}
</table>
Note how it can only be used for tables of this particular width. So in this form it is not even a real solution for the initial problem.
A:
Thanks to Paulo Almeida for providing the two essential hints in his answer:
The table — including labels — should be built in the view.
forloop.first can be used to put the labels in <th>s.
table = [
['', 'Foo', 'Bar', 'Barf'],
['Spam', 101, 102, 103],
['Eggs', 201, 202, 203],
]
<table>
{% for row in table %}
<tr>
{% for cell in row %}
{% if forloop.first or forloop.parentloop.first %} <th> {% else %} <td> {% endif %}
{{ cell }}
{% if forloop.first or forloop.parentloop.first %} </th> {% else %} </td> {% endif %}
{% endfor %}
</tr>
{% endfor %}
</table>
I guess the answer to my implicit question — how the values of multidimensional arrays can be accessed from the template — is that this is not possible.
|
André Villas-Boas has lodged a complaint with the head of referees Mike Riley about the performance of the match officials in Chelsea's Premier League defeat at Manchester United.
Villas-Boas was angered by the manner of his first defeat as manager of Chelsea, which was also his first reverse in a league game for 17 months.
Chelsea went 2-0 behind at Old Trafford on Sunday to goals which replays showed should not have stood as both scorers had been offside, with United finishing 3-1 winners.
Villas-Boas said: "[I am] very, very unhappy with a poor performance from the referees, which in the end had a decisive role in the result, and I don't take it very, very lightly. You expect the linesman to do his job.
"I already went further ahead with the situation by speaking to the correct people. We all feel very, very down when the referee had such an impact on the result."
Chelsea were also let down by their own wastefulness in front of goal, with Fernando Torres producing one of the worst misses in Premier League history. Villas-Boas refused to comment on his £50m striker's state of mind, saying: "It's not a question about an individual player. I'm very, very happy with how the team responded. Everybody is strong and with the correct frame of mind."
However, the Chelsea manager suggested Torres would have to wait for the chance to redeem himself, revealing he planned to pack his team with youngsters for Wednesday's Carling Cup third-round tie with Fulham.
"It gives us a very good idea of where we stand regarding the young players that we are trying to bring through," said Villas-Boas, who announced that Josh McEachran, Romelu Lukaku, Oriol Romeu and Ryan Bertrand will all start. Didier Drogba will lead the line after recovering from his head injury. |
Ignoring occasional flare-ups such as supernovas, the farthest star we can reliably see with the naked eye is the obscure V762 Cassiopeiae, which is just visible under dark skies and is around 16,300 light years away. The most distant well-known star, meanwhile, is Deneb, the brightest star in the constellation of Cygnus, the Swan. It lies a still impressive 2,600 light years away and is the 19th brightest star in the sky, suggesting it is around 200,000 times more luminous than the Sun. |
todocvcd.com is worth $136,800.00
2.88SEO Rating
todocvcd.com has alexa rank of #60,629 in the world, with roughly 15870 daily unique visitors. This website has a Google PageRank of 3/10. It is a domain having .com extension and is hosted in Spain. todocvcd.com estimated worth is: $136,800.00 and have a daily income of around $190.00. As no active threats were reported recently, todocvcd.com is SAFE to browse. |
Living wage calculations for communities across BC decreased significantly this year, according to a new report released today from the Living Wage for Families Campaign. Even though costs are increasing steeply for rent and other basic necessities, the cost of living for families with children is lower in 2019 thanks to the provincial government’s new child care policies.
The living wage is the hourly amount that each of two working parents with two young children must earn to meet their basic expenses (including rent, child care, food and transportation) once government taxes, credits, deductions and subsidies are taken into account.
The 2019 living wage for Metro Vancouver is $19.50 per hour, down from $20.91 in 2018. About 30 per cent of Metro Vancouver two-parent families with two children have incomes below the 2019 living wage according to the most recent Statistics Canada data available.
The Province’s recent investments in child care are reducing out-of-pocket costs for BC families by thousands of dollars this year. In Metro Vancouver, the living wage family saves a total of $8,213 on their child care expenses, a 45-per-cent reduction compared to 2018. These savings come from two programs: the income-tested Affordable Child Care Benefit ($7,013) and the universal Child Care Fee Reduction Initiative ($1,200).
“The living wage calculation shows the connection between wages and government policy in shaping our standard of living,” says Iglika Ivanova, CCPA-BC senior economist and report co-author. “Public investments in child care affordability are helping families with young children weather large increases in the cost of rent and other basic necessities in 2019 and lower the hourly wage they need to earn to meet core budgetary needs."
Without BC’s new child care investments, the living wage amounts would have increased considerably. For example, two parents with two children in Metro Vancouver would have had to each earn $22.47 per hour in 2019 to cover their basic expenses—a shocking 7.5 per cent increase over the 2018 living wage.
The median monthly rent for a three-bedroom unit in Metro Vancouver rose by $103 in 2019 to $1,703, a whopping 6.4-per-cent increase. Shelter continues to be the most expensive item in the living wage budget and the fastest growing in many communities.
“The BC government’s child care investments are a major win for families with children,” says Halena Seiferling, Living Wage for Families Campaign Organizer. “We are also pleased to see the government’s new employment standards legislation, which will provide workers and their families with greater economic security. However, without sustained public investment in key family expense areas the living wage decline we see this year will be short-lived and families will continue to struggle.
“We call on the provincial government to take similar bold measures in other policy areas, especially on the high and rapidly growing cost of rent. Maximum rent increases should be tied to the unit rather than to the tenant to protect housing affordability and existing affordable housing stock must be protected.”
Over 140 companies and organizations in 18 BC communities—employing more than 20,000 workers and covering many thousands more contracted service workers—have been certified as Living Wage Employers over the past ten years. These include the District of Central Saanich, SAP Vancouver, the John Howard Society of the Central and South Okanagan, Integris Credit Union, Modo Cooperative and the City of Vancouver. Eight local governments and one school district have passed living wage policies. |
Q:
How to set blind input field to validate form?
im trying to set up a blind input field with php that will check and make sure the input field is empty and if it is not empty it will not send the message that it is set up to send but I've run into several problems with placement and wording of this here is my code any input would greatly be appreciated.
<?php
// Set email variables
$email_to = 'email@example.com';
$email_subject = 'Website Message';
// Set required fields
$required_fields = array('fullname','email','comment');
$fakes = array('Email1');
// set error messages
$error_messages = array(
'fullname' => 'Please enter a Name to proceed.',
'email' => 'Please enter a valid Email Address to continue.',
'comment' => 'Please enter your Message to continue.'
);
// Set form status
$form_complete = FALSE;
// configure validation array
$validation = array();
// check form submittal
if(!empty($_POST)) {
// Sanitise POST array
foreach($_POST as $key => $value) $_POST[$key] = remove_email_injection(trim($value));
foreach($fakes as $fake)
if($fake == 'Email1') if(!check_for_content($_POST[$fake])) die;
else {
// Loop into required fields and make sure they match our needs
foreach($required_fields as $field) {
// the field has been submitted?
if(!array_key_exists($field, $_POST)) array_push($validation, $field);
// check there is information in the field?
if($_POST[$field] == '') array_push($validation, $field);
// validate the email address supplied
if($field == 'email') if(!validate_email_address($_POST[$field])) array_push($validation, $field);
}
// basic validation result
if(count($validation) == 0) {
// Prepare our content string
$email_content = 'New Website Comment: ' . "\n\n";
// simple email content
foreach($_POST as $key => $value) {
if($key != 'submit') $email_content .= $key . ': ' . $value . "\n";
}
// if validation passed ok then send the email
mail($email_to, $email_subject, $email_content);
// Update form switch
$form_complete = TRUE;
}
}
}
function validate_email_address($email = FALSE) {
return (preg_match('/^[^@\s]+@([-a-z0-9]+\.)+[a-z]{2,}$/i', $email))? TRUE : FALSE;
}
function remove_email_injection($field = FALSE) {
return (str_ireplace(array("\r", "\n", "%0a", "%0d", "Content-Type:", "bcc:","to:","cc:"), '', $field));
}
function check_for_content($fake = FALSE) {
return (preg_match('[A-Z0-9._%+-]', $Email1))? TRUE : FALSE;
}
?>
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Strict//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-strict.dtd">
<html xmlns="http://www.w3.org/1999/xhtml">
<head>
<!-- Contact Form Designed by James Brand @ dreamweavertutorial.co.uk -->
<!-- Covered under creative commons license - http://dreamweavertutorial.co.uk/permissions/contact-form-permissions.htm -->
<title>Contact Form</title>
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/mootools/1.3.0/mootools-yui-compressed.js"></script>
<script type="text/javascript" src="validation/validation.js"></script>
<script type="text/javascript">
var nameError = '<?php echo $error_messages['fullname']; ?>';
var emailError = '<?php echo $error_messages['email']; ?>';
var commentError = '<?php echo $error_messages['comment']; ?>';
</script>
</head>
<body>
<div id="Contactus">
<p>Chisel Multimedia</p>
<p>275 Roswell Street NE <br />
Marietta GA 30060</p>
</div>
<br />
<div id="formWrap">
<div id="form">
<?php if($form_complete === FALSE): ?>
<form action="<?php echo htmlspecialchars($_SERVER["PHP_SELF"]);?>" method="post" id="comments_form">
<div id="label1" 865style="display:none">
<div class="row">
<div class="label">Email</div>
<!-- end .label -->
<div class="input">
<input type="text" id="Email1" class="detail" name="Emial1" />
</div>
<!-- end .input -->
<div class="context">e.g. John Smith or Jane Doe</div>
<!-- end .context-->
</div>
<!-- end .row -->
</div>
<div class="row">
<div class="label">Your Name</div>
<!-- end .label -->
<div class="input">
<input type="text" id="fullname" class="detail" name="fullname" value="<?php echo isset($_POST['fullname'])? $_POST['fullname'] : ''; ?>" />
<?php if(in_array('fullname', $validation)): ?>
<span class="error"><?php echo $error_messages['fullname']; ?></span>
<?php endif; ?>
</div>
<!-- end .input -->
<div class="context">e.g. John Smith or Jane Doe</div>
<!-- end .context-->
</div>
<!-- end .row -->
<div class="row">
<div vlass="label">Your Email Address</div>
<!-- end .lable -->
<div class="input">
<input type="text" id="email" class="detail" name="email" value="<?php echo isset($_POST['email'])? $_POST['email'] : ''; ?>" />
<?php if(in_array('email', $validation)): ?>
<span class="error"><?php echo $error_messages['email']; ?></span>
<?php endif; ?>
</div>
<!-- end .input -->
<div class="context">abc@bca.com</div>
<!-- end .context-->
</div>
<!-- end .row -->
<div class="row">
<div vlass="label">Your Message</div>
<!-- end .lable -->
<div class="input">
<textarea id="comment" name="comment" class="mess"><?php echo isset($_POST['comment'])? $_POST['comment'] : ''; ?></textarea>
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A:
You have to much errors in your code, that prevents you from getting correct results
<input type="text" id="Email1" class="detail" name="Emial1" />
Pay attention, that name="Emial1", but in php code you check for 'Email1'. Correct one of those.
Next piece:
function check_for_content($fake = FALSE) {
return (preg_match('[A-Z0-9._%+-]', $Email1))? TRUE : FALSE;
}
Using $Email1 variable is just out of place. Regex expression is lack of boundaries. At least it should be
function check_for_content($fake = FALSE) {
return (preg_match('/[A-Z0-9._%+-]/i', $fake))? TRUE : FALSE;
}
And when you calling this function why Not condition?
if($fake == 'Email1') if(!check_for_content($_POST[$fake])) die();
I think it should be vice versa.
Anyway, personally I'll just use something like this:
foreach($fakes as $fake)
if(!empty($_POST[$fake])) { die();}
// dont need 'else'
Also when debugging your php code, make sure you turn on errors, it realy helps
error_reporting(0);
ini_set('display_errors', 0);
|
Women like me have been keeping a secret. It’s a secret so shameful that it’s hidden from friends and lovers, so dark that vast amounts of time and money are spent hiding it. It’s not a crime we have committed, it’s a curse: facial hair.
What can be dismissed as trivial is a source of deep anxiety for many women, but that’s what female facial hair is; a series of contradictions. It’s something that’s common yet considered abnormal, natural for one gender and freakish for another. The reality isn’t quite so clearcut. Merran Toerien, who wrote her PhD on the removal of female body hair, explained “biologically the boundary lines on body hair between masculinity and femininity are much more blurred than we make them seem”.
The removal of facial hair is just as paradoxical – the pressure to do it is recognized by many women as a stupid social norm and yet they strictly follow it. Because these little whiskers represent the most basic rules of the patriarchy – to ignore them is to jeopardize your reputation, even your dignity.
About one in 14 women have hirsutism, a condition where “excessive” hair appears in a male pattern on women’s bodies. But plenty more women who don’t come close to that benchmark of “excessive” still feel deeply uncomfortable about their body hair. If you’re unsure whether your hair growth qualifies as “excessive” for a woman, there’s a measurement tool that some men have developed for you.
In 1961, an endocrinologist named Dr David Ferriman and a graduate student published a study on the “clinical assessment of body hair growth in women”. More specifically, they were interested in terminal hairs (ones that are coarser, darker and at least 0.5cm/0.2 inches in length) rather than the fine vellus hairs. The men looked at 11 body areas on women, rating the hair from zero (no hairs) to four (extensive hairs). The Ferriman-Gallwey scale was born.
It has since been simplified, scoring just nine body areas (upper lip, chin, chest, upper stomach, lower stomach, upper arms, upper legs, upper back and lower back). The total score is then added up – less than eight is considered normal, a score of eight to 15 indicates mild hirsutism and a score greater than 15 moderate or severe hirsutism.
The Ferriman-Gallwey scale for the measure of hirsutism
Illustration: Mona Chalabi Photograph: Mona Chalabi
The Ferriman-Gallwey scale for the measure of hirsutism
Illustration: Mona Chalabi Photograph: Mona Chalabi
Most women who live with facial hair don’t refer to the Ferriman-Gallwey scale before deciding they have a problem. Since starting to research hirsutism, I’ve received over a hundred emails from women describing their experiences discovering, and living with, facial hair. Their stories loudly echo one another.
Because terminal hairs start to appear on girls around the age of eight, the experiences start young. Alicia, 38, in Indiana wrote, “kids in my class would be like, ‘Haha look at this gorilla!’”, Lara was nicknamed “monkey” by her classmates while Mina in San Diego was called “sasquatch”. For some girls, this bullying (more often by boys) was their first realization that they had facial hair and that the facial hair was somehow “wrong”. Next, came efforts to “fix” themselves.
Génesis, a 24-year-old woman described her first memories of hair removal. “In fourth grade, a boy called me a werewolf when he saw my arm hairs and upper lip hairs … I cried to my mom about it … she bleached my lower legs, my arms, my back, my upper lip and part of my cheeks to diminish my growing sideburns. I remember it itched and burned.”
After those first attempts come many, many more – each with their own investment in time, money and physical pain. The removal doesn’t just make unwanted hair go away, it raises a whole new set of problems, particularly for women of color. Non-white skin is more likely to scar as a result of trying to remove hair.
Instead of reading or finishing homework on the car drives to school growing up, I would spend the entire length of the drive obsessively plucking and threading my mustache. Every day. – Rona K Akbari, 21, Brooklyn
On average, women with facial hair spend 104 minutes a week managing it, according to a 2006 British study. Two-thirds of the women in the study said they continually check their facial hair in mirrors and three-quarters said they continually check by touching it.
The study found facial hair takes an emotional toll. Forty percent said they felt uncomfortable in social situations, 75% reported clinical levels of anxiety. Overall, they said that they had a good quality of life, but tended to give low scores when it came to their social lives and relationships. All of this pain despite the fact that, for the most part, women’s facial hair is entirely normal.
If I know I have visible facial hair, I’m much more reserved in social situations. I try to cover it up by placing my hand on my chin or over my mouth. And I’m thinking about it constantly. – Ashley D’Arcy, 26
Meanwhile, my 95-year-old demented, deaf and blind Italian aunt sits in a nursing home, and whenever I visit, she points to and rubs her chin, which is her way of communicating to take care of the hair situation. That’s how I know she’s still in there and she cares. I hope someone returns the favor in 40 years. – Julia, 54
There are, however, some medical conditions which can cause moderate or severe hirsutism, the most likely of which is polycystic ovary syndrome, or PCOS, which accounts for 72-82% of all cases. PCOS is a hormonal disorder affecting between eight and 20% of women worldwide. There are other causes too, such as idiopathic hyperandrogenemia, a condition where women have excessive levels of male hormones like testosterone, which explains another 6-15% of cases. .
But many women who don’t have hirsutism, who don’t have any medical condition whatsoever, consider their hairs “excessive” all the same. And that’s much more likely if you’re a woman of color.
The original Ferriman-Gallwey study, like so much western medical research at the time, produced findings that might not apply to women of color (the averages were based on evaluations of 60 white women). More recent research has suggested that was a big flaw, because race does make a big difference to the chances that a woman will have facial hair.
In 2014, researchers looked at high-resolution photos of 2,895 women’s faces. They found that, on average, the white women had less hair than any other race and Asian women had the most. But ethnicity mattered too – for example, the white Italian women in the study had more hair than the white British women.
The percentage of females with at least some upper lip hair by race. Source: Javorsky et al, 2014 Illustration: Mona Chalabi Photograph: Mona Chalabi
But more than a gender thing, for me my hair was about race/ethnicity. My hairiness really solidified how different I was from my peers. I grew up in the suburbs of Dallas. And although my school was pretty diverse, the dominant beauty norm was to be blonde and white. – Mitra Kaboli, 30, Brooklyn
These numbers might be helpful to women like Melissa who said her facial hair meant “I felt inferior, I was a ‘dirty ethnic’ girl”.
But giving reassurance to ethnic minorities probably isn’t why this research was undertaken. The study was funded by Procter & Gamble, the consumer goods company worth $230bn which sells, among other things, razors for women. They know that female hair removal is big business.
Over the years, as women showed more of our bodies – as stockings became sheer and sleeves became short, there was pressure for these new exposed parts to be hairless. Beginning in 1915, advertisements in magazines like Harper’s Bazaar began referring to hair removal for women. Last year, the hair removal industry in the US alone was valued at $990m. The business model only works if we hate our hair and want to remove it or render it invisible with bleach (a norm just as unrealistic as hairlessness – brown women rarely have blonde hair).
When did we sign up to an ideal of female hairlessness? The short answer is: women have hated our facial hair for as long as men have been studying it. In 1575, the Spanish physician Juan Huarte wrote: “Of course, the woman who has much body and facial hair (being of a more hot and dry nature) is also intelligent but disagreeable and argumentative, muscular, ugly, has a deep voice and frequent infertility problems.”
These signposts are strictest when it comes to our faces, and they extend beyond gender to sexuality too. According to Huarte, masculine women, feminine men and homosexuals were originally supposed to be born of the opposite sex. Facial hair is one important way to understand these distinctions between “normal” and “abnormal”, and then police those boundaries.
Scientists have turned their sexist and homophobic expectations of body hair to racist ones, too. After Darwin’s 1871 book Descent of Man was published, male scientists began to obsess over racial hair types as an indication of primitiveness. One study, published in 1893, looked for insanity in 271 white women and found that women who were insane were more likely to have facial hair, resembling those of the “inferior races”.
These aren’t separate ideas because race and gender overlap – black is portrayed in mass media as a masculine race, Asian as feminine. Ashley Reese, 27, wrote “part of my self-consciousness about my facial hair might also tie into some ridiculous internalized racism about black women being less inherently feminine”. While Katherine Parker, 44, wrote, “It makes me feel very confused about my gender.”
Some women are pushing back. Queer women – those who are questioning heterosexual and cisgender norms – are already thinking outside of the framework that shames female facial hair. Melanie, a 28-year-old woman in Chicago explained that as a queer woman “there is less of a prescription for what I should embody as a woman, what attraction between my partner and I looks like, which has helped immensely in coming to terms with my facial hair”.
Social media accounts like hirsute and cute, happy and hairy and activists like Harnaam Kaur are resisting these norms too, by shamelessly sharing images of hairy female bodies. And even women who aren’t rejecting these standards outright, feel deeply ambivalent about them. “I understand, on a rational level, how inherently misogynistic it is to expect women to be constantly ripping hair out of themselves, hair that grows naturally, wrote one woman who, like many I heard from, asked to remain anonymous. “But I can’t bring myself to accept it and let it grow.”
Another wrote: “It’s one thing to be a little heavy, or short, or both. But facial hair? That’s pushing it.”
I’m not about to judge any woman for removing her facial hair. Despite knowing that I don’t need “help”, I still go to see a beauty “therapist” each month. I pay huge sums so she can zap me with a laser that damages my hair follicles. I’ve signed up for a solution, even though I know that the problem doesn’t really exist. I lie there wincing with each shock as she asks me about my weekend and says “Honey, are you sure you don’t want me to do your arms too? They’re very hairy.” |
Systemic juvenile xanthogranuloma with fatal outcome.
Juvenile xanthogranuloma is a benign and self-limited disease which usually appears in the skin of children. Visceral involvement has been rarely reported, as has fatal outcome in some affected individuals. We report a case of systemic juvenile xanthogranuloma in a female newborn with mainly skin, bone marrow, and liver involvement, leading to death at the age of 2 months. |
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Memories & Candles
“I am so sorry to hear of Margie's passing. I will always remember her beautiful smile and how happy she always seemed to be. I love all of you and...Read More »
”
1 of 13 | Posted by: Christy Beverly Bennett - WV
“I am so sorry to hear of Margie passing. I loved her so much. I love the way she smiled and the way she loved her family. If you need anything at...Read More »
”
2 of 13 | Posted by: Christy Beverly Bennett - WV
“So sorry I loved Marg she was a good friend and I know she is in a good place now
Barbara
Betty
Randy
Pauline
”
3 of 13 | Posted by: Barbara Caldwell - Welch, WV
“prayers and thoughts are with all of you. Cherish the memories, they will always be there.
”
4 of 13 | Posted by: Summer (McKinney) Williams - SC
“So sorry for your lose, Margie was a find lady and she was always friendly and always spoke even when she didn't feel good. We will be thinking of...Read More »
”
5 of 13 | Posted by: Charles & Judy Morgan - Fanrock,, WV
“Carolyn,I remember your mom's laugh and smile. She loved you so much. I am sorry for your loss, I pray for God to comfort you and your family at...Read More »
”
6 of 13 | Posted by: Jeannie Cline - Crab Orchard, WV
“Remembering those good times we had with Margie at Connie's home.Her smiling face, The jokes & laughs we shared. I have her reciepe for Christmas...Read More »
”
7 of 13 | Posted by: Cheryl & Sarah Gibson - TN
“Gordon and Connie, so sorry about your mom. She was a sweet person and she was always laughing. Our thoughts and prayers. ...Read More »
”
8 of 13 | Posted by: Penny McKinney - Little River, SC
“Margie could always light up a room,I was honored to serve as her pastor for several years, this family is special to me,and she will be deeply...Read More »
”
10 of 13 | Posted by: Pastor Scott Cline - Brenton, WV
“Margie was unique. She was a friend to everyone. She had that wonderful laugh that brightened your day and a personality that won your heart. ...Read More »
”
12 of 13 | Posted by: Jo Ann and Gerald Scott - WV
“The staff of Calfee Funeral Service would like to extend our deepest sympathies to your family during this difficult time.
”
13 of 13 | Posted by: Calfee Funeral Service Staff
Margie Marie Vaughan Thomas Cook, 78, went to be with the angels Thursday, January 17, 2013 following a sudden illness.
Born July 29, 1934 in McDowell County, she was the daughter of the late Green and Myrtle Suttles Vaughan.
Margie enjoyed traveling, cooking on Sunday for her family, having a good time with her friends, and being with her family; especially her grandchildren. She attended Indian Creek Community Church.
In addition to her parents, she was preceded in death by two husbands, Leroy Thomas and Willis Cook; one son, Roger Thomas; a great-grandson, Zachary Clay; and several brothers and sisters.
Those left to cherish her memory are a son, Gordon Thomas and wife, Tammy of Lynchburg, VA; three daughters, Carolyn Steele and husband, Tommy of Lynco, Connie Deel of Beckley and Cindy Clay of Pineville; two brothers, Frank Vaughan of Goshen, IN and Billy Ray Vaughan of Seattle, WA; a sister, Emilene Sparks of Pineville; eleven grandchildren, Sabrina Richardson, Christy Laxton, Rickey Clay, Jennifer Deel, Tee Jaye Thomas, Kevin Steele, Robert Clay, Dale Thomas, Danielle Larkins, Kaitlyn Thomas, and Emilee Thomas; and seventeen great-grandchildren.
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Memories & Candles
I am so sorry to hear of Margie's passing. I will always remember her beautiful smile and how happy she always seemed to be. I love all of you and if anyone needs anything at all please feel free to contact me.
So sorry for your lose, Margie was a find lady and she was always friendly and always spoke even when she didn't feel good. We will be thinking of you during this difficult time.
Posted by: Charles & Judy Morgan - Fanrock,, WV - Friend Jan 20, 2013
Carolyn,
I remember your mom's laugh and smile. She loved you so much. I am sorry for your loss, I pray for God to comfort you and your family at this time. God Bless all the family.
Love and Prayers, Kenny and Jeannie Cline
Posted by: Jeannie Cline - Crab Orchard, WV - Friend Jan 19, 2013
Remembering those good times we had with Margie at Connie's home.Her smiling face, The jokes & laughs we shared. I have her reciepe for Christmas candy, I made it this year,Thanks Margie. Keeping all of you in my prayers during this difficult time. May God wrap his arms around each & everyone of you as God welcomes her to her permanent home in Heaven! She loved her family so much, You all meant the world to her.
Margie could always light up a room,I was honored to serve as her pastor for several years, this family is special to me,and she will be deeply missed, may god wrap His arms around all those left to grieve her passing
Share A Story
Life Stories provides friends and families a forum to post their favorite stories and memories of Mrs. Margie Cook ensuring the precious experiences are never forgotten. Share joyful times, post a photo that captures the moments you cherish, and allow others to reply, relive and remember.
I was a neighbor of George and Opal Thomas below Baileysville. As a child, along with my brothers and sister, we were very close to Jerry, Nathan and Hassel. We thought of their kin as ours. Margie and Roy visited his oldest brother frequently and we thought nothing of visiting whenever they did. We played with Margie as often as with her children(who were younger than we). She was always cheerful and joking. She was skinny when young and would make fun of herself. She once told Opal, who was making a dress for her, that if she needed to check the measurements just to try it on the nearest fence post. She and Roy had it hard in those years, but Margie was always cheerful.
I hope all of her family feel the eternal comfort of her and God's love.
Posted by
Tamara Shields
from Clinchco, VA - friend
on
February 19, 2013 |
Keating, Corey : Travel Grant - 2016-2017
This summer I plan to return to Hanoi to expand my research on the growing contemporary music scene in Vietnam's northern capital city. In many ways, this trip will be an extension of my previous visit in July of 2015, made possible by the generosity of the Mario Einaudi Center. During my time in Vietnam, I had many fortunate opportunities to meet with musicians, composers, and scholars who were eager to discuss their musical traditions. I hope to further cultivate these relationships, as many of these individuals have extended very generous offers for musical training and assistance with scholarly research, as well as facilitating introductions to Vietnamese master-artists who are not affiliated with any institution. |
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Media Release: Australian National Committee welcomes appointment of new UN Women Executive Director
Newsroom
11 July 2013
Media Release: Australian National Committee welcomes appointment of new UN Women Executive Director
UN Secretary General Ban Ki-Moon appoints Ms Phumzile Mlambo-Ngcuka from South Africa as the new Executive Director of UN Women
The Australian National Committee for UN Women today welcomed the UN Secretary-General’s appointment of Ms Phumzile Mlambo-Ngcuka as the new Executive Director of UN Women.
Ms Phumzile Mlambo-Ngcuka from South Africa brings to the position extensive experience in advocating for women’s issues in a range of roles. She was the first woman to hold the position of Deputy President of South Africa from 2005 to 2008.
Acting Executive Director of the Australian National Committee for UN Women, Rebecca Bromhead, welcomed the announcement and paid tribute to the organisation’s former and founding Executive Director Michelle Bachelet.
“The National Committee looks forward to the leadership of Ms Phumzile Mlambo-Ngcuka in this critical time for UN Women,” Ms Bromhead said.
“Ms Mlambo-Ngcuka’s experience and passion for women’s rights and advocacy will bring enthusiasm and energy to the role.
“The National Committee looks forward to working with the Executive Director and her team to achieve strong outcomes for women across the world.
“On behalf of the National Committee I would like to express our gratitude for former Executive Director Ms Michelle Bachelet’s stewardship during the establishment of UN Women and wish her every future success.
“I would also like to thank Acting Executive Director Lakshmi Puri for her work during the transitional period.” |
Tuesday, April 03, 2012
One wonders, indeed. The Washington Capitals have players on their roster who are not strangers to winning. Once upon a time, Mike Green and Brooks Laich were members of a Calder Cup champion in Hershey that endured a Game 7 in the semi-finals played against a Portland Pirates team that loaded up on ringers – Ryan Getzlaf and Corey Perry, who each played more than 50 games in Anaheim in 2005-2006, as well as Dustin Penner, who had another 19 games with the Ducks that season – and had Bobby Ryan, a second overall draft pick and future 30-goal scorer in the NHL.
The Caps have John Carlson and Karl Alzner, veterans of a Calder Cup team in Hershey that came back from 0-2 down in the Finals against Texas in 2010 to win going away in the next four games. And there is Michal Neuvirth, a goalie who won two straight Calder Cups and in doing so not only defeated the likes of Vancouver’s Cory Schneider (in the 2009 Calder Finals) and Los Angeles’ Jonathan Bernier (in the 2010 Calder semis), but improbably authored shutouts in Games 6 and 7 in the second round of the 2009 playoffs against perhaps Hershey’s most hated rival, the Wilkes-Barre/Scranton Penguins.
And no doubt there are examples of winning among other players on this Capitals roster. Just not in Washington.
And as the Caps find themselves holding onto their own playoff destiny this season by the flimsiest of grips, it begs the question of “why?” Why does one read, after Game 80 of the regular season, such quotes as these from players after last night’s 4-2 loss to the Tampa Bay Lightning?...
"We obviously let off the gas, and there was a lot of bad turnovers and sloppy play. That’s the sign of a team that, in my opinion, thinks they had it a little bit too good. It was a big-time error by us. We never should’ve had that game be where it was."
"Not being able to get a single point tied with three minutes left, it’s disappointing to say the least. We need to make sure that we’re closing out teams or finding ways to make sure that we’re getting points."
"We didn’t come out the way we needed to and took it for granted that it was a team that wasn’t in the playoffs and we had a chance to make [the postseason]. We got a little pressure off ourselves from being out of the playoffs to in the playoffs by just having Buffalo lose. With Florida only a few points [away], you start to think about all these things, ‘We win this tonight we’re in third place’ and you get ahead of yourself. We just didn’t come out and pound them like we should have."
"Missed assignments a bit and it ends up in your net. When you’ve got guys like Stamkos on the ice, you’ve got to be extra careful. We battled back, but it’s not good enough."
If there was a “Stanley Cup” for quotes, the Caps would have as storied a history as the Montreal Canadiens. How can a team that has any serious aspirations for a championship at this level be saying the same things in April that they were saying in November? Talk of bad turnovers, sloppy play, missed assignments. This isn’t the shakedown cruise for the team that early games in the season can be. This is the fight for their playoff lives.
How is it that the same players who have shown an ability to meet adversity and beat it at one level seem thoroughly unprepared or unable to do the same at this level? Put in starker terms, what is the difference between Hershey and Washington? Maybe it is the difference between an organization in which winning is not so much expected (it is) as demanded and one where… well, where it isn’t. There appear to be two entirely different cultures two hours apart, and one wonders if the traits inculcated in one appear, with time, to fade away in the other.
Some of these guys need to find their inner “Hershey” and right quick. Or it is going to be just another “Washington” sort of spring in these parts.
The Washington Capitals took a lead, lost it in the blink of an eye, teased their fans one more time with a late tying goal, then gave up a pair in the last 63 seconds off the stick of Steven Stamkos to lose to the Tampa Bay Lightning, 4-2, last night in Tampa.
It was there for the taking. A team playing out the string, starting a goaltender who had allowed ten goals on the last 71 shots he faced over two games, and the Caps themselves with an opportunity to turn the heat up on the two teams directly in front of them – the Florida Panthers and Ottawa Senators. After a scoreless first period, it appeared as though the Caps might do just that, take charge of their destiny, when Alexander Semin rifled a slap shot past Lightning goalie Dwayne Roloson moments after hitting a post with an open net in front of him.
Five minutes later, the lead was a memory. Teddy Purcell scored on a power play by taking a page from the Alex Ovechkin songbook. Purcell was lurking along the left wing wall as the Lightning zipped passes back and forth to pull the Caps defenders out of their penalty killing box. When the puck found its way to Steven Stamkos’ stick on the right side, Purcell stepped out from the wall, and Stamkos found him through a clot of players in the left wing faceoff circle. Purcell snapped the puck through goalie Michal Neuvirth’s pads to tie the game.
Victor Hedman gave the Lightning a lead at the end of a play that seemed all too typical of the Caps’ season. Nate Thompson outdueled Roman Hamrlik for the puck in the left wing corner, then moved it out to the point to Hedman. The defenseman fired a shot at the Capitals’ net, and with Tom Pyatt getting position on defenseman Mike Green to set a screen, Neuvirth never saw the puck until after it hit a pad and trickled in to give Tampa Bay the lead.
Jason Chimera solved Roloson to tie the game with less than four minutes left in regulation when Roloson could not control a shot from the right win faceoff circle by Alexander Semin. The shot bled through to the other side of the net, where Chimera was lurking. He backhanded the puck into the open net, and the game was tied, giving Caps fans hope of at least one standings point coming out of this.
That hope was dashed as the clock wound down to a minute to play. Brian Lee and Brett Clark worked the puck at the top of the offensive zone while the other Lightning packed themselves in on Michal Neuvirth. Clark let fly, and Neuvirth made a pad save. But Steven Stamkos was open for the rebound, and he quickly stuffed it in to give the Lightning the lead one last time. Stamkos added an empty net goal with two seconds left, and the Caps found themselves where they started the evening – in eighth place trying to fight off the Buffalo Sabres.
Other stuff…
-- A team fighting for its playoff life should not get outworked by a team writing in its tee times in pen, but that’s what happened on the Hedman goal and on the first Stamkos goal. The Caps were outworked for the puck, outworked for position.
-- 12 of 18 Caps skaters were on ice for Lightning goals, Dennis Wideman and Troy Brouwer on for three of them.
-- One could give Dwayne Roloson a lot of credit for this win for the Lightning. Certainly the folks awarding the game’s three stars did (he was first star). But the Caps didn’t exactly make things hard on him for long stretches of this game, taking shots from long range and getting no traffic in front of him. Still, it was a fitting way for Roloson to wind up in what could be the last time he faces the Caps.
-- Sixty seconds of a season… Stamkos gives the Lightning the lead on his rebound goal with 63 seconds left. Alex Ovechkin has a chance to tie it up with a deftly tipped puck from the low slot with nine seconds left. Roloson makes the save. The puck goes the other way; Stamkos fights through a check along the wall by Wideman, scores the empty netter. Stamkos 2 – Ovechkin 0.
-- It was, to be charitable, an interesting game officiated by Kelly Sutherland and Frederick L’Ecuyer. It was reminiscent of the pre-lockout sort of games when impedance fouls were rarely called.
-- But there was the instance of Matt Hendricks (6’, 211) “cross-checking” Victor Hedman (6’6”, 229) to the ice. One could almost see John Biebe telling Tree Lane, “no, you’re the big guy.”
-- “Nice try” just isn’t enough. His line looked good on the right side of the score sheet – six shots, nine shot attempts, four hits, and a blocked shot. But the left side of the score sheet… no goals, no assists, and a minus-2. Alex Ovechkin is paid for what is on the left side of the score sheet, not the right side.
-- First game back from a long absence, a player might get by on adrenaline. Second game back, the rust might show a little more clearly. It did for Nicklas Backstrom last night. He was not bad, but neither was he sharp. All of that is reasonable to expect. But the third time will have to be the charm on Thursday when the Caps play for what could be their season against Florida.
-- In a losing effort, congratulations are still in order for Jason Chimera, who reached the 20-goal mark for the first time in his career. His previous career high was 17 goals in the 2005-2006 season in Columbus.
-- Brooks Laich is really sputtering on faceoffs. Last night it was 1-for-4 in the offensive zone, 1-for-7 in the defensive zone. On the other hand, boy, did he take one for the team right in the boot. It looked grim the way he went off, but make it back he did.
-- I must have missed the changeover when Vincent Lecavalier became a role player. He looks old and out of place out there. He did little things well, but are his days as someone you game-planned for over?
-- On a per-minute basis, Mike Knuble was a shooting machine out there. Seven shot attempts (two on goal) in just over ten minutes of ice time.
In the end, while it was Stamkos being clutch late, this game was likely decided on a sequence late in the second period with the Caps nursing a 1-0 lead. Alex Ovechkin muscled in against a Tampa defender and got of a shot in deep that Roloson shrugged away. Ovechkin followed the play, and the puck came to his stick again along the left wing wall. He circled out and appeared ready to send off a wrist shot from the left wing faceoff dot. But he spied Mike Green pinching in from the weak side and sent the puck across. Green had only to finish the play to put the Caps up 2-0 and change the complexion of the game. Green fired, Roloson got across and gloved the puck down.
Less than two minutes later, Tampa scored two goals. Instead of a 2-0 lead at the second intermission – Washington came into the game 23-0-1 when leading after two periods – Tampa led, 2-1. Washington, while among the better teams in winning games after trailing at the second intermission, still had won only seven games all season is such situations. There is no wonder Mike Green swung his stick at the glass in frustration after failing to convert. Then again, it was all too typical of the Caps’ season – an inability to cash in when the goods were there for the taking.
The other stuff
Pictures, logos, and the occasional quotes used here are the intellectual property of other folks (unless otherwise noted) of considerably more productive imagination than the author of the original stuff read here, which is our very own. |
Adoption of healthy lifestyles among Chinese cancer survivors during the first five years after completion of treatment.
Objectives: The number of cancer survivors is increasing as a result of advances in detection and treatment. Lifestyle is a significant modifiable factor in the development of cancer. Most studies on healthy lifestyles have been conducted in Western countries. Cultural influences on the pursuit of healthy lifestyles among Chinese cancer survivors remain largely unexplored. The objectives of this qualitative study are to explore the experiences of Chinese cancer survivors in adopting healthy lifestyles, with a focus on their goals, the challenges they face, and the influences of Chinese culture. Design: Thirty-two Chinese breast and colorectal cancer survivors in their first five years after treatment were recruited from a hospital in Hong Kong to participate in eight focus groups. Qualitative content analysis was adopted to analyse the data. Results: The adoption of a healthy lifestyle was a strategy through which the participants exercised choice to restore balance in their health after developing cancer. Diet, exercise, psychological well-being, the use of traditional Chinese medicine (TCM) and health/dietary supplements, and attending medical consultations/follow-up visits were the behaviours adopted by the participants, with the goal of improving their health, controlling their cancer and preventing relapse, and managing the residual physical symptoms of their illness. In adopting a healthy lifestyle, the participants encountered challenges such as a lack of reliable and practical instructions from healthcare professionals. Chinese cultural beliefs concerning the nature of food, TCM, minimizing social disturbances, and collaborative control influenced their lifestyle. Conclusions: The cancer survivors adopted a range of healthy lifestyles but encountered challenges. Clarifying the principles of food choice while addressing Chinese beliefs regarding therapeutic food and the use of TCM, clarifying queries about conflicting information, and developing plans according to the needs, and competing demands of survivors can facilitate collaborative control between healthcare professionals and cancer survivors. |
Saturday, July 23, 2011
... I had taken a recovery day yesterday.... I had ridden a TT bike.... I had worn a TT helmet.... I had not drank from my bottle 3 times.... I had put my head down 2cm more.... I had tucked my braid into my skinsuit.... I had not worn my non aero mtb gloves.... I had dug that little bit deeper.... I had not eased off before the finish because I thought I was already at the finish.... I had not been caught behind a lorry.
These are all the excuses that I could come up with for why I didn't win today, in my first ever proper TT (I've done a few uphill and very short ones, but none that was longer than 10min). This is what happened: The weekend came up and I am down for a 3 hour ride. I've trained every day since Tuesday (see Strava), with Thursday a bit lighter (only 1 hour endurance on the turbo trainer due to rain), but I was slowly getting tired. I needed a motivation to keep going hard. In the absence of a road race nearby I went to do the next best thing, the 10-mile TT organized by Sorrento in Kilpeddar, only about an hour by bike from my place. On Friday evening I tried out Ryan's Merida TT bike to see if I could use it, but it's a bit too big and I felt a 15min spin on it was not long enough to get used to it, and the riding a total of about 3hours on an ill-fitting TT bike seemed not the best idea, so I decided to use my own road bike instead. Unfortunately Ryan also had his TT helmet with him in the Portaferry 3 day - only to find out that no TT equipment was allowed.
Anyhow, on an amazingly beautiful and summery warm Saturday morning (if Ireland was just always like this in the summer!) Cait and I made our way to Kilpeddar Village, where the TT was being held. Going towards Bray from Enniskerry you could barely believe you were in Ireland, with views of the glistening sea below, deep green forests and beautiful views. Summer is here! (at least for today).
Cadel Evans riding into the yellow jersey in today's individual TT of the TdF
The course was basically 7km southwards on the N11, turning via 2 roundabouts either side of the tunnel and returning northwards for the remaining 9km on the N11 back to Kilpeddar village. There was a record of 24min to be beaten, to be rewarded with a bonus of 150Euro sponsored by the Women's Commission for the person who breaks the previous record (24min flat), on top of the winner's prize money, but that was fairly far back in my mind, because I was pretty tired and this was just meant to be motivation to put in some hard training. But I had a good start and got myself into a good rhythm. My Garmin told me I was good on time for matching the record time (probably went out too hard). I could see my minute girl, in the distance. I was fine going into the first roundabout of the turning point, but unfortunately for me, a lorry had just pulled in in front of me from the next entry and I got stuck behind this lorry between the two roundabouts. I was happy when the lorry went straight at the 2nd roundabout and I could speed up again leaving it at the 3rd exit. I had lost some time, but my minute girl was still in my sight and I chased her, and I eventually caught her. I caught another few of the earlier started girls and when I looked again, I was STILL pretty much on time for matching the record. I left the N11 for the final km to the finish and went hard. I saw the people and thought I was done, easing off, when I realized that these were just spectators and that the finish line was another hundred meters or so in front, digging in deep again. When I looked at my Garmin, I knew it would come down to a matter of seconds if I had broken the previous record or not. I had not expected that. And the worst of it all was that I knew I had left some seconds on the road.
In the end my time was 24min and 1 second and 42 hundredth of a second. The winning time was 23 min, 59 sec and 78 hundredth of a second (with full TT gear), a difference of 1.54 seconds.......
....... between me ...... (Photo by Paddy Doran)
...... and the winner Sandra Fitzgerald (Photo by Paddy Doran).
That's less than 2 flippin' seconds between about 200 Euro and me..... Argh! What if.... (refer to excuses above). 1-Mississippi, 2-Mississippi. That's it.
But I learned my lesson. You should always give it your best, even if you don't expect to do well and even if you're tired. Or I could just buy myself some speed (TT bike, helmet, aero wheels, gloves).
Oh, and mountain bikers can be good time trialists too (see Cadel Evans - he got himself into the yellow jersey in today's Tour de France individual TT!). Well done Cuddles, it's about time we're being taken seriously!
Tuesday, July 19, 2011
For the first time since I race in the Elite category, I was going to be in the country when the Irish National Champs were on - all previous years I would have been in Germany on that day, racing in the German Champs (as you race in the national champs of your nationality), but the German champs were ran in June, which I missed due to a shoulder injury acquired at the pre-ride for the Offenburg World Cup the week before the German Nationals.
Photo by Vc Glendale
To be eligible for a National Champs title, you have to have had that nationality from the start of the year. I have received Irish citizenship only a few weeks ago, so although I am now Irish, I would not yet be eligible for the title this year. Nonetheless I was allowed to take part in the race as a non-contender. It would also make for an interesting show-down between Cait, who was last year's national champ and I. She's been nipping at my ankles for the last few weeks now and beaten me in a couple of club races and some of my Strava records, so I was excited to measure myself against her in a proper race situation.
Photo by Cieran Maunsell
I was happy I was allowed to take part, because the race was organized by Team WORC in one of my favourite race venues, Killruddery, which is private land, so it's off-limits to mtbers outside the event. The course was great, almost all of it twisty, windy singletrack, featuring a fun bombhole and the "tombstone" drop (although this was taken out of the course later on). After a short grassy start you entered never-ending tight singletrack, so on pre-ride day I made sure to learn the corners well. The tight, twisty singletrack is something that suits Cait, but there was very little climb in the course and the flat suits me. So no advantage either way.
Photo by Cieran Maunsell
On race day there was an awful wind and a few spits of rain before 5 Elite ladies and a Junior lined up at the start for a total of 4 laps. I must have missed the 30sec warning and was about to grab my bottle again for one last sip when we were told "Go! Go now!". Until I got going, Claire had sprinted off into the front, followed by Cait, Ciara and then me. I overtook Ciara before entering the singletrack and then stuck to Cait's wheel. The speed was comfortable and I was enjoying myself. On one of the short climb sections both Cait and I overtook Claire. A few times Cait managed to open a small gap, especially when I messed up on some of the technical rocky sections, but I made sure I didn't loose her out of my view too much.
Photo by Cieran Maunsell
In the second lap then I caught back up to her on the twisty singletrack section and was pondering where it would be a good idea to attack, deciding that probably on the fireroad to the tombstone forest or the short climb within the tombstone forest would be a good idea. But just as I was pondering that, Cait dropped her chain and had to stop to put it back on. I didn't hesitate and took my chances, overtook her and went full gas to open up a gap. Whenever I looked back, I could see Cait behind me, about the same distance as I had been behind her in the 1st lap. In the 3rd lap the gap increased, and I took the last two laps a little easier, keeping an eye out for Cait behind me, ready to speed up again if needs be, but she didn't catch back up.
So I won the race, but as I'm not eligible for the National Champs title this year, it went to Cait, who came in just a minute behind me. Next year! ;)
Thanks to the officials allowing me to race, thanks to Team WORC to have put together such a nice challenging course, to Stew for his help on the day and to all my sponsors, particularly Cycleways, ZipVit and KCNC for their loyalty and ongoing support. |
A dual role of Erk signaling in embryonic stem cells.
Erk signaling plays a critical role in maintaining the pluripotency of mouse embryonic stem cells (ESCs). Inhibition of Mek/Erk signaling by pharmacologic Mek inhibitor promotes self-renewal and pluripotency of mouse ESCs. However, knockout of Erk1/2 genes compromises the self-renewal and genomic stability of mouse ESCs. In this review, we summarize recent progress in understanding the role of Erk signaling in pluripotency maintenance, discuss the dual role of Erk in mouse ESCs, and provide explanations for the conflicting data regarding Mek inhibition and Erk knockout. Remaining questions and the prospects of Erk signaling in pluripotency maintenance are also discussed. |
Q:
How to use tap with observable
I get user details with flatMap and I also need to get a stream of user image bytes:
this.authStore.jwtContainer$.pipe(
tap(j => {
foo = this.userService.getUserImageDataUrl(j.userImageObjectKey).subscribe(i => foo = i);
console.log(j);
}),
flatMap(x => this.userService.getUserById(x.userLoginId)))
.subscribe(z => this.user = z);
This code works, but I seem to have a double subscribe situation going on, is there a way to have both calls fire async based on jwtContainer subscription?
A:
Yes. You can use forkJoin to combine all the observables and have them fired parallely:
this.authStore.jwtContainer$.pipe(
flatMap(x =>
forkJoin([
this.userService.getUserImageDataUrl(x.userImageObjectKey),
this.userService.getUserById(x.userLoginId)])))
.subscribe(([image,user]) => this.user = user);
Note that forkJoin will have to wait for all its observables to be completed before it can emit the final aggregated results in arrray.
|
<?xml version="1.0" encoding="UTF-8"?>
<bpmn2:definitions xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:bpmn2="http://www.omg.org/spec/BPMN/20100524/MODEL" xmlns:bpmndi="http://www.omg.org/spec/BPMN/20100524/DI" xmlns:camunda="http://activiti.org/bpmn" xmlns:dc="http://www.omg.org/spec/DD/20100524/DC" xmlns:di="http://www.omg.org/spec/DD/20100524/DI" xsi:schemaLocation="http://www.omg.org/spec/BPMN/20100524/MODEL BPMN20.xsd" id="Definitions_1" exporter="camunda modeler" exporterVersion="2.6.0" targetNamespace="http://www.example.org/bpmn">
<bpmn2:collaboration id="Collaboration_1" name="Default Collaboration">
<bpmn2:participant id="Process_Engine" name="Process Engine" processRef="extension-attributes-for-user-tasks"/>
</bpmn2:collaboration>
<bpmn2:process id="extension-attributes-for-user-tasks" name="extension-attributes-for-user-tasks" isExecutable="true">
<bpmn2:startEvent id="StartEvent_1">
<bpmn2:outgoing>SequenceFlow_2</bpmn2:outgoing>
</bpmn2:startEvent>
<bpmn2:userTask id="UserTask_1" name="Do something">
<bpmn2:extensionElements>
<camunda:properties>
<camunda:property value="2" name="escalation1"/>
<camunda:property value="15" name="escalation2"/>
<camunda:property value="15" name="escalation3"/>
</camunda:properties>
<camunda:taskListener class="com.camunda.bpm.demo.extension_attributes_for_user_tasks.EscalationStartTaskListener" event="create"/>
</bpmn2:extensionElements>
<bpmn2:incoming>SequenceFlow_2</bpmn2:incoming>
<bpmn2:outgoing>SequenceFlow_3</bpmn2:outgoing>
</bpmn2:userTask>
<bpmn2:sequenceFlow id="SequenceFlow_2" name="" sourceRef="StartEvent_1" targetRef="UserTask_1"/>
<bpmn2:endEvent id="EndEvent_2">
<bpmn2:incoming>SequenceFlow_3</bpmn2:incoming>
</bpmn2:endEvent>
<bpmn2:sequenceFlow id="SequenceFlow_3" name="" sourceRef="UserTask_1" targetRef="EndEvent_2"/>
</bpmn2:process>
<bpmndi:BPMNDiagram id="BPMNDiagram_1" name="Default Collaboration Diagram">
<bpmndi:BPMNPlane id="BPMNPlane_1" bpmnElement="Collaboration_1">
<bpmndi:BPMNShape id="BPMNShape_1" bpmnElement="Process_Engine" isHorizontal="true">
<dc:Bounds height="200.0" width="1000.0" x="10.0" y="10.0"/>
</bpmndi:BPMNShape>
<bpmndi:BPMNShape id="BPMNShape_StartEvent_1" bpmnElement="StartEvent_1">
<dc:Bounds height="36.0" width="36.0" x="60.0" y="93.0"/>
<bpmndi:BPMNLabel>
<dc:Bounds height="6.0" width="6.0" x="78.0" y="131.0"/>
</bpmndi:BPMNLabel>
</bpmndi:BPMNShape>
<bpmndi:BPMNShape id="_BPMNShape_UserTask_2" bpmnElement="UserTask_1">
<dc:Bounds height="80.0" width="100.0" x="146.0" y="71.0"/>
</bpmndi:BPMNShape>
<bpmndi:BPMNEdge id="BPMNEdge_SequenceFlow_2" bpmnElement="SequenceFlow_2" sourceElement="BPMNShape_StartEvent_1" targetElement="_BPMNShape_UserTask_2">
<di:waypoint xsi:type="dc:Point" x="96.0" y="111.0"/>
<di:waypoint xsi:type="dc:Point" x="146.0" y="111.0"/>
</bpmndi:BPMNEdge>
<bpmndi:BPMNShape id="_BPMNShape_EndEvent_2" bpmnElement="EndEvent_2">
<dc:Bounds height="36.0" width="36.0" x="296.0" y="93.0"/>
</bpmndi:BPMNShape>
<bpmndi:BPMNEdge id="BPMNEdge_SequenceFlow_3" bpmnElement="SequenceFlow_3" sourceElement="_BPMNShape_UserTask_2" targetElement="_BPMNShape_EndEvent_2">
<di:waypoint xsi:type="dc:Point" x="246.0" y="111.0"/>
<di:waypoint xsi:type="dc:Point" x="296.0" y="111.0"/>
</bpmndi:BPMNEdge>
</bpmndi:BPMNPlane>
</bpmndi:BPMNDiagram>
</bpmn2:definitions> |
Magneto magnetic torch
Why is no one ever around to hold the torch when you really need it? Here’s a solution: the Magneto. Featuring a rugged aluminium body, this nifty item comes with three super-bright LEDs and magnets at both ends, one in the base (so you can attach it to a metal surface, such as a car, while you work), and another in the head (allowing you to pick up light metal tools or stray nuts, bolts and screws). The telescopic, rotating head can extend to 546 mm. Price: about R130. Contact Tevo on 0861 778 888 or visit www.tevo.co.za |
Q:
What is the difference between calling the function without parentheses and with parentheses
What is the difference between calling the function without parentheses and with parentheses on onPressed or Ontap?
I just know that void function can't be called with parentheses on onPressed.
floatingActionButton: FloatingActionButton(
onPressed: _incrementCounter,
tooltip: 'Increment',
child: Icon(Icons.add),
),
_incrementCounter has void return type
void _incrementCounter() {
setState(() {
_counter++;
});
}
But I didn't find any proper documentation.
A:
_incrementCounter inside onPressed is a function reference, which basically means it is not executed immediately, it is executed after the user clicks on the specific widget.(callback)
_incrementCounter() is a function call and it is executed immediately.
Therefore, inside onPressed you can either pass a function reference or an anonymous function that will act as a callback.
floatingActionButton: FloatingActionButton(
onPressed: _incrementCounter,
tooltip: 'Increment',
child: Icon(Icons.add),
),
or
floatingActionButton: FloatingActionButton(
onPressed: () {
// Add your onPressed code here!
},
tooltip: 'Increment',
child: Icon(Icons.add),
),
The is not something specific to dart, it is also done in javascript and many other languages:
What is the difference between a function call and function reference?
Javascript function call with/without parentheses
|
Guaiacoxypropanolamine derivatives of capsaicin: a new family of beta-adrenoceptor blockers with intrinsic cardiotonic properties.
A series of guaiacoxypropanolamine derivatives of capsaicin was synthesized by replacing the phenolic OH of N-nonanoylvanillamide with epichlorohydrin, followed by cleavaging the obtained epoxide compound with alkylamines. Intravenous injection of these propanolamine derivatives (1 mg/kg) in normotensive Wistar rats induced a transient fall in blood pressure but significantly reduced the heart rate for more than 30 min. These derivatives (10(-8)-10(-6) M) inhibited isoproterenol (10(-10)-10(-5) M)-induced positive chronotropic and inotropic effects in isolated guinea pig atrium. On the other hand, these derivatives (10(-5)-10(-4) M) exhibited a positive cardiotonic effect that is independent of intrinsic sympathomimetic effects. Investigation of the structure-activity relationship of these derivatives revealed that the position of the oxypropanolamine side chain and substituents of the 4-OH position play significant roles in imparting their pharmacological effects. Of the derivatives tested, the most effective one was compound 9. In conclusion, the results obtained from in vitro and in vivo studies suggested that these derivatives and compound 9 may be expected to be beta-adreneoceptor blocking agents with nonadrenergic positive chronotropic and inotropic properties. |
Multiple wavelength stabilization on a single optical cavity using the offset sideband locking technique.
We implemented a compact, robust, and stable device for simultaneous frequency stabilization of lasers with different wavelengths used for the cooling and trapping of Yb atoms in an optical lattice clock. The lasers at 399, 556, and 759 nm are locked to a single ultra-stable cavity using the offset sideband locking technique, a modified version of the Pound-Drever-Hall method. For the most demanding stabilization here, the 556 nm laser, this system exhibits a 300 Hz linewidth for an integration time of 80 ms. We observed a long-term drift of less than 20 kHz per day at 759 nm that is suitable for operating the lattice laser with a light shift uncertainty below 1×10<sup>-18</sup>. We successfully tested the system for operating the clock during a typical working day by simultaneously locking the three lasers to the cavity. |
Effects of dietary lead and zinc on fetal organ growth.
In order to further understand the effects of ingested lead (Pb) on the fetus and the possible interaction of the trace element zinc (Zn) with Pb, groups of rats with dated pregnancies were fed 0, 10, 50, 100, 200 or 500 mg/L of Pb in water throughout pregnancy. Diet was provided ad libitum. A group pair fed with the 200 mg/L of Pb group and a group fed both Zn and Pb, 200 mg/L, were also studied. Placental weight remained constant, but cell division and total protein level were decreased while cell size increased markedly. Fetal carcass and liver weight, cell division, and protein were decrease while cell growth was unchanged. Brain weight decreased while cell division, growth, and protein were unchanged. Kidney weight, cell division and protein level were unchanged but cell growth was decreased. Organ dry weight varied with wet weight while the percentage of water was unchanged. Whether pair feeding and Zn supplements improve carcass and liver weight is questionabel. |
On Saturday (Sept. 1), a pod of 22 short-finned pilot whales turned up at Avalon State Park Beach near St. Lucie on Florida's east coast. Volunteers and environmental officials responded to the scene to try to keep the whales — the largest of which was more than 25 feet (7.6 meters) long — hydrated and shaded.
But 17 died or had to be euthanized. The remaining five, all young whales, were brought to a rehab center at Florida Atlantic University's (FAU) Harbor Branch Oceanographic Institute. There, they were treated for malnutrition, dehydration, stress and infection, FAU officials said.
One of the whales died Monday around noon after a rapid decline in health, according to FAU; a necropsy, or animal autopsy, was performed Tuesday. The other four were taken to SeaWorld Orlando early Wednesday and placed in a quarantine area, where they will be cared for and tube-fed, a statement from the marine park explained. The caretakers hope to return the whales to the wild eventually, but a statement from FAU said the outlook for their survival "remains guarded."
Authorities are investigating the cause of the stranding and expect to get the dead whales' autopsy results in the next several weeks. Some officials speculate that one of the dominant adult whales became ill, stranded itself and the rest of the pod followed. Pilot whales live in pods of between 15 to 50 individuals and they tend to stick together — and that means even when one of the whales becomes ill or injured.
For instance, in May 2011, more than 16 pilot whales stranded in the lower Florida Keys. "They tend to strand in pods, they stick together, if one is sick, the whole pod is going to strand," Anne Biddle, media relations director for the Marine Mammal Institute, told LiveScience at the time. And in 2003, some 25 pilot whales were stranded in the Keys.
Scientists say many factors could cause a mass stranding of whales, including disease, loud noises (from oil and gas exploration, Navy sonar, and even natural events like earthquakes), toxins or simple confusion. |
Out of ColorControl
OPI Sheer Tints Collection
This spring, there’s just an overload of OPI collections being pumped out consecutively
OPI Sheer Tints is a collection of TINTED TOP COATs
Comes in 4 basic colours – yellow, pink, purple & blue
They are meant to create additional dimensions to your existing colours,
multiplying the colours you already own
Other than being just a top coat, phoenixbeautylounge came up with several nail art designs you can play with Sheer Tints which will be explored below..
NAIL ART with OPI Sheer Tints
Here’s another quick nail art you can do easily with OPI Sheer Tints:
credits: mllrdesign.com
***
Melva suggests that the Sheer Tints can also be a colour on it’s own
Apply 3 thin layers to give a jelly finish
You will get the jello colours that you have been looking for so hard last summer 2013
I would think that this collection is so versatile that it’s probably one of the best collections I’ve seen so far from OPI in 2014 |
Abstract
The Malay Royal Institutionhas existed for centuries in the social system of the administration of Malay states in Malaysia. In this monarchy system, the sovereign has the absolute power in the state administration. However, during British colonization, the Sultan had to refer to the Resident as advisor except on matters related to Islam and Malay customs. The situation continued until the Malayan Union was introduced in 1946 when the Sultan was only authoritative on the Malay customs and Islam, while the state administration was held by the Governor. When the Malayan Union Constitution was replaced by the Malay Federation in 1948, the Sultan was given the rights to rule the country in accordance with the Constitutional Monarch. This study aims to analyze the perception of undergraduates at public higher education institutions regarding the Malay Royal Institutionin the Federal Constitution within the context of ethnic relations in Malaysia. It involved 422 respondents selected from 4 public higher education institutions in Malaysia. The data obtained through surveys was analysed using SPSS program. Descriptive statistics such as frequency was used to describe the background of the respondents and to analyze the students’ perception. The findings show that the acceptance level of the students on the position of Malay Royal Institutionsin the constitution is relatively and generally high. Based on the findings, several recommendations are put forward to address issues related to the position of the Malay Royal Institution. This study also contributes to the field of knowledge as the methodology employed was quantitative which was different from previous studies which used qualitative methods.
Nazri Muslim. 2015. Level of Acceptance among Public Institution of Higher Learning towards the Institution of Malay Rulers According to Federal Constitution of Malaysia. Mediterranean Journal of Social Sciences (6): 153-164. |
Q:
passing variables / models in mvc 4 c#
Ok so i have 2 models
Model Clients
[Table("Clients")]
public class Clients
{
[Key]
[DatabaseGeneratedAttribute(DatabaseGeneratedOption.Identity)]
public int ClientID { get; set; }
public string Client { get; set; }
public string Address { get; set; }
public string State { get; set; }
public string City { get; set; }
public string County { get; set; }
public int Zip { get; set; }
public string Phone { get; set; }
public string LogoLocation { get; set; }
public string ContactName { get; set; }
public string ContactPhone { get; set; }
public string ContactEmail { get; set; }
public int Authorized { get; set; }
public string Note { get; set; }
public string Comment { get; set; }
public virtual ICollection<Countys> Countys { get; set; }
}
Model Countys
[Table("Countys")]
public class Countys
{
[Key]
[DatabaseGeneratedAttribute(DatabaseGeneratedOption.Identity)]
public int CountyID { get; set; }
public int ClientID { get; set; }
public string County { get; set; }
public string Note { get; set; }
public string Comment { get; set; }
public virtual ICollection<TownShips> Townships { get; set; }
}
** My Countys Index Controller at this time **
public ActionResult Index(int id)
{
var cnty = from r in db.Clients
where r.ClientID == id
select r;
if (cnty != null)
{
try
{
return View(cnty);
}
catch (Exception ex)
{
}
}
return HttpNotFound();
}
My view is a standard generated view :
@model OilNGasWeb.ModelData.Clients
@{
ViewBag.Title = "Index";
}
<h2>County's for </h2>
<p>
@Html.ActionLink("Create New", "Create",new { id = Model.ClientID },null)
</p>
<table>
<tr>
<th>
@Html.DisplayNameFor(model => model.Countys.First().County)
</th>
<th>
@Html.DisplayNameFor(model => model.Countys.First().Note)
</th>
<th>
@Html.DisplayNameFor(model => model.Countys.First().Comment)
</th>
</tr>
</Table>
EXCEPTION :
System.InvalidOperationException: The model item passed into the dictionary is of type 'System.Data.Entity.Infrastructure.DbQuery1[OilNGasWeb.ModelData.Clients]', but this dictionary requires a model item of type 'OilNGasWeb.ModelData.Clients'. at System.Web.Mvc.ViewDataDictionary1.SetModel(Object value) at System.Web.Mvc.ViewDataDictionary..ctor(ViewDataDictionary dictionary) at System.Web.Mvc.WebViewPage1.SetViewData(ViewDataDictionary viewData) at System.Web.Mvc.RazorView.RenderView(ViewContext viewContext, TextWriter writer, Object instance) at System.Web.Mvc.BuildManagerCompiledView.Render(ViewContext viewContext, TextWriter writer) at System.Web.Mvc.ViewResultBase.ExecuteResult(ControllerContext context) at System.Web.Mvc.ControllerActionInvoker.InvokeActionResult(ControllerContext controllerContext, ActionResult actionResult) at System.Web.Mvc.ControllerActionInvoker.<>c__DisplayClass1a. <InvokeActionResultWithFilters>b__17() at System.Web.Mvc.ControllerActionInvoker.InvokeActionResultFilter(IResultFilter filter, ResultExecutingContext preContext, Func1 continuation) at System.Web.Mvc.ControllerActionInvoker. <>c_DisplayClass1a.<>c_DisplayClass1c.b_19() at System.Web.Mvc.ControllerActionInvoker.InvokeActionResultWithFilters(ControllerContext controllerContext, IList`1 filters, ActionResult actionResult) at System.Web.Mvc.Async.AsyncControllerActionInvoker.<>c_DisplayClass25.<>c_DisplayClass2a. b_20() at System.Web.Mvc.Async.AsyncControllerActionInvoker.<>c_DisplayClass25. b_22(IAsyncResult asyncResult);
A:
If your view needs a Clients object (which includes an enumeration of Countys objects), then you need to pass a Clients object to your view:
public ActionResult Index(int id)
{
var client = (from r in Clients
where r.ClientID == id
select r).SingleOrDefault();
if (client != null)
{
return View(client);
}
return HttpNotFound();
}
And in the view you'd declare a Clients model:
@model OilNGasWeb.ModelData.Clients
Then in the view you would have access to the information about the Clients object as well as the (potentially empty) list of Countys objects it contains. Essentially what's "wrong" with your code is that the view is logically in the context of a Clients (it's really hard not to just say "Client", you might want to work on the class names), but all you're giving it is an enumeration of Countys objects.
Note: For safer code, you'll probably want to initialize that enumeration to whatever its value should be, in case it isn't populated in the event that it's an empty list. You'd do this in the class' constructor. For example, if you want to initialize it to a List<Countys> then the constructor might be something like this:
public Clients()
{
Countys = new List<Countys>();
}
That way the property is never null, at worst it's an empty list which is a lot easier to manage in the view.
|
Q:
Tomcat warning: "Setting property 'showServerInfo' to 'false' did not find a matching property"
I'm trying to change my webapp so that it doesn't provide any details about the server type or version when an error is generated, as described in this page. So in the META-INF/context.xml file of my webapp I have added an ErrorReportValve like this:
<Host name="localhost" appBase="webapps"
unpackWARs="true" autoDeploy="true">
...
<Valve className="org.apache.catalina.valves.ErrorReportValve"
showReport="false"
showServerInfo="false" />
...
</Host>
But when I start tomcat I get this error message:
Feb 27, 2015 11:48:26 PM org.apache.tomcat.util.digester.SetPropertiesRule begin
WARNING: [SetPropertiesRule]{Context/Valve} Setting property 'showReport' to 'false' did not find a matching property.
Feb 27, 2015 11:48:26 PM org.apache.tomcat.util.digester.SetPropertiesRule begin
WARNING: [SetPropertiesRule]{Context/Valve} Setting property 'showServerInfo' to 'false' did not find a matching property.
(I am running tomcat 7.0.52 on Ubuntu 14.04.2 LTS)
Can anyone suggest why the ErrorReportValve seems to be missing properties?
A:
The problem is my version of tomcat is too old - the properties weren't included until version 7.0.54:
http://tomcat.apache.org/tomcat-7.0-doc/changelog.html
|
An effective method for the preparation of a block copolymer offers one the opportunity to modify the copolymer with monomers which are normally incompatible. Thus the process permits the chemical union of two incompatible macromolecules which would otherwise be difficult to link. A successful process for synthesis of a block copolymer requires a reliable polymerization process which is not hindered by the usual problems of linking already formed polymer chains.
It was the hope that a phase transfer catalyzed (PTC) reaction would lend itself to the synthesis of desirable polymers and free the process from the use of anhydrous aprotic solvents, which led me to exploit the particular characteristics of a PTC reaction for the purpose at hand. In nucleophilic displacement step-growth polymerizations, for example in the synthesis of polyethers and polycarbonates, these characteristics are as follows: (a) the reaction is very fast, reaching 100% yield and high molecular weight (mol wt) in a few minutes; (b) the polymer's mol wt does not depend strongly on the ratio between the nucleophilic and electrophilic reactants as in conventional step polymerizations; and, (c) the obtained polymer almost always contains electrophilic species as chain ends, independent of the reaction yield and reactant ratio.
It is hypothesized that the etherification reaction occurs in the organic phase according to a mechanism similar to that of interfacial polycondensation. The concentration of reactive bisphenolate in the organic phase is controlled by the concentration of the PTC and is very low in comparison with that of the electrophilic monomer. I eventually came to realize that PTC reactions can be exploited as a simple method for the synthesis of telechelic polymers containing electrophilic chain ends. These polymers with functional electrophilic end groups are useful polymeric materials because they can be further used as macroinitiators for cationic polymerization and for synthesis of ordered and block copolymers by condensation polymerization in the presence of a PTC.
The particular interest of this invention is to tailor an (A)(B) type block copolymer of polyarylene polyether ("PAPE") segments so that the block copolymer exhibits desired properties, for example, the ability to withstand thermal degradation at a temperature in the range from above 100.degree. C. to about 200.degree. C.
This invention is more particularly related to block copolymers formed by combining a PAPE segment A having phenolic (Ph) or thiophenolic (TPh) chain ends with a PAPE segment B having haloallylic chain ends. Segment A consists essentially of polymers of dihydroxybenzene, dihydroxynaphthalene, and diphenols, all referred to herein as dihydric phenols ("DHP"), and the corresponding sulfur (thio) compounds referred to as dihydric thiophenols ("DHTP"), which polymers have a Mn (number average mol wt) less than about 10,000, hence termed oligomers. One or the other DHP and DHTP, or both, are referred to herein as "DH(T)P" for brevity. Such oligomers are defined herein as polymers containing from 2 to about 100 repeating units each having the formula--DH(T)P.sub.1 --DH(T)P.sub.2 --, where DH(T)P.sub.1 and DH(T)P.sub.2 each represents the residue of a DH(T)P. These oligomers contain at least three phenyl or thiophenyl rings which may have inert substituents, each ring linked to another through an O, Si, C or S atom. Such DHP and DHTP oligomers, also, poly[DH(T)P], or [DH(T)P].sub.n, are terminated at each end (hence "di-terminated") with a phenol ("Ph") or thiophenol ("TPh") group respectively, which group may also have inert substituents. For brevity, "di-(T)Ph-terminated" refers herein to either or both oligomers which are Ph- and TPh-terminated respectively, the preparation of which oligomers is described in detail in my copending U.S. patent application Ser. No. 586,678 filed Mar. 6, 1984 the disclosure of which is incorporated by reference thereto as if fully set forth herein.
Specific alternating block copolymers and regular (chain extended) polymers and details of their preparation, analyses of the copolymers obtained, and a discussion of various aspects of their preparation and results of the analyses, are provided in an article titled "Functional Polymers and Sequential Copolymers by Phase Transfer Catalysis. 6. On the Transfer Catalyzed Williamson Polyetherification as a New Method for the Preparation of Alternating Block Copolymers" by Virgil Percec, Brian C. Auman and Peter L. Rinaldi, Polymer Bulletin 10, 391-396 (1983), and in another article titled "Functional Polymers and Sequential Copolymers by Phase Transfer Catalysis, 1.-Alternating Block Copolymers of Unsaturated Polyethers and Aromatic Poly(ether sulfone)s" by Virgil Percec and Brian C. Auman, Makromol. Chem. 185 617-627 (1984), the disclosures of which articles and relevant portions of the references cited therein are incorporated by reference thereto as if fully set forth herein. |
All India Netaji Revolutionary Party
All India Netaji Revolutionary Party, was a political party in India. AINRP was led by V.P. Saini, who is also the president of the Netaji Research Foundation. The party was founded in connection with the 5th All India Netaji Convention 1999. The party was formed in order to work the ideology of Netaji Subhas Chandra Bose. The party demanded a through investigation into what happened to Netaji Subhas Chandra Bose, about whom the circumstances of his death are still unclear.
AINRP was critical of the collaboration of the left (Communist Party of India (Marxist) and All India Forward Bloc) with the Indian National Congress. In the Lok Sabha elections of 1999 V.P. Saini was launched as a candidate in the constituency of Hoshiarpur in Punjab. Saini only got 942 votes (0,16%).
Later Saini joined All India Forward Bloc, and became the secretary of the central committee. In 2003 Saini visited North Korea as part of an AIFB-delegation from Punjab.
References
External links
Article in Indian Express
Another article in Indian Express
Article in Indian Express about the foundation of the party
Category:Defunct political parties in Punjab, India
Category:Memorials to Subhas Chandra Bose |
**Cite this article as:** Mohsenpour B, Ahmadi A, Mohammadi Baneh A, Hajibagheri K, Ghaderi E, Afrasiabian Sh, Azizi S. Relation between serum zinc levels and recurrent urinary tract infections in female patients: A case-control study. *Med J Islam Repub Iran*. 2019 (22 Apr);33:33. https://doi.org/10.34171/mjiri.33.33
↑ What is "already known" in this topic: {#box1}
========================================
According to the results of this study, It seems that serum zinc level is a risk factor for recurrent urinary tract infections.
→ What this article adds: {#box2}
=========================
There is a relationship between low serum zinc level and urinary tract infection. Therefore, a clinical trial is required for people with UTIs.
Introduction {#s1}
============
Urinary tract infections (UTIs) are more common in women and become more prevalent with age ([@R1]-[@R3]). Prevalence of UTIs is 10% among people aged 4--7 years old while it is about 52% in people aged 18--26 years old ([@R4], [@R5]). Recurrence is one of the complications of the disease, and its prevalence is different. In a follow-up study, the recurrent disease was observed once in 27% of patients and twice in 2.7% of UTI patients. Recurrent UTIs are defined as incidences of UTIs at least twice in six months or three or more times in one year ([@R6]).
Zinc is a micronutrient, and its deficiency can increase the risk of infectious diseases ([@R7]). Zinc is involved in the regulation of the host immune system, and its moderate deficiency leads to the dysfunction of the immune system. Zinc is necessary for the development and activity of T lymphocytes, and its deficiency leads to decreased levels of cellular immunity ([@R8]). Host cells can change the cytoplasmic and lysosomal level of zinc to make a response to the bacteria; in addition, the accumulation of zinc in phagolysosome and macrophages helps to control pathogens. The bacterial poisoning caused by zinc can be a defense mechanism used by macrophages to clear the infection ([@R9]). Zinc deficiency is associated with bad outcomes in response to bacterial infections and sepsis ([@R10]). So, it seems that zinc levels can be a risk factor for recurrent UTIs. This study compared serum zinc levels between women with and without recurrent UTIs.
Methods {#s2}
=======
This case-control study was conducted after aprooval by the Ethics Committee of Kurdistan University of Medical Sciences (MUK.REC.1392.158). Considering a 1% error rate of 0.05, mean zinc level of 105.9 (±23.7) in healthy subjects and 95.4 (±21.8) in patients, and second type error of 10%, sample size was calculated as 48 in each group ([@R10]). To enroll the subjects into the two groups, they were briefed about the objectives of the study and informed consent was obtained. The study participants included women with recurrent UTIs who referred to Tohid Hospital or the Clinic of Infectious Diseases. Initial screening was performed by an infectious diseases specialist who examined patients to detect signs and symptoms of UTIs.
UTIs are defined as: presence of dysuria, or/and frequency, and suprapubic pain or/and tenderness, or/and costovertebral tenderness, or/and fever, or/and chills with WBC equal or more than 10 /hpf in centrifuged urine smear and presence of organism compatible with UTI more than 100000 in urine culture. After initial screening, the inclusion criteria were defined as recurrent UTIs or UTI at least twice in the last six months, or UTI at least three times over the past year, and being older than 12 years. It is worth noting that only incidences were enrolled into the study (i.e. those who had not been treated for recent infection). Considering the exclusion criteria, we did not enroll those who consumed zinc in the past year, immunocompromised patients, dialysis patients, patients receiving corticosteroid therapy, patients with cancer, and patients with malabsorption.
The control group consisted of those referred to clinics to undergo routine tests or to accompany patients and consented to participate in this study. They underwent primary screening, which was performed by an infectious disease specialist; in the absence of UTI symptoms or a history of past UTIs, they were selected as members of the control group. The control group matched with the cases in terms of age and place of residence (urban/rural) by group matching. To ensure the absence of UTIs, participants in the control group underwent urinalysis and a urine culture test. The exclusion criteria used for the control group were similar to those used for test group.
Blood samples (5 cc) were taken from the subjects who met the inclusion criteria; to measure the serum level, the collected blood samples were sent to the laboratory. The subjects in the control group underwent the same procedure and the tests were performed by one laboratory. To measure the serum zinc levels, a zinc calorimetry test was performed using the BT 3000 auto analyzer device (made in Italy) and the Bio rex kit (made in the UK).
Statistical analysis {#s2-0-1}
--------------------
After entering the collected data into SPSS ver. 18, charts and tables were used to describe the variables. A Chi-square test and Fisher test were used to compare qualitative variables between the two groups; moreover, the t-test and Pearson correlation coefficient were used to compare quantitative values. Then, multiple linear regression was used to determine factors affecting serum zinc levels. The significance level was set at 0.05.
Results {#s3}
=======
The mean age of the participants in the test group and the control groups was 53.37 (±19.2) and 52.70 (±19.33) years, respectively (p=0.865). The mean serum zinc levels of the participants from urban and rural areas were 86.78 (±17.32) and 86.75 (±20.6) microgram/deciliter, respectively (p=0.995). There was a weak correlation between age and serum zinc level (r=-0.205, p=0.045). Mean serum zinc level of the test group and the control group was 96.83 (±11.25) and 76.72 (±17.06) microgram/deciliter (p=0.001), respectively.
The results of multiple linear regression ([Table 1](#T1){ref-type="table"}) shows that the level of zinc reduced with age; in addition, the group with recurrent UTIs had lower zinc levels than the control group (p=0.010, R^2^=0.377).
###### Relationship between serum zinc level and other variables after adjusting for other factors using a regression model
--------------------------------------- --------------------------- ----------------------- ------------- -------------- -------- --------
Model Non-standard coefficients Standard coefficients p 95% CI for B
Beta Coefficient Std. Error Beta Coefficient Lower bound Upper bound
Constant coefficient 1 07.82 6.75 \<0.001 94.40 121.23
Age (years) -0.198 0.076 -0.215 0.010 -0.34 -0.04
Location (urban / rural) -0.384 4.35 -0.007 0.930 -9.02 8.25
Group (recurrent infection / control) -20.23 2.87 -0.579 0.001 -25.95 -14.52
--------------------------------------- --------------------------- ----------------------- ------------- -------------- -------- --------
Decreased serum zinc levels was related to increased age and recurrent infections after adjusting for other variables.
Discussion {#s4}
==========
According to the results of this study, people with recurrent UTIs had lower serum zinc levels than the control group; this difference was also observed after adjusting for age and location. Javadinia et al. ([@R11]) showed that serum zinc levels were significantly lower in children with UTIs than in the control group. In our study, we observed the same result. This confirms that serum zinc levels in patients with UTIs are lower than healthy individuals. It should be noted that our study was conducted on patients with recurrent UTIs, which was different from Javadinia et al. ([@R11]). The results of our study showed that serum zinc levels decreased with age and it was observed in both groups, which indicates a reverse relationship between age and serum zinc level. Given the fact that with aging there is an increase in the incidence of UTIs, it can be hypothesized that, in addition to various causes for the increased incidence of UTIs in old age, zinc deficiency can also be regarded as one of the reasons.
Hancock et al. ([@R12]) indicated that the antimicrobial and anti-biofilm effects of zinc on urinary tract pathogens, E.coli, and Klebsiella was investigated; it was observed that the divalent zinc was able to inhibit the mechanisms of biofilm formation by the studied organisms in order to apply its antimicrobial mechanism. Therefore, it might be concluded that frequent recurrences may be due to the formation of biofilm in the urinary tract or stones in the urinary system.
Combination of some antibiotics with zinc results in a synergistic effect against organisms and might be effective in decreasing recurrent infections. Zinc is a cofactor of more than 200 enzymes that are necessary for the metabolic activity of cells. Additionally, zinc has an important role in immune system physiology and is important for the development and activity of T lymphocyte. Decreased serum zinc levels cause decreased immune system activity. Zinc has an important role to play in the production of gamma interferon, interleukin-2, and interferon alpha. Zinc is necessary for radical detoxification and antioxidant defenses. In patients with UTIs, oxidative stress is increased and antioxidant levels is decreased. Markers of oxidative stress such as malondialdehyde in UTIs is increased; whereas, serum cations such as Cu, Ca, and Zn are decreased ([@R13]).
The above-mentioned conclusions are consistent with the results of our study. The administration of antibiotics together with an appropriate level of serum zinc could result in a synergistic ability to eradicate organisms in the urinary system.
This study had some limitations. We could not follow patients with low levels of serum zinc. Follow up of these patients can clearly define the possible relation of zinc deficiency and recurrent UTI.
Conclusion {#s5}
==========
It can be suggested that recurrent UTIs are associated with low serum zinc levels. Therefore, it is recommended to conduct a clinical trial to study the effects of zinc supplementation on patients with recurrent UTIs and to determine their effectiveness in the prevention or treatment of recurrent UTIs.
Acknowledgments {#s6}
===============
This paper was extracted from a thesis belongs to Samaneh Azizi and would like to thank Kurdistan University of Medical Sciences and Research Deputy of Kurdistan University of Medical Sciences for financial support.
Conflict of Interests {#s7}
=====================
The authors declare that they have no competing interests.
|
Introduction {#S1}
============
Characterizing chromosome folding is fundamental to better understand gene expression and how it possibly constrains genome evolution. Chromosome conformation capture (3C) methods, and notably its high-throughput sequencing-based derivatives such as Hi-C, 5C and 4C[@R1], have significantly contributed to current understanding of genome architecture revealing that chromosome folding is driven by at least two independent mechanisms. On the one hand, the mutually exclusive associations between transcriptionally active or inactive loci generate the so-called A and B compartments[@R2]. On the other hand, chromatin loops are formed between regulatory sequences and between convergent CTCF binding sites, the latter through cooperative action between cohesin and the DNA-binding protein CTCF[@R3]. The interplay between compartmentalization and CTCF-cohesin looping results in complex hierarchies of folding domains[@R4],[@R5], among which topologically associating domains (TADs)[@R6]--[@R8] stand out as preferential functional units[@R9]. The involvement of CTCF in loop formation has been demonstrated using global depletion experiments[@R10],[@R11], as well as targeted deletions and inversions of CTCF sites leading to loss of looping interactions[@R12]--[@R14]. The underlying mechanisms are however still incompletely understood. An influential hypothesis is that CTCF-mediated interactions occur as cohesin extrudes chromatin loops until it is blocked by CTCF bound to DNA in a defined orientation[@R15]. According to this hypothesis, ectopic insertion of CTCF sites could result in newly established loops onto endogenous CTCF sites, depending on their mutual orientation. Whether this actually occurs and how it modifies interactions within TADs has however not been demonstrated.
In 3C, detection of spatial proximity relies on formaldehyde crosslinking followed by digestion and ligation of crosslinked chromatin[@R1]. Crosslinking and ligation are sources of potential experimental bias, raising the question of whether structures detected by 3C methods actually exist in living cells[@R16]--[@R19]. 3C crosslinking frequencies are assumed to be proportional to absolute chromosomal contact probabilities and used to build mechanistic physical models of chromosome folding[@R20],[@R21], including the loop-extrusion model[@R14],[@R15],[@R22],[@R23]. However, formal proof of this assumption is missing. Independent techniques such as DNA fluorescence in situ hybridization (DNA FISH)[@R6],[@R24], genome architecture mapping (GAM)[@R25], native 3C[@R26] and split-pool recognition of interactions by tag extension (SPRITE)[@R27] have also detected loops, TADs and compartments. Nevertheless, these methods still involve substantial biochemical manipulation of cells, and employ either crosslinking or ligation.
An alternative approach to study chromosomal contacts without crosslinking and ligation is recruiting an ectopic DNA modifying enzyme to specific genomic locations, and detecting chemically modified DNA at sequences that physically interact with the recruitment sites. Three previous studies have provided proof of principle for such an approach using a modified version of DamID[@R26],[@R28],[@R29]. In DamID, the bacterial DNA adenine methyltransferase Dam is fused to a DNA-binding protein resulting in adenine methylation within GATC motifs in the neighborhood of the protein DNA binding sites[@R30]. Methylated GATCs (GmATC) are specifically digested by the DpnI restriction enzyme, allowing to determine DNA binding locations of the fusion protein after normalization for nonspecific methylation by freely diffusing Dam. Methylation at distal chromosomal sites interacting with the viewpoint in 3D can also be observed[@R26],[@R28],[@R29] if interaction-specific methylation is significantly higher than nonspecific methylation. However, previous studies detected methylated DNA with semi-quantitative PCR readouts and analyzed interactions of one viewpoint with a limited number of restriction sites, similar to early 3C experiments[@R31]. This and the lack of formal schemes to convert methylation states into contact probabilities have prevented these versions of DamID from reaching the resolution and throughput needed to detect TAD boundaries and CTCF loops. Thus to date no crosslinking- and ligation-free method is available to study chromosome interactions in the context of contacts made by all other surrounding genomic sequences. Remarkably, evidence that CTCF-associated loops exist is based exclusively on crosslinking methods.
Here we present DamC, a new modified version of DamID coupled to physical modelling of DNA methylation kinetics. In DamC, Dam is recruited to ectopically inserted Tet operators (TetOs) through fusion to the reverse tetracycline receptor (rTetR). Methylated DNA is detected by high-throughput sequencing allowing the identification of chromosomal contacts at high genomic resolution across hundreds of kilobases around viewpoints. Modelling of this process shows that experimental output in DamC is proportional to chromosomal contact probabilities, providing a theoretical framework for the interpretation of data.
Using DamC we provide the first crosslinking- and ligation-free validation of structures identified by 3C methods. By comparing DamC with 4C-seq and Hi-C at hundreds of genomic locations in mouse embryonic stem cells (mESC), we confirm the existence of TADs and CTCF loops. We also show that the scaling of contact probabilities measured in DamC is the same as in 4C and Hi-C, providing evidence in favor of current interpretations of 3C-based data in terms of physical models of chromosome folding. We additionally demonstrate that ectopic insertion of CTCF sites can lead to the formation of new loops with endogenous CTCF-bound sequences and alter sub-TAD contacts. This shows that chromosome structure can be manipulated by inserting short ectopic sequences that rewire interactions within TADs.
Results {#S2}
=======
DamC: Methylation-based detection of chromosomal contacts *in vivo* {#S3}
-------------------------------------------------------------------
Based on the results of previous studies[@R26],[@R28],[@R29], we reasoned that fusing Dam to rTetR and inserting an array of TetOs in the genome would ensure targeted, inducible recruitment of large numbers of Dam molecules to a specific genomic viewpoint in the presence of doxycycline (Dox) ([Figure 1a](#F1){ref-type="fig"}, left). In the absence of Dox, rTetR-Dam would not bind to the viewpoint, allowing accurate estimation of nonspecific methylation ([Figure 1a](#F1){ref-type="fig"}, right) and precise background correction. Coupled to high-throughput sequencing, this strategy could provide 4C-like, 'one vs. all' profiles[@R32] of contact probabilities from the TetO viewpoint ([Figure 1a](#F1){ref-type="fig"}) across large genomic distances and at high genomic resolution (one GATC every \~250bp on average). Inserting multiple TetO arrays separated by large genomic distances would allow interrogating chromosomal interactions in parallel from many viewpoints ([Figure 1b](#F1){ref-type="fig"}). We refer to this method as 'DamC'.
To test this approach, we employed female mESCs carrying an array of 256 TetOs at the 3' end of the *Chic1* gene within the X inactivation center[@R33]. We transfected an X0 subclone of these cells with an rTetR-Dam expression plasmid and measured methylation after 24 hours[@R34]. Quantification of the methylated GmATCs by high-throughput sequencing revealed significantly higher methylation upon Dox induction compared to the uninduced control over approximately 300kb around the TetO viewpoint ([Figure 1c](#F1){ref-type="fig"}). Thus, targeted recruitment of Dam leads to increased methylation in *cis* over long genomic distances, consistent with previous observations using semi-quantitative methods for the detection of methylation[@R26],[@R28],[@R29]. Since methylation is determined by the interplay between methyltransferase activity and passive demethylation during DNA replication, we reasoned that it should be possible to model this process and derive chromosomal contact probabilities from sequencing readouts.
DamC enrichment is proportional to chromosomal contact probabilities {#S4}
--------------------------------------------------------------------
The methylation level of a single GmATC is determined by a dynamic interplay between methylation (by freely diffusing or TetO-bound Dam) and passive demethylation by DNA replication, when a fully methylated GmATC becomes two hemi-methylated sites that are essentially not detected in DamID[@R35] ([Figure 2a](#F2){ref-type="fig"}). To identify experimental quantities that are directly proportional to chromosomal contact probability, we generated a physical model describing the time evolution of methylation at an arbitrary genomic distance from the TetO viewpoint ([Supplementary Note 1](#SD2){ref-type="supplementary-material"}) through rate equations ([Figure 2b](#F2){ref-type="fig"}) which take into account that methylation by TetO-bound Dam only occurs in the presence of Dox ([Figure 1a](#F1){ref-type="fig"}). Methylation rates are allowed to depend on local biases (e.g. chromatin accessibility or mappability). Under the assumptions that methylation is faster than demethylation[@R35] and the duration of an experiment, we found that contact probabilities between the GATC site and the TetO viewpoint are directly proportional to a measurable quantity. This quantity, which we refer to as 'DamC enrichment', is simply the relative difference between methylation levels in the presence and absence of Dox ([Figure 2c](#F2){ref-type="fig"}). Thus, DamC can directly measure chromosomal contact probabilities.
For a given contact probability between the GATC site and the TetO viewpoint, the model predicts that DamC enrichment depends on: 1) the nuclear rTetR-Dam concentration, 2) the rTetR-Dam binding affinity for the TetO array, and 3) the average nonspecific binding affinity of rTetR-Dam for endogenous genomic sites ([Figure 2c](#F2){ref-type="fig"}). DamC enrichment does not depend on local methylation biases and therefore should not be affected by differential accessibility or mappability, provided that interactions with the TetO viewpoint can increase methylation at the GATC site (i.e. local methylation is not saturated in the absence of Dox). In real experiments, where binding affinities are fixed, the main determinant of DamC enrichment is the nuclear concentration of rTetR-Dam. In particular, DamC enrichment should be maximal when rTetR-Dam concentration is around the nuclear concentration of TetO viewpoints ([Supplementary Figure 1a](#SD1){ref-type="supplementary-material"}), and negligible when the concentration is very high or very low ([Figure 2d](#F2){ref-type="fig"}). This does not depend on the particular values of the affinity constants ([Supplementary Figure 1b](#SD1){ref-type="supplementary-material"}) and implies that maximal DamC enrichment occurs at different Dam concentrations depending on the number of viewpoints. Thus, modeling predicts that accurate control of rTetR-Dam nuclear concentrations is needed to perform DamC with optimal signal-to-noise ratio.
DamC from hundreds of genomic viewpoints validates model predictions {#S5}
--------------------------------------------------------------------
To test model predictions and measure chromosomal interactions using DamC, we established mESCs allowing to control the rTetR-Dam nuclear concentration. We first created a stable cell line expressing rTetR fused with enhanced green fluorescent protein (EGFP), Dam, and the mutant estrogen ligand-binding domain ERT2. ERT2 ensures cytoplasmic localization of the fusion protein in the absence of 4-hydroxy-tamoxifen (4-OHT), preventing constitutive GATC methylation. It also enables to control its nuclear level by changing 4-OHT concentrations in the culture medium ([Figure 3a](#F3){ref-type="fig"}) as confirmed by increasingly nuclear accumulation of EGFP upon increasing 4-OHT doses ([Supplementary Figure 2a](#SD1){ref-type="supplementary-material"}).
To measure chromosomal interactions in a wide variety of randomly selected genomic contexts in parallel, we further inserted arrays of 50 TetOs (each spanning approx. 2.7kb) using the piggyBac transposon system[@R36] ([Figure 3c](#F3){ref-type="fig"}). This resulted in clonal mESC lines carrying at least 100 TetO arrays, judging from EGFP accumulation in nuclear foci in the presence of 4-OHT and Dox ([Figure 3c](#F3){ref-type="fig"}). We further selected one polyclonal population carrying a total of 890 TetO array insertions and one clonal line with 135 insertions (**Supplementary Table 1 and 2**), as determined by mapping piggyBac insertion locations ([Methods](#S11){ref-type="sec"}). To quantitatively measure rTetR-Dam nuclear concentrations as a function of 4-OHT concentration, we analyzed nuclear protein extracts using mass spectrometry. Combining the proteomic ruler strategy with parallel reaction monitoring (PRM) ([Methods](#S11){ref-type="sec"}, [Supplementary Figure 2b-c](#SD1){ref-type="supplementary-material"} and **Supplementary Table 3**) we estimated nuclear rTetR-Dam concentrations to vary gradually between approximately 3 and 25 nM, and 1 and 6 nM in the polyclonal and pure clonal lines, respectively, when increasing the 4-OHT concentration from 0.1 to 500 nM ([Figure 3b](#F3){ref-type="fig"}).
We performed DamC after treating cells overnight with different doses of 4-OHT in the presence and in the absence of Dox. Experiments were performed using a custom next-generation sequencing library preparation protocol that includes unique molecular identifiers (UMI) and increases the coverage of methylated GATC sites genome-wide, thus maximizing proportionality between methylation levels and sequencing readout ([Supplementary Figure 2d-e](#SD1){ref-type="supplementary-material"}, [Methods](#S11){ref-type="sec"}). We quantified DamC enrichment in the immediate vicinity of TetO viewpoints, and plotted it as a function of rTetR-Dam concentration quantified by mass spectrometry ([Figure 3d](#F3){ref-type="fig"}). For the polyclonal line we considered the 100 insertions with highest signal-to-noise ratios, corresponding to the most abundant insertions. In the pure subclone, all insertions showed similar enrichment levels except five, possibly as a consequence of recombination of the TetO array or high levels of transcription at the insertion point preventing TetO binding. These insertions were discarded from analysis and their coordinates are provided in the [Methods](#S11){ref-type="sec"} section.
Consistent with model predictions, in the polyclonal mESC line maximum enrichment occurs at \~3 nM corresponding to \~860 viewpoints ([Figure 3d](#F3){ref-type="fig"}, upper panel). Model fitting returned an estimate of 0.4 nM for the specific rTetR-TetO binding constant, in the range of *in vitro* measurements[@R37], and 17nM for the average non-specific binding constant. Again in line with the model, enrichment in the clonal line carrying \~7-fold less viewpoints (130) was compatible with maximal enrichment occurring at \~7-fold lower rTetR-Dam nuclear concentration (0.4 nM). These results provide a validation of the DamC model and support the interpretation of the DamC enrichment in terms of contact probabilities. They additionally highlight that in our experimental system, maximal DamC enrichment in cells with \~100 insertions is observed in a range of rTetR-Dam nuclear concentrations corresponding to 0.1-1 nM 4-OHT ([Supplementary Figure 2f](#SD1){ref-type="supplementary-material"}). In the following analysis, reads from these two conditions were pooled to maximize read coverage.
DamC reveals the existence of TADs and loops *in vivo* {#S6}
------------------------------------------------------
Under optimal 4-OHT concentrations (0.1-1 nM pooled), zooming into individual TetO viewpoints in the clonal line with 130 insertions revealed significant DamC enrichment over hundreds of kilobases around each viewpoint ([Fig. 4a](#F4){ref-type="fig"}). Since biological replicates were highly correlated ([Supplementary Figure 3a](#SD1){ref-type="supplementary-material"}), we analyzed merged data. DamC enrichment profiles showed remarkable agreement with 4C performed using the same TetO arrays as viewpoints and DpnII as primary restriction enzyme ([Figure 4a](#F4){ref-type="fig"} and [Methods](#S11){ref-type="sec"}). DamC enrichment was systematically concentrated within TAD boundaries detected in Hi-C ([Figure 4a](#F4){ref-type="fig"}) and steeply decayed across TAD boundaries by roughly a factor two, in excellent agreement with 4C ([Figure 4b](#F4){ref-type="fig"}). Only a minor fraction of TetO insertions occurred in close proximity (\<1kb) to either an active regulatory element or a CTCF site ([Supplementary Figure 3b](#SD1){ref-type="supplementary-material"}). Also in these cases, DamC enrichment profiles highly overlapped with 4C ([Figure 4c](#F4){ref-type="fig"}) and revealed looping interactions between endogenous convergent CTCF sites ([Figure 4c](#F4){ref-type="fig"}, **left**), which were confirmed using the partner CTCF sites as reciprocal viewpoint in 4C ([Supplementary Figure 3c](#SD1){ref-type="supplementary-material"}). The targeted TetO insertion at the 3' end of *Chic1*[@R33] ([Figure 1c](#F1){ref-type="fig"}) allowed measuring chromosomal interactions within the the well-characterized *Tsix* TAD[@R6],[@R38]. In accordance with 4C, DamC recapitulated the previously observed CTCF-mediated interactions between *Chic1*, *Linx* and *Xite/Tsix*[@R6], as well as the boundaries of the *Tsix* TAD ([Supplementary Figure 3d](#SD1){ref-type="supplementary-material"}). Additional DamC and 4C profiles are plotted in [Supplementary Figure 4](#SD1){ref-type="supplementary-material"} and bedGraph tracks are available online (Data Accessibility section).
We next investigated whether despite evident global similarities, DamC and 4C showed local differences. We defined a deviation score measuring differences between DamC and 4C interaction profiles within windows of 20 DpnI/II restriction fragments (5kb on average) ([Supplementary Figure 3e](#SD1){ref-type="supplementary-material"}). Most dissimilar windows were enriched in active chromatin marks ([Supplementary Figure 3f](#SD1){ref-type="supplementary-material"}), although local differences between DamC and 4C within these regions are relatively mild ([Supplementary Figure 3e](#SD1){ref-type="supplementary-material"}). We reasoned that local discrepancies might be due to the fact that the methylation signal highly correlates with chromatin accessibility measured by DNase I sensitivity ([Supplementary Figure 3g](#SD1){ref-type="supplementary-material"}). Correction by nonspecific methylation generally normalizes for chromatin accessibility in the DamC enrichment, unless GATC sites are highly methylated in the absence of Dox preventing further increases when interacting with the TetO viewpoint in +Dox conditions. However, only 0.05% of GATC sites within DNase I hypersensitive regions were saturated ([Methods](#S11){ref-type="sec"}), and masking DNase I hypersensitive sites did not increase the overall similarity between DamC and 4C profiles ([Supplementary Figure 3h](#SD1){ref-type="supplementary-material"}). Thus local differences between the two techniques are not due to saturated methylation levels, and are possibly due to experimental factors not described by the DamC model and thus not accounted for in the calculation of DamC enrichment.
In summary, crosslinking- and ligation-free measurements of contact probabilities using DamC quantitatively agree with 4C, confirm the existence of TAD boundaries and show that crosslinking and ligation do not significantly distort the detection of chromosomal interactions.
piggyBac-TetO insertions do not perturb chromosome structure {#S7}
------------------------------------------------------------
Next, we set off to understand whether insertion of TetO/piggyBac cassettes themselves could perturb local chromosome structure. We compared TetO insertion sites with the corresponding WT loci in Hi-C experiments ([Supplementary Figure 5a](#SD1){ref-type="supplementary-material"}) using a modified version of deviation score defined in [Supplementary Figure 3d](#SD1){ref-type="supplementary-material"} describing differences in virtual 4C profiles extracted from Hi-C data ([Supplementary Figure 5b](#SD1){ref-type="supplementary-material"}). Deviation scores between WT cells and cells carrying TetO arrays were similar to those between Hi-C replicates at random genomic locations, and significantly smaller than those between different WT loci ([Supplementary Figure 5b](#SD1){ref-type="supplementary-material"}). Finally, 4C profiles obtained with and without TetO viewpoints were indistinguishable ([Supplementary Figure 5c](#SD1){ref-type="supplementary-material"}). Thus, piggyBac-mediated insertion of TetO arrays does not lead to measurable perturbations on chromosome structure.
*In vivo* detection and manipulation of CTCF-mediated interactions {#S8}
------------------------------------------------------------------
Loops between convergent CTCF sites are a defining feature of chromosome architecture. However, it is unclear whether new loops can be established between endogenous and ectopically inserted CTCF sites. Early 3C observations suggested that ectopic sequences containing CTCF sites can change the surrounding chromosomal interactions[@R39],[@R40]; however, experimental resolution in 3C did not allow to resolve single CTCF loops, and inserted sequences contained additional regulatory regions. Since piggyBac-TetO constructs alone do not perturb chromosome structure, we further engineered them to insert ectopic CTCF sites in the genome and detect resulting structural modifications without confounding effects.
Starting from the founder rTetR-GFP-Dam-ERT2 mESC line described in [Figure 3a](#F3){ref-type="fig"}, we randomly introduced modified piggyBac cassettes where the TetO array is flanked by three CTCF sites oriented outwards ([Figure 5a](#F5){ref-type="fig"}). To test if ectopically inserted CTCF sites could establish loops with endogenous CTCF sites ([Figure 5b](#F5){ref-type="fig"}), we selected one clone carrying 91 insertions, for which we could map insertion positions and genomic orientations (**Supplementary Table 4**), and performed 4C and DamC with 0.1-1 nM 4-OHT.
Interaction profiles from TetO-CTCF viewpoints displayed prominent distal peaks ([Figure 5c](#F5){ref-type="fig"}) detected by both DamC and 4C. We used the PeakC algorithm, developed to analyze 4C profiles[@R41], to identify distal preferential interactions. Using stringent thresholds ([Methods](#S11){ref-type="sec"}) and excluding viewpoints within 1kb from an endogenous CTCF site ([Supplementary Figure 3b and Supplementary Figure 6a](#SD1){ref-type="supplementary-material"}), we detected 38 specific interactions separated by at least 20 kb from single TetO-CTCF viewpoints (\~0.5 distal peaks per insertion site on average, [Supplementary Figure 6b](#SD1){ref-type="supplementary-material"}). Of those, 74% contained one or more bound CTCF sites based on ChIP-seq datasets in mESC[@R11], predominantly (79%) convergent with the ectopic CTCF insertion ([Figure 5d](#F5){ref-type="fig"}). As a comparison, in the cell line harboring TetO viewpoints without CTCF we detected only 0.1 peaks per insertion site ([Supplementary Figure 6b](#SD1){ref-type="supplementary-material"}), of which 58% contained one or more bound CTCF sites ([Figure 5d](#F5){ref-type="fig"}). These correspond to endogenous CTCF loops since in virtually all these cases, the TetO was located between 1 and 20 kb away from an endogenous CTCF. Thus, peaks in the TetO-CTCF line are likely to coincide with new loops established by ectopic CTCF sites. Insertions without distal peaks predominantly correspond to TetO-CTCF cassettes integrated either in CTCF 'deserts', or conversely close (\<30kb) to the nearest endogenous convergent CTCF site and in regions with many endogenous CTCF sites ([Supplementary Figure 6c](#SD1){ref-type="supplementary-material"}), resulting in short-distance loops that are difficult to distinguish in 4C and DamC profiles. Additional TetO-CTCF DamC and 4C profiles are plotted in [Supplementary Figure 7](#SD1){ref-type="supplementary-material"} and bedGraph tracks are available online (Data Accessibility section).
We then performed Hi-C in the TetO-CTCF line and compared it to the data obtained from TetO-only mESCs (see [Figure 4a](#F4){ref-type="fig"}), where insertion locations are different. Since TetO-CTCF insertions are heterozygous, the Hi-C readout is confounded by the presence of a WT allele. Nevertheless, in a fraction of insertions showing prominent distal CTCF peaks in 4C and DamC, we could detect the formation of new structures in Hi-C and notably new loops ([Figure 6a](#F6){ref-type="fig"}, arrows and [Supplementary Figure 6d](#SD1){ref-type="supplementary-material"}) leading to increased partitioning of interactions within TADs and the appearance of sub-TAD boundaries ([Figure 6b](#F6){ref-type="fig"}). Ectopic CTCF insertion also reinforced pre-existing interactions between convergently oriented sites ([Figure 6a](#F6){ref-type="fig"}, arrowheads), possibly by bringing them closer by effect of the new loops. Even insertions without prominent distal CTCF peaks could be associated with new structures ([Figure 6c](#F6){ref-type="fig"}) reminiscent of stripes predicted by the loop extrusion model[@R15] and recently observed in Hi-C data at endogenous locations[@R42]. Consistent with the loop extrusion model interpretation, the stripe shown in [Figure 6c](#F6){ref-type="fig"} occurred at a location where the three ectopic CTCF sites landed close to a cluster of Nipbl sites, and far from the nearest convergent CTCF sites (\~800 kb). Formation of an ectopic CTCF-associated stripe also resulted in modifications of intra-TAD chromosomal interactions ([Figure 6d](#F6){ref-type="fig"}).
Finally, to formally prove that new structures are induced by ectopic CTCF binding sites (rather than the piggyBac-TetO cassette), we removed the three CTCF sites by Cre-assisted recombination using two flanking LoxP sites ([Supplementary Figure 6e](#SD1){ref-type="supplementary-material"}). DamC performed in one mESC clone where CTCF sites had been excised at both loci shown in [Figure 6a-b](#F6){ref-type="fig"} ([Supplementary Figure 6f](#SD1){ref-type="supplementary-material"}) revealed that removal of CTCF sites led to loss of distal interactions ([Figure 6e](#F6){ref-type="fig"}).
In summary, DamC identifies chromatin loops formed through specific long-range chromatin interactions. Additionally, our data shows that ectopically inserted CTCF sites can establish new loops with endogenous CTCF sites and stripes, leading to modified partitioning of interactions within TADs.
Quantitative properties of chromosome folding *in vivo* {#S9}
-------------------------------------------------------
Given the high similarity between DamC and 4C both with and without CTCF sites at the viewpoint, we next asked whether DamC and 3C-based techniques measured the same scaling of interaction probabilities. We pooled all viewpoints from TetO-only and TetO-CTCF lines and plotted the data as a function of genomic distance from the viewpoints. For distances between 15 kb and 1 Mb, fitting both DamC and 4C with a power law resulted in decay exponents around 0.9, in excellent agreement with Hi-C from the same cells and viewpoints ([Figure 7a](#F7){ref-type="fig"}), and in accordance with previous measurements in similar genomic ranges[@R14],[@R43].
Below \~10 kb, both DamC and 4C showed a gentler decay as recently observed in Hi-C experiments on yeast chromosomes[@R44]. In Ref.[@R44] this was attributed to crosslinking artefacts but DamC, showing the same behavior, argues against this explanation. The leveling-off of contact probabilities at short genomic distances can be explained in terms of a simple coarse-grained polymer model with a persistence length of \~2.5 kb ([Figure 7b](#F7){ref-type="fig"}, [Supplementary Note 1](#SD2){ref-type="supplementary-material"}). We cannot formally rule out alternative explanations such as experimental factors not accounted by the DamC model and thus not normalized in the calculation of enrichment. One such scenario could be that the spacing between GATC sites imposes an effective capture range of few kb, consistent with micrococcal nuclease-based Micro-C experiments showing that yeast chromatin is flexible at lower scales[@R45]. However, in the absence of Micro-C measurements on mammalian chromatin, we can safely assume that DamC provides an upper limit to the persistence length of chromosomes *in vivo* of approximately 2.5kb.
Discussion {#S10}
==========
In this work we provide the first *in vivo*, high resolution, systematic measurements of chromatin contacts that do not require crosslinking nor ligation using DamC. An essential feature of this method is that its experimental output is directly proportional to contact probabilities. This is supported by rigorous modeling of methylation kinetics ([Figure 2](#F2){ref-type="fig"}), providing a rational basis to quantitatively interpret sequencing results. Importantly, DamC confirms that contact frequencies drop across TAD boundaries approximately by a factor 2, in accordance with 4C ([Figure 4b](#F4){ref-type="fig"}) and previous estimations based on Hi-C[@R15],[@R46]. Such modest decrease raises the question of how TAD boundaries can functionally insulate enhancers and promoters from a biophysical point of view, although they might represent an optimal compromise between enriching and depleting interactions between regulatory sequences within and across boundaries, respectively[@R9].
DamC detects chromosomal contacts on short spatial distances, since GATC motifs can only be methylated if Dam directly binds DNA. We estimate a detection range of \<10nm, given that the expected physical size of the rTetR-EGFP-Dam-ERT2 fusion protein does not exceed 3 nm[@R47]. Decreases in interaction frequencies at TAD boundaries, as well as increases due to CTCF loops, therefore closely match what a promoter would 'experience' through its bound protein complexes. Interestingly DamC also picks up 'non-specific' interactions due to random collisions within the chromatin fiber to the same extent as 4C and Hi-C ([Figure 4a](#F4){ref-type="fig"}, [Supplementary Figure 4](#SD1){ref-type="supplementary-material"}). Thus, random collisions do occur *in vivo*, despite not being detected in crosslinking-free analysis of chromosome folding using native 3C[@R26].
Scaling of crosslinking probabilities measured in Hi-C data are at the core of physical models developed to explain chromosome folding and infer its mechanistic determinants[@R15],[@R38],[@R48]--[@R50], including the highly influential loop extrusion model. Importantly, DamC confirms scaling exponents measured in 4C and Hi-C ([Figure 7](#F7){ref-type="fig"}). Since DamC enrichment is proportional to actual short-range contact probabilities, our measurements provide strong evidence in favor of chromosome folding models based on Hi-C. Scaling analysis at short genomic distances additionally suggests that mouse chromosomes might have a persistence length of approximately 2.5kb, corresponding to \~40 nm assuming a linear density of \~60 bp/nm[@R38].
The finding that loops can be established *de novo* upon insertion of CTCF binding sites and can be detected *in vivo* ([Figure 5](#F5){ref-type="fig"}-[6](#F6){ref-type="fig"}) confirms earlier reports[@R39],[@R40] and argue that chromosome structure at the TAD level can be manipulated in a 'gain of function' manner by adding new structures. New structures formed upon ectopic insertion of three CTCF sites can significantly modify intra-TAD interactions and can result in the formation of new boundaries within pre-existing TADs ([Figure 6b and 6d](#F6){ref-type="fig"}). Remarkably, we could only detect newly formed interactions within pre-existing TAD boundaries, possibly due to the fact that TAD boundaries are particularly enriched in clusters of CTCF sites[@R7],[@R9] providing efficient barriers to loop extrusion.
A limitation of DamC is that it requires genetic manipulation to insert genomic viewpoints and to stably express rTetR-Dam-ERT2 allowing accurate control of nuclear Dam concentration. This prevents DamC in its current form to be considered as an alternative to 3C-based methods in routine experimentation. However DamC can be performed by transiently nucleofecting cells with a Dam-TetR expression plasmid, which ensures low expression levels ([Figure 1c](#F1){ref-type="fig"}), and future implementations based on TAL effector proteins (similar to TALE-ID[@R26]) or catalytically inactivated Cas9 could overcome the need for targeted insertion of TetO arrays.
The current TetR-based implementation of DamC might nevertheless be beneficial in situations where 4C cannot be used, notably to detect chromosomal interactions in a tissue-specific context by expressing the rTetR-Dam fusion under a tissue-specific promoter[@R51] and starting from small numbers of cells[@R52]. Contrary to 3C methods where one ligation event per allele can be retrieved at most, in the course of a DamC experiment (\~18 hours) several GATCs might be contacted by a TetO viewpoint depending on the temporal dynamics of chromosome structure. Based on our previous measurements of the dynamics of the TetO array at the *Chic1* locus[@R53] as well as recent data from other chromosomal locations[@R54],[@R55], several contacts might be created and disassembled in 18 hours. If *n* GATC sites are methylated in this time window, DamC would in principle require *n* times less cells than 4C to build similar contact profiles. In this manuscript we analysed \~10 thousand cell equivalents per 4C and DamC experiment, but scaling down cell numbers in DamC will be an interesting future development.
In summary, by coupling a methylation-based readout with physical modeling, DamC enables systematic and quantitative crosslinking- and ligation-free measurements of chromatin interaction frequencies. Our experiments provide an orthogonal validation of 3C-based findings, including TADs and endogenous as well as ectopically induced CTCF loops, and demonstrate that 3C methods do not significantly distort the detection of chromosomal interactions.
Methods {#S11}
=======
Physical modeling {#S12}
-----------------
Detailed descriptions of the physical model of methylation kinetics in DamC, as well as of the polymer model with persistence length are available as a separate file ([Supplementary Note 1](#SD2){ref-type="supplementary-material"}).
Cell culture and sample collection {#S13}
----------------------------------
All cell lines are based on feeder-independent PGK12.1 female mouse embryonic stem cells (mESC), kindly provided by Edith Heard's laboratory. The founder cell line in our study is an X0 sub-clone of the PGKT2 clone described in (Masui et. al. 2011), carrying the insertion of a 256x TetO array within the 3'UTR of the *Chic1* gene on chromosome X and the additional deletion of the *Linx* promoter[@R6] Cells were cultured on gelatin-coated culture plates in Dulbecco Modified Eagle's medium (Sigma) in the presence of 15% foetal calf serum (Eurobio Abcys), 100 µM β-mercaptoethanol, 20 U/ml leukemia inhibitory factor (Miltenyi Biotec, premium grade) in 8% CO2 at 37°C. Cells were tested for mycoplasma contamination once a month and no contamination was detected. After insertion of the rTetR-Dam vector (see below), cells were cultured in the presence of 250 µg/mL hygromycin. To induce nuclear translocation of the rTetR-Dam fusion protein to the nuclei, mESC were trypsinized and directly seeded in culture medium containing 4-hydroxy-tamoxifen (4-OHT) at the concentrations indicated in the main text for 18 hours. Binding of the Dam fusion protein to the TetO arrays was induced by simultaneously adding 2.5 μg/ml doxycycline (Dox).
Generation of cell lines expressing rTetR-Dam and carrying random insertions of TetO arrays {#S14}
-------------------------------------------------------------------------------------------
The rTetR-EGFP-Dam-ERt2 construct was cloned into a pBroad3 backbone (Invivogen) carrying a mouse Rosa26 promoter. We used a modified rTetR based on the rtTA-M2 transactivator in Ref.[@R58], which has substantially decreased affinity for the Tet operator in the absence of Dox. The construct was randomly integrated in the PGKT2 X0 subclone by co-transfecting 5x10^5^ cells with 3 μg pBROAD3-rTetR-ICP22-EGFP-EcoDam-Ert2 and 0.2 μg of pcDNA3.1hygro plasmid using Lipofectamin 2000 (Thermo Fisher Scientific). After 10 days of hygromycin selection (250 μg/ml), one clone (\#94.1) expressing low levels of EGFP was selected and expanded for subsequent experiments. To obtain large numbers of viewpoints for DamC experiments, stable random integrations of arrays of Tet operator (TetO) sites were introduced in the \#94.1 mESC clone using the piggyBac transposon system. A mouse codon optimized version of the piggyBac transposase[@R36] was cloned in frame with the red fluorescent protein tagRFPt (Evrogen) into a pBroad3 vector using Gibson assembly cloning (pBroad3_hyPBase_IRES_tagRFPt). 5x105 cells were co-transfected with 0.2ug of pBroad3_hyPBase_IRES_tagRFPt and 1µg of a piggyBac donor vector containing an array of 50 TetO binding sites using Lipofectamin 2000 (Thermo Fisher Scientific). Cells with high levels of RFP were FACS sorted two days after transfection and seeded at three serial 10x dilutions in 10-cm dishes to ensure optimal density for colony picking. To identify clones with high numbers of TetO integration sites, cells were screened for large numbers of nuclear EGFP accumulation foci using live-cell imaging (see below) in the presence of 500nM 4-OHT and 2.5ug/ml Dox. One polyclonal population (\#94.1_2.7) and one subclone (\#94.1_2.7_pureclone3) was further expanded.
To introduce CTCF binding sites flanking the TetO viewpoints, the piggyBac donor vector was modified as follows. Three CTCF binding motifs (TGGCCAGCAGGGGGCGCTG, CGGCCAGCAGGTGGCGCCA and CGACCACCAGGGGGCGCTG) were selected based on high CTCF occupancy in ChIP-seq experiments[@R11] and cloned into the piggyBac donor vector in an outwards direction with respect to the TetO array, including 100bp of their surrounding endogenous genomic sequence (chr8:13461990-13462089, chr1:34275307-34275419 and chr4:132806684-132806807, respectively). The three CTCF binding motifs were flanked by two LoxP sites for CRE assisted recombination. 5x10^5^ \#94.1 were co-transfected with 0.2ug of pBroad3_hyPBase_IRES_rfp and 1µg of the modified piggyBac donor vector using Lipofectamin 2000. Cells with high levels of RFP were FACS sorted two days after transfection and seeded at three serial 10x dilutions in 10-cm dishes for colony picking. Clones with \>50 of integration sites were identified through accumulation of EGFP at nuclear TetO foci in the presence of 500nM 4-OHT and 2.5ug/ml dox. One clone (\#94.1_216_C3) was further selected for analyis.
Transient transfection {#S15}
----------------------
To transiently express rTetR-Dam for the proof-of-principle experiment in [Figure 1d](#F1){ref-type="fig"}, the PKGT2 X0 subclone was transiently transfected with pBroad3-rTetR-EGFP-Dam-ERt2 using the Amaxa 4D-Nucleofector X-Unit and the P3 Primary Cell 4D-Nucleofector X Kit (Lonza). 5x106 cells were resuspended in 100 µl transfection solution (82ul primary solution, 18ul supplement 1, 2μg pBroad3-rTetR-EGFP-Dam-ERt2) and transferred in a single Nucleocuvette (Lonza). Nucleofection was done using the protocol CG109. Transfected cells were directly seeded in pre-warmed 37°C culture medium containing 10nM 4-OHT +/- 2.5 μg/ml Dox. Genomic DNA was collected 18 hours after transfection. Sequencing libraries were prepared as previously described[@R34],[@R59].
Mapping of piggyBac insertion sites {#S16}
-----------------------------------
2µg of genomic DNA were fragmented to an average of 500bp by sonication (Covaris S220, duty cycle: 5%, peak power: 175W, duration: 25sec). End-repair, A-tailing and ligation of full-length barcoded Illumina adapters were performed using the TruSeq DNA PCR-free kit (Illumina) according to the manufacturer guidelines with the exception that large DNA fragments were not removed. 750ng of libraries for each sample were pooled together, and fragments of interest were captured using biotinylated probes against the the piggyBac inverted terminal repeats (ITRs) sequence and the xGen Hybridisation Capture kit (IDT) according to the manufacturer protocol (probes concentration of 2.25pmol/µl). Following the capture, libraries were amplified for 12 cycles using the Kapa Hi-fi polymerase and the following primers: 5'-AATGATACGGCGACCACCGAGAT, 5'-CAAGCAGAAGACGGCATACGAGA. Final libraries were purified using AMPure XP beads (1:1 ratio), quality controlled and sequenced on the NextSeq500 platform (paired-end 300 cycles mid-output) for a total of 8x10^8^ paired-end reads per sample on average.
Capture probe sequences are as follows:
ITR3-1 \[Btn\]ATCTATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTT,
ITR3-2 \[Btn\]TCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAA,
ITR5-1\[Btn\]TTAACCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAA,
ITR5-2 \[Btn\]ATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAA ACTCAAAATTTCTTCTATAAAGTAACAAA.
Genotyping of CTCF integration sites by PCR {#S17}
-------------------------------------------
We designed primers binding to endogenous genomic DNA sequence outside the piggyBac 3' ITR based on the genomic position of mapped piggyBac insertion sites. We then amplified the junction between the ITR and the genome using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) with one genomic primer and a T7 promoter primer (5'TAATACGACTCACTATAGGG3') flanking the piggyBac CTCF integration cassette (see [Supplementary Figure 4d](#SD1){ref-type="supplementary-material"}). PCR products were purified and Sanger sequenced. For the verification of CTCF integrations shown in [Figure 4](#F4){ref-type="fig"} and [Supplementary Figure 4e](#SD1){ref-type="supplementary-material"} on chromosome 6 and chromosome 10, the following genomic primers were used: Ch6_flxCTCF_11F (5'AGGCATTCTGTCCAACTGGT3') and Chr10_flxCTCF_13F (5'TGTTGAGCATCTATCACATTCCTTA3').
Excision of CTCF sites using Cre recombinase {#S18}
--------------------------------------------
In order to excise ectopically inserted CTCF sites from the \#94.1_216_C3 clone, 5x10^5^ cells were transfected with 0.5 μg of pIC-Cre[@R60] using Lipofectamine 2000 (Thermo Fisher Scientific). After 4 days under G418 selection (300 ug/ml), single colonies were expanded and genotyped following the procedure described above.
Live-cell Imaging {#S19}
-----------------
Gridded-glass-bottom dishes (Mattek) were coated with 2 ug/ml recombinant mouse E-cadherin (R&D Systems, 748-EC) in PBS at 4°C overnight. 5x10^5^ cells were seeded in full medium one day before imaging supplemented with 4-OHT and Dox as indicated above. Cells were imaged with a Nikon Eclipse Ti-E inverted widefield microscope (Perfect Focus System with real time drift correction for live cell imaging) operating in TIRF mode using a CFI APO TIRF 100x/1.49 oil objective (Nikon). A 488nm, 200mW Toptica iBEAM SMART laser was used as excitation source. Cells were maintained at a constant temperature of 37°C and 8% CO2 within an incubation box. Images were collected with an Evolve™ 512 Delta EMCCD high speed Camerang using Visiview (Visitron). Background subtraction (150-pixel rolling ball radius) and maximum intensity projections were performed in ImageJ.
Nuclear volume measurements {#S20}
---------------------------
3x10^6^ Cells from the \#94.1_2.7 mESC clone were cultured in gelatin-coated 6-well plates in full medium, dissociated 5 minutes at room temperature with Accutase (GIBCO), then centrifuged 4 minutes at 950 rpm and resuspended in 500 µl of culture medium. 25-µl droplets of cell suspension were spotted on coverslips previously coated with poly-L-lysine, let adsorb on ice for 5 minutes and washed gently once with 1X PBS. Cells were then permeabilized on ice for 5 min in 1X PBS and 0.5%Triton X-100, and coverslips were stored in 70% EtOH at -20C. Nuclei were counterstained with 0.2 mg/ml DAPI and Z-stack images were acquired using a Zeiss Z-1 microscope equipped with a 40x oil immersion (NA=1.3) (voxel size 0.227x0.227x0.73 µm). Z-stacks were then deconvolved using the Huygens software (20 iterations of the CMLE algorithm). To segment individual nuclei, we binarized DAPI images based on a single intensity threshold based on the fact that image histograms of all Z-stacks were bimodal (threshold = 7000 in 32-bit images). The volumes of binary 3D objects was then calculated using the 3DObjectCounter plugin in FIJI/ImageJ, excluding objects on the edges of each Z-stack.
Preparation of nuclear extracts {#S21}
-------------------------------
Cell nuclei were extracted as previously described[@R61]. Briefly, 10^7^ mES cells were seeded in ES medium (see above) supplemented with the appropriate concentration of 4-OHT on a gelatin coated 15 cm^2^ dish. The next day, cells were harvested using trypsin and washed twice in ice cold PBS. Next, cells were carefully resuspended in 500μl ice cold Buffer A1 (10mM HEPES pH7.9, 10mM KCl, 1.5mM MgCl2, 0.34M sucrose, 10% glycerol, 0.1% Triton-X 100, 1mM DTT, 1mM PMSF) to obtain nuclei. After incubating for 5 minutes on ice, extracted nuclei were washed twice with buffer A1.
Mass spectrometry {#S22}
-----------------
Nuclear extracts were dissolved in 400 µL 50mM HEPES pH 8.5 in 8.3M guanidine hydrochloride. All the samples were heated at 95°C for 5 min, sonicated using Bioruptor® sonication device and supplemented with 5mM TCEP and 10mM CAA. To reduce sample complexity, lysates were diluted to 6M guanidine hydrochloride and transferred onto 100kDa molecular weight cut-off Amiconultra-0.5 centrifugal filter units. Samples were concentrated for 2 x 15 minutes at 14kG followed by refill of the filter with 6M guanidine hydrochloride in 50mM HEPES pH 8.5 and 3 x 45 minutes at 14kG followed by refill of the filter with 1M guanidine hydrochloride in 50mM HEPES pH 8.5. For digestion 10µg of Lys-C (Wako Chemicals) and 10µg of trypsin (Thermo Fisher) were added to each sample and incubated over night at 37°C. In the morning additional 10µg of trypsin was added, incubated for 3h and acidified using TFA.
To estimate nuclear proteins copy numbers samples were desalted using SEP-PAK (Waters) and subjected to high pH offline fractionation on a YMC Triart C18 0.5 x250 mm column (YMC Europe GmbH) using the Agilent 1100 system (Agilent Technologies). 96 fractions were collected for each experiment and concatenated into 48 fractions as previously described[@R62]. For each LC-MS analysis, approximately 1 µg of peptides were loaded onto a PepMap 100 C18 2 cm trap (Thermo Fisher) using the Proxeon NanoLC-1000 system (Thermo Fisher). On-line peptide separation was performed on the 15 cm EASY-Spray C18 column (ES801, Thermo Fisher) by applying a linear gradient of increasing ACN concentration at a flow rate of 150 nL/min. An Orbitrap Fusion Tribrid (Thermo Fisher) or an Orbitrap Fusion LUMOS Tribrid (Thermo Fisher) mass spectrometers were operated in a data-dependent mode. The top 10 most intense precursor ions from the Orbitrap survey scan were selected for higher-energy collisional dissociation fragmentation and analyzed using the ion-trap.
Mass spectrometry data processing {#S23}
---------------------------------
Maxquant version 1.5.3.8 was used to search raw mass spectrometry data using default settings[@R63],[@R64] against the mouse protein sequences from Uniprot database (release 2017-04). The label free quantification (LFQ) algorithm was used for quantification. The protein groups table were loaded to Perseus software[@R65] (version 1.5.0.0) filtered for potential contaminants and reverse hits. Protein copy numbers per cell were calculated using the Protein ruler plugin of Perseus by standardization to the total histone MS signal[@R56] (Wisniewski et al., 2014). The LFQ values were normalized using same normalization for all samples. To estimate cytoplasmic contamination "GOCC slim name" annotations provided in Perseus were used. Exclusively cytoplasmic proteins were defined as being associated with GOCC terms "cytoplasm" or "cytosol" and not associated with terms "nucleus", "nuclear", "nucleoplasm" and "nucleosome". Exclusively nuclear proteins were defined as being associated with GOCC terms "nucleus", "nuclear", "nucleoplasm" and "nucleosome" and not associated with terms "cytoplasm" or "cytosol". The cytoplasmic contamination was estimated using a ratio of summed LFQ intensity between exclusively cytoplasmic proteins and exclusively nuclear proteins in samples with and without nuclear extraction.
Parallel reaction monitoring (PRM) data acquisition and analysis {#S24}
----------------------------------------------------------------
To select peptides for PRM assays, the rTetR-Dam-EGFP-ERT2 construct was enriched using magnetic ChromoTek\'s GFP-Trap beads and analyzed using shotgun data-dependent acquisition LC-MS/MS on an Orbitrap Fusion Lumos platform as decribed above. For PRM analysis the resolution of the orbitrap was set to 240k FWHM (at 200 m/z), the fill time was set to 1000 ms and ion isolation window was set to 0.7 Th. For LC-MS analysis of samples derived from a polyclon carrying 890 TetO array insertions, approximately 1 µg of peptides were loaded onto a PepMap 100 C18 2 cm trap (Thermo Fisher) using the Proxeon NanoLC-1000 system (Thermo Fisher). On-line peptide separation was performed on the 15 cm EASY-Spray C18 column (ES801, Thermo Fisher) by applying a linear gradient of increasing ACN concentration at a flow rate of 150 nL/min. Whereas for LC-MS analysis of samples derived from a polyclon carrying 100 TetO array insertions, approximately 1 µg of peptides were on-line separated on a 50 cm µPACTM cartridge (PharmaFluidics) by applying a linear gradient of increasing ACN concentration at a flow rate of 300 nL/min using the Proxeon NanoLC-1000 system (Thermo Fisher).The acquired PRM data were processed using Skyline 4.135[@R66]. The transition selection was systematically verified and adjusted when necessary to ensure that no co-eluting contaminant distorted quantification based on traces co-elution (retention time) and the correlation between the relative intensities of the endogenous fragment ion traces, and their counterparts from the library. As a loading control the mean of total MS1 signal was estimated using RawMeat v2.0b1007.
DamC library preparation {#S25}
------------------------
DamC experiments are based on a newly developed DamID-seq NGS library preparation protocol to maximize the proportionality between methylation levels and sequencing readout ([Supplementary Figure 2c](#SD1){ref-type="supplementary-material"}). One crucial issue in the calculation of enrichment as in [Figure 2c](#F2){ref-type="fig"} is that small fluctuations in -Dox methylation in the denominator can be amplified into large fluctuations in enrichment levels. GATC sites must therefore be equally and robustly represented in the DamID sequencing library irrespective of their methylation level. From this perspective, the principal limitation of the original DamID protocol[@R59] for our present application was its dependence on the genomic distance between two GmATC sites, resulting in large adaptor-ligated molecules and as a consequence in a strong bias towards densely methylated regions. In our optimized protocol, GmATC sites are sequenced independently of the neighboring GATC methylation status resulting in a \~30% increase in GmATC coverage at equivalent sequencing/read depth ([Supplementary Figure 2e](#SD1){ref-type="supplementary-material"}). In addition, we introduced unique molecular identifiers (UMIs) allowing a precise enrichment quantification after excluding PCR duplicates from the sequencing data.
Overall, the DamC library construction protocol can be divided in 3 parts: 1) ligation of UMI adapters with a "one-tube" strategy, 2) integration of the second sequencing adapter, followed by 3) a final PCR amplification. Briefly, 3x10^6^ cells were harvested using trypsin after 18 hours of induction with tamoxifen +/- doxycyclin. Genomic DNA was extracted using the Qiagen blood and tissue kit adding 250U of RNaseA in step 1. Genomic DNA was eluted in 80ul ddH2O. DNA concentration was measured using the Qbit DNA Broad Range kit. Genomic DNA (350ng input) was treated with Shrimp Alkaline Phosphatase treatment (NEB, 1U), followed by DpnI digestion (ThermoFisher Scientific, 10U), A-tailing (0.6mM final dATP, 5U Klenow exo-, ThermoFisher Scientific), and UMI adapters ligation (30U T4 DNA ligase, PEG4000, ThermoFisher Scientific) performed within the same tube and buffer (Tango 1X, ThermoFisher Scientific) by heat inactivating each enzymatic step followed by adjustment with the reagents required for the next step. UMI adapters were made by annealing the following oligos: 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC\*T and 5'-pGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT. Ligation reactions were treated with Exonuclease I (20U, ThermoFisher Scientific) then purified using AMPureXP beads (1:0.8 ratio, Agencourt) and the second sequencing adapter (5' TGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNN\*N 3', IDT) was tagged using heat denaturation and second strand synthesis (5U T4 DNA Polymerase, ThermoFisher Scientific). The tagging reaction was purified using AMPure XP beads (1:1 ratio) followed by a final library amplification (12 cycles) using 1U of Phusion polymerase, 2μl 10 μM DAM_UMIindex_PCR (5' AATGATACGGCGACCACCGAGATCTACA\*C 3'), and 2μl 10μM NEBnext indexed primer (NEB). Final libraries were purified AMPure XP beads (1:1 ratio) and QCed using Bioanalyser and Qbit. DamC libraries were sequenced on a NextSeq500 (75 cycles single-end) with a custom sequencing protocol (dark cycles at the start of read1 to \"skip\" the remaining DpnI site TC sequence). Samples index were determined using index1 read, and UMI sequence using index2 read. Detailed number of total and valid reads can be found in **Supplementary Table 5**.
4C-seq {#S26}
------
4C sample preparation was performed as previously described[@R67]. Briefly, 10^7^ cells were cross-linked in 2% formaldehyde for 10 minutes and quenched with glycine (final concentration 0.125M). Cells were lysed in 150 mM NaCl/50 mM Tris-HCl (pH 7.5)/5 mM EDTA/0.5% NP-40/1% Triton X-100. The first digest was performed with 200 U DpnII (NEB), followed by ligation at 16° C with 50 U T4 DNA ligase (Roche) in 7 mL. Ligated samples were de-crosslinked with Proteinase K (0.05 ug/uL) at 65° C, purified, and digested with 50 U Csp6I (Thermo Fisher Scientific) each, followed by ligation with 100 U T4 DNA ligase in 14 mL and purification. Resulting products were directly used as PCR template for genomic dedicated 4C viewpoints. Primers for PCR were designed using guidelines described previously[@R67]. We obtained the following read counts: \#94.1_2.7 cell line (135 TetO insertions only), 5.7x10^6^ valid reads in total (+Dox, two replicates). For the \#94.1_216_C3 line (TetO-CTCF), 3.5x10^6^ valid reads on average per sample; for the experiments shown in [Supplementary Figure 3c and 4c](#SD1){ref-type="supplementary-material"}, we obtained an average of 3.2x10^6^ reads per sample. Detailed number of total and valid reads can be found in **Supplementary Table 5**.
In vitro Cas9 digestion of 4C templates {#S27}
---------------------------------------
In order to detect chromosomal interactions directly from the genome integrated TetO platform, viewpoint primers were designed to amplify directly from the DpnII fragments contained in the TetO sequence. The 2.7 kb TetO platform contains a total of 50x contiguous repeats of the same TetO DpnI/II viewpoint. To prevent PCR amplification and sequencing of TetO repeats due to tandem ligation of two or more TetO DpnII fragments in a given 4C circle, an in vitro Cas9 digestion was performed on the 4C templates. Cas9 was targeted into the TetO repeats in between viewpoint primers using a single gRNA. In vitro transcribed gRNA template was obtained using the Megashortscript T7 transcription kit (Invitrogen). gRNA was purified with 4 × AMPure purification (Agencourt). Purified Cas9 protein was kindly provided by N. Geijsen. Cas9 was pre-incubated with the sgRNA for 30 min at 37 °C. Subsequently, 4C template DNA was added to the pre-incubated gRNA-Cas9 complex and incubated for 3--6 h at 37 °C for digestion. Cas9 was inactivated by incubating at 70 °C for 5 min.
Hi-C library preparation {#S28}
------------------------
6x10^6^ mESC were harvested and diluted in 1x PBS to final 1x10^6^ cells/ml, then crosslinked with 1% formaldehyde and quenched with 0,125M glycine for 5 min at RT. After two 1x PBS washes, cells pellets were obtained by centrifugation, snap frozen and stored at -80°C. Pellets were thawed on ice and resuspended in 500 ul lysis buffer (10 nM Tris-HCl pH8.0, 10 nM NaCl, 0.2%NP40, 1x Roche protease inhibitors) and left 30 min on ice. Cells were then pelleted by centrifugation (954 x g, 5 min, 4°C), washed once with 300 ul 1x NEB2 buffer and nuclei were extracted by 1 h incubation at 37°C in 190 ul 0.5%SDS 1xNEB2 buffer. SDS was neutralized by diluting the sample with 400 ul NEB2 buffer and adding 10% Triton X-100. After 15 min of incubation at 37°C, nuclei were pelleted, washed once in PBS and resuspended in 300 ul NEB2 buffer. 400U of MboI (NEB, 25 000 units/ml) were added and incubated at 37°C overnight. The next day, nuclei were pelleted again, resuspended in 200 ul fresh NEB2 buffer and additional 200U of MboI were added for two more hours before heat inactivation at 65°C for 15 min. 43 ul of end-repair mix (1.5 μL of 10 mM dCTP; 1.5 μL of 10mM dGTP; 1.5 μL of 10 mM dTTP; 37.5 μL of 0.4 mM Biotin-11-dATP (Invitrogen) and 1 μL of 50U/μL DNA Polymerase I Large Klenow fragment (NEB) were added to the nuclear suspension, incubated at 37°C for 45 min and heat inactivated at 65°C for 15 min. The end repair mix was exchanged with 1.2 ml of ligation mix (120μL of 10X T4 DNA Ligase Buffer; 100 μL of 10% Triton X-100; 6 μL of 20 mg/mL BSA; 969μL of H2O) plus 5 ul of T4 ligase (NEB, 2000 units/ml) and ligation was performed at 16°C overnight. Nuclei were reconstituted in 200 ul fresh NEB2 buffer followed by RNA digestion in 0.5 mg/ml RNAse A for 10 min at 37°C. Samples were de-crosslinked with Proteinase K at 65°C overnight and DNA was purified using phenol/chloroform. 2 ug of DNA sample were sonicated using Diagenode Bioruptor Pico. MyOne Streptavidin T1 (Life Technologies \# 65601) magnetic beads were used to capture biotinylated DNA followed by A-tailing. Adapter ligation was performed according to NEB Next Ultra DNA Library prep kit instructions. Two independent PCR reactions with multiplex oligos for Illumina sequencing were performed and pooled for the final PCR clean-up by magnetic AMPure bead (Beckman Coulter) purification. The final libraries were eluted in nuclease-free water, QCed by Bioanalyzer and Qubit. HiC libraries were sequenced on a Illumina Nextseq500 platform (2x42bp paired end). We obtained an average of 3.5x10^8^ valid reads per sample (TetO-only and TetO-CTCF cells, -Dox, two biological replicates each). Detailed number of total and valid reads can be found in **Supplementary Table 5**.
Sequencing data processing and data analysis {#S29}
--------------------------------------------
### DamC analysis {#S30}
All samples were aligned to mouse mm9 using qAlign (QuasR package[@R68]) using default parameters. PCR duplicates were removed using a custom script. Briefly, reads were considered PCR duplicates if they map to the same genomic location and have the same 8-bp UMI sequence. We quantified the number of reads mapped to each GATC that could be uniquely mapped using qCount (QuasR package[@R68]). The *query* object we used in qCount was a GRanges object containing the uniquely mappable 76-mers GATC loci in the genome shifted upstream (plus strand) or downstream (minus strand) by 5 base-pairs (3 dark cycles + *GA*, see the *'*Bulk DamID-seq Library preparation' paragraph in the [Methods](#S11){ref-type="sec"} section). Each sample was then normalized to a common library size of 10M reads and a pseudo-count of 0.2 was added. Prior to calculating DamC enrichments, a running average over 21 restriction fragments was performed and the mean value was assigned to the central GATC. Enrichment was then calculated as in [Figure 2c](#F2){ref-type="fig"}: E=(\[+Dox\]-\[-Dox\])/\[-Dox\] where \[+Dox\] and \[-Dox\] are the normalized and running-averaged number of reads in the presence and absence of Dox, respectively. We defined the DamC signal to be saturated if it satisfies the following criteria: 1) it belongs to the top highest 25% genome wide both in +Dox and -Dox samples, and 2) the ratio between +Dox and -Dox methylation is close to 0.5, i.e. belongs to the \[0.45, 0.55\] quantile of all ratios genome wide. Coordinates of excluded viewpoints in the clonal cell line with TetO integrations are: chr6:25758950, chr8:26653938, chr8:96714938, chr11:33429300, and chr11:51411650.
### 4C analysis {#S31}
Mapping of 4C reads was performed as described for DamC, with the exception of UMI de-duplication since 4C libraries did not include UMIs and quantification was done by counting the reads mapped exactly to the GATC sites. The two restriction fragments immediately flanking the piggyBac-TetO cassette were excluded from subsequent analyses.
### Hi-C Analysis {#S32}
Hi-C data were analysed using HiC-Pro version 2.7.10 with *\--very-sensitive \--end-to-end \--reorder* option. Briefly, reads pairs were mapped to the mouse genome (build mm9). Chimeric reads were recovered after recognition of the ligation site. Only unique valid pairs were kept. Contact maps at a given binning size were then generated after dividing the genome into equally sized bins and applying iterative correction[@R70] on binned data.
### Fit of scaling plots {#S33}
Average normalized Hi-C counts, DamC enrichment or 4C counts were calculated for all pairs of loci separated by logarithmically binned distance intervals. The binning size in logarithmic scale (base 10) was 0.1. Curves were fitted in log-log scale using the lm function in R.
### Fitting the DamC model to DamC experiments as a function of 4-OHT concentration {#S34}
The DamC enrichment depends on the rTetR-TetO specific and nonspecific dissociation constants, the concentration of TetO and the nuclear rTetR-Dam concentration ([Supplementary Note 1](#SD2){ref-type="supplementary-material"}). In addition, it depends on the actual contact probability between the genomic location where it is calculated and the TetO viewpoint. In [Figure 3d](#F3){ref-type="fig"} we calculated the DamC enrichment at the closest fragments to the 100 TetO viewpoint with higher signal to noise ratio in the polyclonal line. We assumed that the contact probability between the TetO array and the closest fragment is \~1, and fitted the model to the experimental data using the other parameters with the *NonlinearModelFit* function in Mathematica. The constraints that the dissociation constants and the concentration of TetO are positive were imposed. The goodness of the fit was evaluated using the adjusted R^2^ (0.73). In the clonal line, we assumed that the specific dissociation rTetR-TetO does not change compared to the polyclonal line and by setting the concentration of viewpoints to 135 per cell, we fitted the nonspecific dissociation constant using the *NonlinearModelFit* function in Mathematica. Model fitting resulted in an estimate of 5nM for the average non-specific binding constant accounting for rTetR and Dam interactions with GATC sites genome-wide. The goodness of the fit was evaluated using the adjusted R^2^ (0.68).
### ChromHMM {#S35}
In order to assign chromatin states, we used the ChromHMM software[@R57] with four states. We used histone modifications as in **Supplementary Table 6**. The four states correspond to active (enriched in H3K36me3, H3K27ac, H3K4me1 and H3K9ac), poised (H3K36me3, H3K27ac, H3K4me1, H3K9ac and H3K27me3), inert (no enrichment) and heterochromatic (H3K9me3) states.
### Deviation scores {#S36}
Given a set of restriction fragments (or genomic bins) {x~i~} belonging to a window \[a,b\], the deviation score is defined as $$Dev\left( {a,b} \right) = 2\frac{\sqrt{< \left( {f - g} \right)^{2} >_{\lbrack{a,b}\rbrack}}}{< \left| f \right| + \left| g \right| >_{\lbrack{a,b}\rbrack}}$$ Where *f* and *g* are data vectors (e.g. DamC enrichment, 4C or virtual 4C counts) and \< \>~\[a,b\]~ represents the average in the window \[a,b\]. If two profiles are identical in the window \[a,b\], then the deviation score is zero; increasing deviation from zero indicates increasing dissimilarity.
### PiggyBac-TetO integration site mapping {#S37}
Paired-end reads (see '[Mapping of piggyBac insertion sites](#S16){ref-type="sec"}' above) were trimmed to 50 bp using a custom script. Read1 and Read2 were mapped separately to the piggyBac-TetO sequence using QuasR (qAlign). Only hybrid pairs with one of the reads mapping to array were kept. The second reads from hybrid pairs were mapped to the mouse genome (build mm9) using QuasR (qAlign). Reads were then piled up in 25 bp windows using csaw (windowCounts function). Integration sites can be identified because they correspond to local high read coverage. Local coverage was calculated by resizing all non-zero 25-bp windows up to 225 bp (expanding by 100 bp upstream and downstream). Overlapping windows were then merged using reduce (from GenomicRanges) thus resulting in a set of windows {*w~i~*}. The size distribution of *w~i~* is multimodal, and only *w~i~* from the second mode on were kept. For each *w~i~* we estimated the coverage *c~i~* as the number of non-zero 25-bp windows. Only *w~i~*'s where the coverage were higher than 16 were considered. The exact position of the integration sites were then identified with the center of *w~i~*.
### Determination of the orientation of TetO-CTFC insertions {#S38}
In order to determine the orientation of ectopically inserted TetO-CTCF sites, we exploited the fact that the three CTCF sites are oriented within the piggyBac casette in the 3' ITR - 5' ITR direction. If the genomic position of the 5' ITR is upstream of the 3' ITR, then CTCF sites are in the reverse orientation (- strand), and vice versa. To determine the relative orientation of the 3' and 5' ITRs in the genome, we used only reads that run through the junction between the ITRs and the genome. More precisely, we extracted reads that contain an exact match to 30bps of the ITRs (3' and 5' ITR separately), trimmed the ITR sequence and mapped the reads to the mouse genome using qAlign (from QuasR). We quantified the reads at single bp resolution using scanBam. Only integration sites where both 5' and 3' ITRs are mapped are kept. This resulted in 91 integration sites (**Supplementary Table 4**).
### Z-score analysis of Hi-C data {#S39}
In order to identify and exclude 'noisy' interactions in Hi-C maps we used a custom algorithm named 'Neighborhood Coefficient of Variation' (van Bemmel *et al*., under revision). Since the chromatin fiber behaves as a polymer, the contact probability of a given pair of genomic loci *i* and *j*, is correlate to that of fragments i+N and j+N if N is smaller (or on the order of) than the persistence length of the chromatin fiber. Hence, a given pixel in a Hi-C map can be defined as noisy if its numerical value is too different from those corresponding to neighboring interaction frequencies. To operatively assess the similarity between neighboring interactions, we calculated the coefficient of variation (CV) within a 10x10 pixel square centered on every interaction and discarded all pixels whose CV is larger than a certain threshold. Given that the distribution of the coefficient of variation of Hi-C samples in this study is multimodal with the first component terminating around CV=0.6, we set the CV threshold to 0.6. Discarded interactions appear as grey pixels in the differential Hi-C maps. For differential analysis between TetO-CTCF and wt samples, we calculated the difference between distance-normalized Z-scores calculated for each individual map[@R71]. The Z-score is defined as (obs-exp)/stdev where (obs) is the Hi-C signal for a given interaction and (exp) and (stdev) are the genome-wide average and standard deviation of Hi-C signals at the genomic distance separating the two loci.
### 4C peak calling {#S40}
In order to call specific interactions in 4C profiles, we used the peakC package[@R41] using the following parameters: qWr = 2.5 and minDist = 20000. peakC was applied to 2 replicates of 4C profiles at single fragment resolution. Peak regions were then extended 1kb upstream and downstream. Overlapping peaks were merged.
Supplementary Material {#SM}
======================
This work is dedicated to the memory of Maxime Dahan. Research in the Giorgetti lab is funded by the Novartis Foundation and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 759366 'BioMeTre'). The Kind lab was funded by the ERC (grant agreement No 678423 'EpiID') and EMBO (LTF 1214-2016 to I). RSG acknowledges support from the European Union's Horizon 2020 research and innovation program under the Marie Sk1odowska-Curie grant agreement no. 705354 and an EMBO Long-Term fellowship ALTF 1086-2015. We would like to thank Peter Cron for cloning TetO-piggyBac plasmids, Sirisha Aluri and Stéphane Thiry for assistance with high-throughput sequencing, Michael Stadler for help with bioinformatics analysis, Stefan Grzybek and Hans-Rudolf Hotz for server supports, Edith Heard and Rafael Galupa (Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, France) for kindly providing PGK cells. We are grateful to Dirk Schuebeler and Rafael Galupa for critically reading the manuscript and Geoffrey Fudenberg for useful comments on scaling behavior. We acknowledge The ENCODE Project Consortium and in particular the Ren and Hardison laboratories for ChIP-Seq data sets in ESC.
**Code availability**
The custom-made codes used to analyze the data can be obtained upon request to L. Giorgetti.
**Data availability**
The sequencing data from this study, including bedgraph files for the visualization of DamC and 4C profiles from all the samples described in the manuscript are available at the NCBI Gene Expression Omnibus, with accession code GEO GSE128017. A UCSC session containing all the DamC and 4C tracks used can be found at [https://genome.ucsc.edu/s/zhan/DamC_publication_2019](https://genome.ucsc.edu/cgi-bin/hgTracks?hgS_doOtherUser=submit&hgS_otherUserName=zhan&hgS_otherUserSessionName=DamC_publication_2019). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE[@R72] partner repository with the dataset identifier PXD013507. Source data for [Figure 1](#F1){ref-type="fig"}, [3](#F3){ref-type="fig"}-[7](#F7){ref-type="fig"} and [Supplementary Figure 1-3, 5 and 6](#SD1){ref-type="supplementary-material"} are available online.
**Author contributions**
JR generated cell lines and performed DamC experiments. YZ wrote the model with assistance from GT and analyzed the data. CV performed 4C in WdL's lab. MK assisted with cell culture and DamC library preparation and performed Hi-C experiments. IG and JK helped with experimental design and data analysis. VI performed mass spectrometry experiments and analysis. TP provided constructs for initial experiments and discussed the data. RSG provided CTCF site sequences and tested CTCF binding in preliminary experiments. EM contributed designing the initial experiments. SAS developed the DamC library preparation protocol and performed piggyBac insertion mapping experiments. LG designed the study and wrote the paper with JR and YZ and input from all the authors.
**Competing Interests Statement**
The authors declare no competing financial interests.
{#F1}
{ref-type="supplementary-material"}).](emss-82706-f002){#F2}
{ref-type="supplementary-material"}). Error bars are s.d. of two biological replicates (independent cell cultures). **c.** Random integration of large numbers of 50x TetO platforms using the piggyBac transposon. Accumulation of EGFP signal to nuclear foci in the presence of Dox (right: max. intensity projection over 10 Z planes) indicates binding of rTetR-Dam to the arrays and allows selecting clones with large numbers of insertions. **d)** Quantification of DamC experiments as a function of rTetR-Dam concentration in cells with 890 (upper panel, blue) and 135 (lower panel, green) TetO viewpoints. Blue data points, mean and s.d. from over the 100 TetO viewpoints with highest enrichment. Green data points, mean and s.d. from over 130 TetO viewpoints (5 viewpoints were excluded due to absence of DamC signal). Red line, model fit to the experimental data.](emss-82706-f003){#F3}
![DamC confirms the existence of TAD boundaries and quantitatively correlates with 4C and Hi-C.\
**a.** Four representative DamC and 4C interaction from the same piggyBac-TetO viewpoints, aligned with Hi-C experiments performed in the same cell line. Dashed lines mark TAD boundaries in mESC detected using CaTCH[@R9]. Hi-C data were binned at 10 kb resolution. DamC was performed using 0.1 and 1 nM 4-OHT (pooled). Data from two biological replicates were pooled for DamC, 4C and Hi-C. **b.**Aggregated plot over 130 TetO viewpoints showing DamC and 4C data aligned to TAD boundaries identified using CaTCH[@R9]. Gray shading: +/- 40 kb uncertainty on boundary definition[@R9]. **c.** Interaction profiles from viewpoints located \<1kb from a CTCF site (left) belonging to a cluster of forward sites (red shading) interacting with reverse CTCF sites (blue shading) and \<1kb from the active promoter of the *Mrs2* gene (right), highlighted in the green shaded area.](emss-82706-f004){#F4}
![DamC-based detection of CTCF loops.\
**a.** Modified piggyBac strategy to insert TetO viewpoints flanked by three CTCF sites oriented outwards. **b.** The TetO-CTCF cassette can insert in the genome in both directions and lead to the formation of interactions with either forward or reverse endogenous CTCF sites. **c.** Three representative interaction profiles obtained using DamC and 4C from TetO-CTCF viewpoints. Asterisks indicate interactions identified by PeakC that overlap with CTCF sites. Shaded boxes indicate the overlap with the genomic positions of endogenous and ectopic CTCF locations. **d.** Left, average number of peaks per viewpoint detected by PeakC at least 20kb away from the viewpoint, in cells with TetO-CTCF or TetO-only insertions. Viewpoints landing within 1kb from an endogenous CTCF site were excluded. Right, percentage of peaks containing a CTCF motif that is bound based on ChIP-seq data[@R11].](emss-82706-f005){#F5}
![Ectopic CTCF insertion leads to the formation of new loops and stripes.\
**a.** TetO-CTCF insertion site giving rise to ectopic loops with convergently oriented endogenous CTCF sites. Top, interaction profiles measured with DamC and 4C are overlaid with the position of CTCF ChIP-seq sites from ref.[@R11] and Nipbl ChIP-seq data from ref.[@R42]. Middle, Hi-C data from the ESC lines carrying wither the heterozygous TetO-CTCF insertion or two wild-type alleles. Bottom, distance-normalized Z-scores, highlighting interactions that are either enriched (red) or depleted (blue) compared to the expected interaction frequency. Arrows, interactions between convergent CTCF sites that are established upon CTCF insertion. Arrowheads, pre-existing interaction that are strengthened after CTFC insertion. Hi-C data are binned at 10 kb resolution. Data from two biological replicates (independent cell cultures) were pooled for DamC, 4C and Hi-C. **b)** Z-score difference between heterozygous CTCF and wild-type cells showing increased partitioning of interactions inside the TAD. Hi-C data were binned at 20kb. Shaded areas correspond to 'noisy' interactions that did not satisfy a quality control filter based on their correlations with immediate nearest neighbors (see [Methods](#S11){ref-type="sec"}). **c.** Same as panel a for an insertion on chromosome 10, occurring in proximity to an isolated cluster of Nipbl binding and giving rise to a stripe-like interaction pattern. **d.** Z-score differences for the locus shown in panel c. **e.** DamC interaction profiles from the same viewpoints as in panel a and c, before and after Cre-mediated excision of ectopically inserted CTCF sites (but not of the piggyBac cassette).](emss-82706-f006){#F6}
{ref-type="supplementary-material"}). The best value of *a* extracted from the fit is \~2.5 kb.](emss-82706-f007){#F7}
[^1]: equal contribution
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Video: Syrian forces leave besieged city
Syria's army suspended days of punishing attacks on the restive city of Homs and began withdrawing its tanks Tuesday just as Arab League monitors visited the area, activists and officials said.
Activists posted video clips on YouTube which appeared to show crowds numbering in the tens of thousands pouring into the streets shortly after the pullback, shouting defiantly that they will not be cowed by the crackdown.
"There are at least 70,000 protesters," Rami Abdelrahman of the British-based Syrian Observatory for Human Rights told Reuters. "They are marching towards the city center and the security forces are trying to stop them. They are firing teargas."
In another amateur video posted online, Syrians shouted "We want international protection" as the team of observers passed through bloodied and rubble-strewn streets.
'We are unarmed people'
Other footage appeared to show residents of Homs' tense Baba Amr district speaking to the Arab monitors.
"We are unarmed people who are dying," one resident shouts. Seconds later, shooting is heard from a distance as someone else screams: "We are being slaughtered here."
Syria has banned foreign journalists, making it impossible to independently verify the source or date of these video clips.
About 60 Arab League monitors — the first Syria's regime has allowed in during its nine-month crackdown on an anti-government uprising — began work Tuesday. They are there to ensure compliance with the League's plan to halt violence against mostly unarmed, peaceful protesters and the pullback in Homs was the first tangible sign President Bashar Assad was implementing any of the terms.
"I am returning to Damascus for meetings and I will return tomorrow to Homs," Sudanese General Mustafa Dabi said. "The team is staying in Homs. Today was very good and all sides were responsive."
Activist reports just before the monitors arrived said up to a dozen tanks were seen leaving Baba Amr but others were being hidden to fashion a false impression of relative normality in the city while observers were around.
"My house is on the eastern entrance of Baba Amr. I saw at least six tanks leave the neighborhood at around 8 in the morning (0600 GMT)," Mohamed Saleh told Reuters by telephone. "I do not know if more remain in the area."
Al Jazeera television showed an estimated 20,000 Syrians in a square in Khalidiya, one of four districts where there has been bloodshed as rebels fight security forces using tanks.
They were whistling and shouting and waving flags, playing music over loudspeakers and clapping. Women were advised to leave because of the risk of bloodshed. But a speaker urged the men to "come down, brothers."
The protesters shouted "We have no one but God" and "Down with the regime." An activist named Tamir told Reuters they planned to hold a sit-in in the square.
"We tried to start a march down to the main market but the organizers told us to stop, it's too dangerous. No one dares go down to the main streets. So we will stay in Khalidiya and we will stay here in the square and we will not leave from here."
After signing on to the plan early last week, Assad's regime had only intensified the violence, rather than easing up, and it was condemned internationally for flouting the agreement. Government troops killed hundreds in just the past week. On Monday, security forces killed at least 42 people, most of them in Homs.
Opposition activist Mohammed Saleh said the heavy bombardment of Homs stopped Tuesday morning and tanks were seen pulling out of the streets. Another Homs-based activist said he saw armored vehicles leaving early Tuesday on a highway leading to the city of Palmyra to the east. He asked that his name not be made public for fear of retribution.
Homs, Syria's third-largest city, has a population of 800,000 and is at the epicenter of the revolt against Assad, located about 100 miles north of the capital, Damascus. Many Syrians refer to Homs as the "capital of the revolution."
"Special forces were able to kill and wound several gunmen and seized some weapons, ammunitions, army uniforms, communication tools and fake identity cards," SANA said, but it did not give a specific casualty count.
SANA also reported that a "terrorist group" had attacked a gas pipeline near Homs but there were no further details immediately available.
In Cairo, an official at the Arab League's operations room said the Sudanese head of the mission to Syria, Gen. Mohamed Ahmed Mustafa al-Dabi, was leading the team of at least 12 observers in Homs Tuesday. The official, who spoke on condition of anonymity because he was not authorized to speak to journalists, gave no further details.
Parts of Homs are defended by the Free Syrian Army, made up of defectors from the regular armed forces, who say they have tried to protect civilians.
The Arab League plan agreed to by Assad last week requires the government to remove its security forces and heavy weapons from city streets, start talks with opposition leaders and allow human rights workers and journalists into the country. Before Tuesday's redeployment of at least some tanks, there had been no sign that Assad was implementing any of the terms, much less letting up on his brutal crackdown.
Assad's opponents fear that the monitors —
who arrived in the country on Monday
after weeks of negotiations with Arab states — will be used as a cloak of respectability for a government that will hide the extent of violence.
The Associated Press, Reuters and msnbc.com's Alastair Jamieson contributed to this report. |
I'd say that the pauldrons are too big...but it's 40K.Great work on the armor decorations and the bolter, looks amazing. |
Introduction
============
Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular abnormalities, fibrosis of the skin, musculoskeletal manifestations, and internal organ involvement ([@B01]). Pulmonary involvement in SSc in the form of cellular or fibrotic nonspecific interstitial pneumonia (NSIP) occurs in 25-90% of patients, depending on the sensitivity of the evaluation ([@B02]-[@B06]), and is a significant cause of morbidity and mortality in this patient population ([@B02]-[@B07]). Consequently, there is great interest to identify which NSIP groups are likely to progress to a more fibrotic pattern that may result in shorter patient survival. In addition, identification of these specific NSIP groups after surgical lung biopsy may allow for optimal treatment approaches. In this regard many have studied biological markers in alveolar as well as in vascular compartments to discover what might relate with the progression of fibrosis or treatment responses, or to tumor recurrence and shortened survival ([@B08]-[@B12]). Because scleroderma-associated fibrotic lung disease is the phenotypic consequence of the interactions between epithelial and mesenchymal components (such as endothelial cells and myofibroblasts), currently much interest is focused on the influence of proliferative factors on growth, activation, and replication of these components. SSc is thought to be a consequence of the aberrant regulation of endothelial tissue, resulting in both vascular damage and subsequent tissue damage. Thus, several interleukins (IL-4, IL-6, IL-8, and IL-13) and growth factors \[transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF-α), insulin-like growth factors (IGFs), basic fibroblast growth factor (bFGF), and interferon gamma (IFN-γ)\] released from inflammatory cells, endothelial cells, fibroblasts, and other cells in the lung have been implicated in the initiation and maintenance of the fibrotic process ([@B13],[@B14]). In addition, a group of nitric oxide (NO) synthase (NOS) isoforms has been targeted as potentially useful vascular markers of dysfunction ([@B15],[@B16]). Among these, constitutively expressed endothelial NO synthase (eNOS) and plasminogen activator inhibitor-1 (PAI-1) have shown promise. In many pathological states, most notably reperfusion injuries, dysregulation of inducible NOS (iNOS) and PAI-1 result in endothelial damage, thus leading to excessive levels of NO. Excessive levels of NO react with superoxides to form peroxynitrite and highly reactive hydroxyl radicals, which in turn result in cell injury and apoptosis ([@B17]). As the histopathological changes in the lungs of patients with SSc are consistent with alveolar and vessel cell damage ([@B18]-[@B20]), we presume that this interaction can be characterized by analyzing the expression of proteins regulating NO synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and the outcome, as well as the relationship between these factors and other clinical data and pulmonary function tests, we studied these markers in 23 SSc-NSIP cases.
Patients and Methods
====================
Between January 2002 and July 2004, 23 consecutive patients with SSc and interstitial lung disease (ILD) shown by high-resolution computed tomography (HRCT) were submitted to an open lung biopsy at the Hospital das Clínicas, Universidade de São Paulo ([@B21]). All patients were women (mean age±SD, 44.89±8.74 years) who fulfilled the diagnostic and subtype criteria for SSc ([@B22],[@B23]). Open lung biopsy was performed by formal thoracotomy avoiding honeycombing areas. All 23 patients signed a free informed consent (No. 0960/08) form, and the study was approved by the Hospital Ethics and Scientific Committees.
Analysis of the clinical records was performed for all patients. The disease duration was established from the first symptom of the disease except for Raynaud\'s phenomenon. Skin thickness was assessed using the modified Rodnan Skin Score (MRSS) ([@B24]), consisting of clinical palpation in 17 body areas on a 0-3 basis and the sum of the scores in all 17 areas. HRCT and pulmonary function tests were performed within a period of up to 3 months before the biopsy. Disease duration (from the onset of Raynaud\'s phenomenon) and MRSS to score cutaneous fibrosis were analyzed. All eligible patients were submitted to blood tests immediately before the start of the study (complete blood count, urinalysis, liver enzymes, renal function tests, and anti-topoisomerase antibody). They were followed monthly before cyclophosphamide infusion with regular blood tests, and the dosage was adjusted if the total leukocyte count fell below 3000/mm^3^. Lung function tests \[diffusing capacity of the lung for carbon monoxide corrected for hemoglobin concentration (DLCO-Hb), forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), and total lung capacity (TLC)\] were performed before (up to 3 months), after 12 months of treatment, and after 3 years from the end of the study. MRSS was scored before treatment, on months 6 and 12 of treatment, and also after 3 years from the end of the study. The primary end point was to evaluate changes in NOS and PAI-1 and to analyze differences between the two groups: cellular SSc-NSIP *vs* fibrotic NSIP.
HRCT
----
All HRCT was performed with 1.0- or 1.5-mm thick sections at supine and full inspiration at 10-mm intervals. A specialized chest radiologist and a pneumologist analyzed the images at three pre-established levels (trachea, carina, and pulmonary veins) for the presence of any signs of ILD: ground glass, consolidation, reticular, honeycombing, and bronchiectasia.
Histological analysis
---------------------
Open lung biopsy was performed by formal thoracotomy avoiding honeycombing areas. Two pathologists specialized in lung diseases, blinded to clinical aspects of the patients, classified the lung specimens according to the new consensus on classification of ILD ([@B25]). Final diagnoses were reached by consensus of the pathologists. Regarding NSIP, the most predominant pulmonary histological pattern was also defined as cellular or fibrosing. As control, normal lung tissue was obtained from 10 individuals (3 males and 7 females), with a median age of 47 years (range, 31 to 60 years) who died suddenly of nonpulmonary causes.
Pulmonary function tests
------------------------
Spirometric analyzes included the assessment of FEV1, FVC, and TLC. DLCO-Hb ([@B26]) was evaluated using a single-breath technique. Results are reported as a percentage of predicted values based on gender, age, and height.
Immunostaining
--------------
A standard peroxidase technique was used, with Harris\'s hematoxylin as the counterstain. All of the antibodies used were biotinylated rabbit polyclonal antibodies. Neuronal NOS (nNOS), eNOS, iNOS, PAI-1, α-smooth muscle actin (α-SMA), IL-4, IL-13, and bFGF polyclonal antibodies (Santa Cruz Biotechnology, Inc., USA) were incubated with tissue sections at a 1:100 dilution. A Max Polymer Novolink amplification kit (Leica, Newcastle Inc., UK) was used for signal amplification and 3,3′-diaminobenzidine tetrachloride (0.25 mg dissolved in 1 mL 0.02% hydrogen peroxide) was used as a precipitating substrate for signal detection. The specificity of primary antibodies was confirmed by appropriate reagent controls (omitting the primary antibody or substituting nonimmune serum for the primary antibody in the staining protocol), which revealed no staining.
Histomorphometry
----------------
Immunohistochemical staining of NOS isoforms, PAI-1, α-SMA, IL-4, IL-13, and bFGF-positive cells in alveolar septa, as well as endothelial, myofibroblast, and smooth muscle cells in terminal bronchiolar arteries, were quantified by stereology at 400× magnification with an eyepiece systematic point-sampling grid with 100 points and 50 lines used to count the fraction of lines overlying the positively stained structures ([@B27]).
We averaged the observations from 10 microscopic fields to obtain the final results, which are reported as a percentage of the stained structures. To control for variation in scoring between our two histologists (ACAJ and ERP), 20% of the stained slides were independently scored by both observers. The coefficient of variance between cell counts for the two observers was \<5%.
Statistical analysis
--------------------
Data are reported as means±SD with 95% confidence intervals. Statistical analysis was performed by ANOVA, followed by appropriate *post hoc* tests, including Bonferroni\'s for multiple comparisons by one-way ANOVA and the Student *t*-test for two variables between groups. The general linear model was used to test the relationship between one continuous variable and several others, and the residuals were examined to ensure that they were approximately normally distributed. Survival analyses were initially done using Kaplan-Meier curves, and final multivariate analyses were done using the Cox proportional hazard model. All analyses were done with SPSS 18.0 (SPSS Inc., USA). A P value \<0.05 was considered to be significant.
Results
=======
Clinical features
-----------------
The clinical features of the 23 patients included in this study are shown in [Table 1](#t01){ref-type="table"}. Age, gender, disease duration, anti-topoisomerase I positivity, MRSS, gastro-esophageal reflux symptoms, esophageal dysmotility, and dyspnea were comparable in both groups. All 23 patients had NSIP in histological examination, and the prevalence of cellular and fibrosing patterns was similar in both groups. Diffuse skin involvement was significantly associated with cellular SSc-NSIP compared to fibrotic NSIP (72 *vs* 33%, P=0.01). All patients studied showed a restrictive lung function pattern characterized by a decrease in TLC (mean values were 81% of predicted in cellular SSc-NSIP and 79% of predicted in fibrotic NSIP) and an increased FEV1/FVC ratio/100 (mean values of 106% of predicted in cellular SSc-NSIP *vs* 108% of predicted in fibrotic NSIP). The mean predicted values of DLCO were significantly decreased in fibrotic NSIP (55%) compared to cellular NSIP (77%) patients ([Table 1](#t01){ref-type="table"}). No difference was found for DLCO/alveolar volume in cellular SSc-NSIP compared to fibrotic NSIP (92 *vs* 70%; P=0.26; [Table 1](#t01){ref-type="table"}).
Morphological features
----------------------
Normal and NSIP histological patterns of alveolar septa and vessels are shown in [Figures 1](#f01){ref-type="fig"}, [2](#f02){ref-type="fig"}, and [3](#f03){ref-type="fig"}, with immunohistochemical staining by nNOS ([Figure 1](#f01){ref-type="fig"}, left panels), eNOS ([Figure 1](#f01){ref-type="fig"}, middle panels), and iNOS ([Figure 1](#f01){ref-type="fig"}, right panels); PAI-1 ([Figure 2](#f02){ref-type="fig"}, left panels), α-SMA ([Figure 2](#f02){ref-type="fig"}, middle panels), and IL-4 ([Figure 2](#f02){ref-type="fig"}, right panels); IL-13 ([Figure 3](#f03){ref-type="fig"}, left panels), and bFGF ([Figure 3](#f03){ref-type="fig"}, right panels). Different immunostaining intensities were exhibited by epithelial, endothelial, myofibroblast, and smooth muscle cells from alveolar septa and vessels in cellular SSc-NSIP histological patterns when compared to normal and fibrotic SSc-NSIP. [Table 2](#t02){ref-type="table"} summarizes the morphometric results. A significant percentage of septal and vessel cells immunostained for iNOS in a cellular SSc-NSIP histological pattern (P=0.001 and P=0.02, respectively). In addition, we found that the level of staining for iNOS related significantly to several factors having to do with the immune response and fibrinolysis regulators. A general linear model analysis demonstrated that staining for septal iNOS related significantly to the staining of septal cells for IL-4 (P=0.03) and to septal IL-13 (P=0.03). All these relationships were significant after allowing for the contribution of the others, and for this analysis we used a multivariable model. In addition, using univariate analyses, staining for vascular iNOS related significantly to staining of vascular eNOS (P=0.009), vascular PAI-1 (P =0.003), and vascular IL-4 (P=0.02). Also, using univariate analysis, septal and vascular iNOS were negatively related, respectively, to bFGF (P=0.02) and α-SMA (P=0.001). In other words, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing bFGF and myofibroblast proliferation, respectively. Other NOS isoforms did not relate to IL-4, IL-13, PAI, and bFGF. [Figure 4](#f04){ref-type="fig"} uses two plots to demonstrate the relationships between staining for septal and vascular iNOS and SSc-NSIP histological patterns. The two box plots demonstrate that the relationship between iNOS and SSc-NSIP histological patterns was very strong. The scatter plots in [Figure 5](#f05){ref-type="fig"} show that there was a strong relationship between staining of septal iNOS and IL-4, IL-13, and bFGF, as well as between vascular iNOS and IL-4, eNOS, PAI-1, and α-SMA. No significant association was found between staining of septal iNOS and eNOS, PAI-1, and α-SMA; equally, no significant association between staining of vascular iNOS and IL-13 and bFGF was found.
{#f01}
{#f02}
{#f03}
{#f04}
{#f05}
Survival analysis
-----------------
Preliminary examination of Kaplan-Meier survival curves demonstrated that, in this study, patients with fibrotic NSIP, septal iNOS \<17.26%, septal IL-13 \<5.46%, vascular nNOS \<7.37%, vascular α-SMA \>63.4%, and vascular IL-4 \<12.79% had approximately the same hazard for survival with a median survival time equal to 49.5 months for all these variables. Thus, we coded overall NSIP histological patterns as a single dummy variable with a value of zero for cellular and a value of one for fibrotic. The results of the Cox model analysis are reported in [Table 3](#t03){ref-type="table"}. After controlling for the SSc-NSIP histological pattern, only three variables were significantly associated with survival time: septal iNOS (P=0.04), septal IL-13 (P=0.03), and septal bFGF (P=0.02). Once these three variables was accounted for, none of the others related to survival. Multivariate analyses showed low risk of death for low septal iNOS, septal IL-13, and septal bFGF expression.
Discussion
==========
We demonstrated higher expressions of iNOS in alveolar and vascular structures in patients with SSc when compared with the normal lung tissue group. Alveolar structures and vessels had a high expression of iNOS in epithelial, endothelial, myofibroblasts, and smooth muscle cells. When total iNOS and NSIP histological patterns were compared, a clear switch was shown in the expression of the iNOS isoform in septal and vascular lesions of patients with SSc. Although the expression data were similar between iNOS in septal and vascular cells, the following features were constant: iNOS was upregulated in epithelial, endothelial, myofibroblasts, and smooth muscle cells from septa and vessels; and the expression of iNOS was more strongly associated with higher pulmonary fibrosis in SSc. One interpretation of the relative pattern of expression and correlation of iNOS is that, as pulmonary lesions in patients with severe pulmonary fibrosis become more extensive, more extrinsic or intrinsic stimuli cause endothelial upregulation of iNOS production and more endothelial injury than in patients with minimal pulmonary fibrosis. This in turn leads to production of sufficient amounts of NO to cause NO-mediated free radical damage to proteins within endothelial and smooth muscle cells in patients with extensive pulmonary damage, which can be recognized as accumulation of these proteins and an increase in the expression of PAI-1. An imbalance in the equilibrium of iNOS and other isoform (nNOS and eNOS) synthesis and the resulting increased production of NO have been reported to be associated with serious cell damage ([@B28]). In the present study, the grades at which greatest endothelial damage occurred were the same as those in which morphological studies from this laboratory have shown evidence of endothelial injury and death ([@B18]). In other situations, such as vascular changes associated with endotoxic shock ([@B29]), and ischemia-reperfusion injuries ([@B30]), endothelial iNOS expression has been associated with endothelial damage mediated by free radicals. All of our data indicate a similar process in patients with extensive pulmonary fibrosis, and this situation contributes to maintenance of the disease. The conclusion that free radical-mediated oxidative injury is involved in the progression of SSc is supported by the increase in iNOS and reduced circulating levels of antioxidants (selenium and ascorbic acid) in these patients. This situation is indicative of the formation of peroxynitrite, nitration of cellular proteins, and cell damage. The switch from upregulation of iNOS in endothelial and smooth muscle cells is not unique to SSc, having been described in other collagen vascular diseases ([@B31]). Increased expression of iNOS by endothelial cells has also been described in patients with systemic lupus erythematosus ([@B32]), but in that study there was no reduction in iNOS expression.
There is a growing body of evidence that cytokines, such as IL-1α, TNF-α, TGF-α, IFN-α, and bFGF, and other local effectors, such as heparin, lipopolysaccharide, and ischemia, might be involved in the regulation of iNOS isoform expression in the endothelium ([@B16],). Our results support the hypothesis that NO production following induction of vascular iNOS contributes to free radical damage previously implicated in the pathogenesis of SSc. One implication of these findings is that general stimulation of NO production in patients with SSc through vasodilatation improves tissue blood flow, and thus cell viability, and could be counterproductive unless therapy is first directed toward selective inhibition of these isoforms. Such selective inhibition can diminish endothelial damage that occurs in progressive pulmonary fibrosis in SSc.
Our study presented clinical and functional impacts. We found an important correlation between pulmonary function tests and high compromise by pulmonary fibrosis in these patients. In order to establish the relevance of these findings to the evolution of the patients, NOS and cytokines were evaluated in the function of survival controlled for histological patterns. Clearly, multivariate analyses showed a low risk of death for low expressions of septal iNOS, septal IL-13, and bFGF.
We conclude that iNOS, IL-13, and bFGF expression in lung parenchyma offers us the potential to control oxidative injury involved in fibrotic progression of SSc, suggesting that strategies aimed at preventing high iNOS synthesis, or local responses to high IL-13 and bFGF cytokines may have a greater impact on SSc. To finalize this conclusion will require greater study in a randomized and prospective trial.
We are grateful to the biologist Sandra de Morais Fernezlian from the Immunohistochemistry Laboratory of the Department of Pathology (Faculdade de Medicina da Universidade de São Paulo) for technical assistance. Research supported by CNPq and FAPESP (Project \#2008/53022-3).
First published online October 15, 2013.
|
LIZ SMITH: Too Close to Call
The Debate Result — It's Still a Race Too Close to Call ... The Fall of Billy Bush — Is That Really Fair? ... Celebrating Manhattan's Legendary New National Landmark, The Stonewall Inn.
“I DON’T necessarily agree with his [Bill Clinton’s] victims. His victims are terrible. He is really a victim himself. But he put himself in that position. But the whole group is a truly unattractive cast of characters ... Paula Jones, Lewinsky, it’s really an unattractive group. I’m not just talking physical.”
Donald Trump to Fox News’ Neil Cavuto in 1998.
Trump in another interview also referred to Clinton’s Inspector Javert (aka Ken Starr) as “a total wacko.”
Donald in 1998.
THE SECOND debate. It changed nothing. Trump supporters say he did brilliantly. Clinton’s insist she won the night. The dial has not moved. What will matter for both candidates is voter turnout on Nov. 8th (The smug over-confidence of Clinton’s advocates continues to concern me deeply.)
Clinton’s best moments came after she left the stage, on board a plane. She was smiling charming — obviously relieved it was over — and ignoring pleas to sit down and fasten up. She declared her staff “all needed a couple of drinks,” and reiterated her mantra — Trump is unfit to hold the highest office in the land. In those unscripted moments she was the very much I woman I have known and met.
I enjoyed Donald’s impersonation of Lurch from "The Addams Family," hovering around and behind Clinton. His facial expressions alone were priceless.
But in truth, the debate itself, what it was compelled to address in the first twenty minutes, and the banana republic threats that came later ("You'd be in prison") were the stuff of nightmares and national disgrace.
THE MOST startling thing (to me) to come out of the weekend frenzy was the indefinite suspension of NBC’s Billy Bush, because he happened to be the reporter caught on tape, chortling with Donald Trump, as the businessman spoke so charmingly about women. Bush did not use the language Trump did, but he didn’t clutch his pearls and flee the trailer, either.
NBC has cooperated with the Trump publicity machine as cravenly as any other network. What — they suddenly disapprove of Donald, but since they can’t chastise him, they bear down on Bush?
Don’t get me wrong. No love here for BB. I find him minimally talented and not an asset to ... anything. And we had our own experiences with him, viewed from afar (like, a table away) at several Hollywood events in years past.
Pat O’ Brien and Billy Bush.
At one — Clive Davis’ annual pre-Grammy gala — he was accompanied by “Entertainment Tonight’s” Pat O’ Brien. Bush (and O’Brien) were obnoxious, obstreperous and entitled. But that was then. (O’Brien eventually sought help in rehab.) I recall that nobody had a kind word for Bush. At least not in observing him in action, on these two separate, perhaps atypical nights. Today, even in a locker room or trailer conversation Billy might be a shining example of propriety. We don’t know.
However, Billy Bush — a cousin to the Bushes who have not spent their lives chatting up actresses with implants for brains and actors with brains no larger than a pea — is not running for office. He has not ever presented himself as a role model. He apologized fully (although he was not exactly a callow youth even back in 2005.) He will not have power over our lives. He will not control the nuclear code. Nobody is going to point to Billy Bush and say, “Grow up like him, kids.” Must we all now fear being in the presence of a powerful person, speaking crassly 11 years ago, because we might lose our jobs today?
Suspending him, possibly for good, is yet another pointless example of political correctness gone oppressive. And as a liberal Dem, I blame liberal Dems for this. It has given the other side endless fodder.
ON June 28th 1969, life changed forever for gay people in the United States, indeed around the word. (Today we refer to the LGBT community, which I find an awkward mouthful, but what the hell.) It was the Stonewall riots, a long-simmering, spontaneous series of battles, which went on for several nights. Within a week these initially mocked confrontations elevated the issues, rights, and massive oppression of gay men and women into a powerful influential, righteously angry movement. (The spirit of finally being free and unafraid, in time led to the more radical Act-Up during the horrors of the AIDS era. They couldn’t have known it at the time, but the brave, very young men and women who went out to the Stonewall one hot night in 1969 for a drink and a good time, saved lives, and in time presided, often in spirit, over same-sex marriages.)
The Stonewall Inn became a historical spot in the hearts and minds of gay people everywhere. On June 28th, of this year, President Obama declared it a genuine historical site.
Now in celebration of the designation, an online celebrity auction will happen on October 25th, running for two weeks. There will be a kick-off reception on Monday October 24th, at the Stonewall itself. Bidders should go to Charitybuzz.com.
By the time this "amusing" headline was published, the gay genie was out of the bottle, and liberation was in the air.
Madonna, Cher, Stevie Nicks, Michael Buble, Demi Lovato, Miley Cryrus, George Clooney, Taylor Swift, Anderson Cooper, Troye Sivan and Andy Cohen are among the many who have donated items. (Many more celebs are expected to donate!) Proceeds will raise funds to help maintain what is now called the Stonewall National Monument (the oval space of several blocks that border the bar.)
Among the organizations involved are Diana Rodgriguez’ Pride Live Nation ... The National Parks Service ... Charity Buzz and Prizeo, another online fundraising entity. (Madonna will part with a pair of shoes, Cher a Philip Treacy hat, Stevie Nicks an autographed tambourine, with the words, “Love to You, Stonewall.”)
This will be an event that so many who danced on the black-and-white checkerboard Stonewall floor — lit from below — to the sounds of The Supremes, Sly and the Family Stone, The Fifth Dimension, The Archies, Credence Clearwater Revival and Jackie DeShannon’s “Put a Little Love In Your Heart” — could never ever have imagined.
LOOK AT This! Here is actress/model Luisana Lopilato, in a still from an
upcoming film. We are not sure of the title. It is either "Predeterminados" or "Los que aman odian." Both are described as in the thriller/drama genre. Luisana is supposed to be a femme fatale of the dangerous film noir type. She certainly looks dangerous! Ms. Lopilato is in real life married to singer Michael Buble. Men, this is what hitting the right notes can get you. Start taking lessons now. |
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Q:
Is there a way to simplify block Cholesky decomposition if you already have decomposed the submatrices along the leading diagonal?
Let's say we have a block matrix $ M =\left( \begin{array}{ccc}
A & B\\
B^{*} & C \end{array} \right)$ where $M$ is positive definite. ($A$ and $C$ are also positive definite.)
There is a formula for carrying out block Cholesky decomposition. See Wikipedia: Block LU decomposition. Summarising we have the following result.
The matrix $M = LU$ can be decomposed in an algebraic manner into
$$L =
\begin{pmatrix}
A^{\frac{1}{2}} & 0 \\
B^{*} A^{-\frac{*}{2}} & Q^{\frac{1}{2}}
\end{pmatrix}$$
where $\begin{matrix}
Q = C - B^{*} A^{-1} B
\end{matrix}$ ($*$ indicates transpose in this case)
Now lets say we have already carried out the Cholesky decomposition for A, and C. So we have already calculated $A^{1/2}$, and $C^{1/2}$ (It is therefore straightforward to calculate the inverses $A^{-1/2}$, and $C^{-1/2}$ using forward substitution).
Rewriting $Q$ in terms of these quantities we now have
$$Q = Q^{1/2}Q^{*/2} = C^{1/2} C^{*/2} - (B^{*} A^{-*/2})(A^{-1/2} B)$ = $(C^{1/2} + B^{*}A^{-*/2})(C^{1/2} - B^{*}A^{-*/2})^{*}.$$
My question is this: Given this set up is it possible to algebraically calculate $Q^{1/2}$ without having to apply Cholesky decomposition to $Q$. Or, in other words, can I use $C^{1/2}$ to help me in the calculation of $Q^{1/2}$.
A:
If A,C are fixed, and B is variable but nice (low-rank), then you want what is called "Cholesky update". If A,B,C are fixed, then probably you should not be picky about how the blocking is done, and you want to use a standard "block Cholesky". I have not found a clear answer for A,C fixed, B variable and not nice (I can ask around, so let me know if that really is your case).
Cholesky update
Rank one updates, chol(A) to chol(A+xx*), are easy and safe. Rank one "downdates", chol(A) to chol(A-xx*), are easy but require a little care: stable algorithms are given in Stewart's Matrix Algorithms Vol 1, Algorithm 4.3.8, p. 347. Chapter 12.5 of Golub–Van Loan has some similar stuff, and Cholesky down-dating in 12.5.4. This function has been widely implemented, and the cholupdate command in matlab dates back to 1979 code from LINPACK.
[0,B*;B,0] is a sum of rank one matrices, and so by updating and downdating those rank one guys, you could probably get what you want, and it might even be faster than chol(Q). However, it can be a lot better to update more ranks at a time.
Apparently this is a common request in machine learning, and M. Seeger wrote a technical report on this problem of low rank updates to a Cholesky factorization, and mentions several common pitfalls, especially as regards to actually doing it with existing software.
A more scholarly (and older) treatment is in section 3 of this article version of Ch. 12.5 of GvL:
Gill, P. E.; Golub, G. H.; Murray, W.; Saunders, M. A. "Methods for modifying matrix factorizations."
Math. Comp. 28 (1974), 505–535.
MR343558
DOI:10.2307/2005923
Davis and Hager in MR1824053 note that algorithm C1 can be used for a reasonably efficient, multiple rank, single pass, update of a dense matrix (and go on to describe sparse techniques).
Note that these mostly do not take advantage of the block structure of [0,B*;B,0], so you might find something better that is more specialized.
Block Cholesky
Blocking the Cholesky decomposition is often done for an arbitrary (symmetric positive definite) matrix. I didn't immediately find a textbook treatment, but the description of the algorithm used in PLAPACK is simple and standard.
In their algorithm they do not use the factorization of C, just of A. That allows them to reduce the problem of chol([A,B*;B,C]) to just chol(A) and chol(Q). The point of the algorithm is that you do not choose A and C to have the same size. You choose A to fit nicely in cache, and do your work at a higher BLAS level. In other words, A is a real block, and C is just leftover garbage you'll need to sweep up next.
In particular, C is discarded and replaced by Q during the algorithm, but chol(Q) is also computed by decomposing Q itself into a block matrix. This means that the algorithm is discarding any information you had about C, so if C is fixed while B varies, this would be quite wasteful.
|
Q:
An inequality for graph nodes and edges
Suppose that we have undirected simple graph $G=(V,E)$ (i.e. without loop), where $V$ is nodes and $E$ is edges.
I would like to prove that if $G$ has some cycle where its length is an even number, then $3|V| \geq 2|E|$.
Can anybody help me with this please?
A:
The complete graph $K_n$ on $n \ge 5$ vertices has a cycle of even length, but $$3|V|=3n \not\ge n(n-1) = 2\binom{n}{2} = 2|E|.$$
|
A possible hormone controlling O2 consumption in the nematode Phocanema decipiens.
The third-stage larva of Phocanema decipiens, which initiates development upon transfer from 5 to 37 degrees, has been used in these experiments. Larvae ligatured at both the head and tail ends lose weight more rapidly during the first 4 hr after transfer to 37 degrees than do normal worms or those ligatured at the front end alone. Larvae ligatured at the tail end exhibit a higher rate of O2 consumption as determined manometrically than do normal larvae. The O2 consumption of strips of body wall is depressed by the addition of an homogenate of the posterior ends of worms, but unaffected by the addition of an homogenate of the anterior ends. These facts are consistent with the release from the tail end of the worm of a hormone which depresses the O2 consumption of the worm. |
Spindle-cell lipoma of the preseptal eyelid.
An 82-year-old woman had experienced progressive enlargement of a long-standing left upper lid mass for 3 weeks. The superior visual field was compromised secondary to mechanical ptosis from this lid lesion. Computed tomography showed a large lid mass with a low density signal, similar to intraorbital fat. The tumor was completely excised. Histopathologic examination showed an encapsulated tumor composed of mature adipose tissue interspersed with fibrovascular septa containing spindle-cells, confirming a diagnosis of spindle-cell lipoma. |
Blockchain beyond the hype: What is the strategic business value in this technology?
Speculation on the value of blockchain is rife, with Bitcoin, the first and most infamous application of blockchain, grabbing headlines for its rocketing price and volatility. That the focus of blockchain is wrapped up with Bitcoin is not surprising.
Its market value surged from less than $20 billion to more than $200 billion over the course of 2017.1 Yet Bitcoin is only the first application of blockchain technology that has captured the attention of government and industry.
Blockchain was a priority topic at Davos; a World Economic Forum survey suggested that 10 percent of global GDP will be stored on blockchain by 2027.2 Multiple governments have published reports on the potential implications of blockchain, and the past two years alone have seen more than half a million new publications on and 3.7 million Google search results for blockchain.
Most tellingly, large investments in blockchain are being made. Venture-capital funding for blockchain start-ups consistently grew and were up to $1 billion in 2017.3 The blockchain-specific investment model of initial coin offerings (ICOs), the sale of cryptocurrency tokens in a new venture, has skyrocketed to $5 billion. Leading technology players are also heavily investing in blockchain:
IBM has more than 1,000 staff and $200 million invested in the blockchain-powered Internet of Things (IoT).
4 Despite the hype, blockchain is still an immature technology, with a market that is still nascent and a clear recipe for success that has not yet emerged. Unstructured experimentation of blockchain solutions without strategic evaluation of the value at stake or the feasibility of capturing it means that many companies will not see a return on their investments. |
Association of Superficial Temporal Artery Dilatation with Headache After Revascularization in Adult Moyamoya Disease.
The underlying mechanisms of headache in adult moyamoya disease (MMD) are not clear. The aim of this study is to clarify the factors that are associated with headache in adult patients with MMD after superficial temporal artery (STA)-middle cerebral artery (MCA) anastomosis. We retrospectively analyzed the cases of 68 adult patients with MMD: 30 with surgery and 38 without surgery. Each STA-MCA anastomosis was performed by the standard technique. Magnetic resonance angiography (MRA) and single photon emission computed tomography were performed perioperatively. We stratified the intensity and frequency of the patients' headaches into 4 ranks. Pre- and postoperative STA diameters were retrospectively measured on digital subtraction angiography (DSA) and/or MRA. In the surgery group, preoperative regional cerebral blood flow (rCBF) laterality and a postoperative rCBF increase >20% showed no significant difference between the patients with and without headache with a univariate analysis. The postoperative STA diameters of the distal branch (DSA) and main trunk (DSA/MRA) in the patients with headache were significantly larger than those of the patients without headache. The rate of postoperative increase of the STA diameters of the distal branch/main trunk was also significantly higher in the patients with headache than those without headache. A multivariate analysis showed that the standard regression coefficient β for sex, a >20% increase of postoperative rCBF, and the increase rate of the STA diameter of the distal branch shown by DSA was 0.37, 0.54, and 0.56, respectively. The results of our analyses revealed that aside from ischemia, the postoperative increase rate of the STA may be a candidate reason for headache, especially in adult patients with MMD. |
Mass spectrometric analysis of a sample requires that the sample be provided in the form of a gas or molecular vapor and then ionized. Ionization may be performed in the mass analyzing portion of a mass spectrometer, i.e., in the same low-pressure region where mass sorting is carried out. Alternatively, ionization may be performed in an ion source (or ionization device) that is external to the low-pressure regions of the mass spectrometer. The resulting sample ions are then transmitted from the external ion source into the low-pressure mass analyzer of the mass spectrometer for further processing. The sample may, for example, be the output of a gas chromatographic (GC) column, or may originate from another source in which the sample is not initially gaseous and instead must be vaporized by appropriate heating means. The ion source may be configured to effect ionization by one or more techniques. One class of ion sources is gas-phase ion sources, which include electron impact or electron ionization (EI) sources and chemical ionization (CI) sources. In EI, a beam of energetic electrons is formed by emission from a suitable filament and accelerated by a voltage potential (typically 70 V) into the ion source to bombard the sample molecules. In CI, a reagent gas such as methane is admitted into the ion source conventionally at a high pressure (e.g., 1-5 Torr) and ionized by a beam of energetic electrons. The sample is then ionized by collisions between the resulting reagent ions and the sample. The resulting sample ions may then be removed from the ion source in the flow of the reagent gas and focused by one or more ion lenses into the mass analyzer. The mass spectrometer may be configured to carry out EI and CI interchangeably, i.e., switched between EI and CI modes according to the needs of the user.
High-pressure CI ion sources have been employed in conjunction with three-dimensional (3D) quadrupole ion trap mass spectrometers, and would also be applicable to two-dimensional (2D, or “linear”) ion trap mass spectrometers (linear ion traps, or LITs). With either 3D ion traps or LITs, the sample is often introduced into the external ion source at an elevated temperature, such as when the sample is the output of a GC column. When the sample is provided at an elevated temperature, it is necessary to heat the ion source to prevent the sample from condensing in the ion source. However, because the ion source in this case is external to the ion trap and the ion trap itself is not utilized for ionization, it is not necessary to also heat the ion trap in this case, which is an advantage of external ion sources. Yet conventional external CI ion sources operate at high pressure as noted above, which is a disadvantage. High pressure CI requires the use of compressed gas cylinders to supply the reagent gas, as well as vacuum pumping stages between the ion source and the very low pressure ion trap. High pressure CI may increase contamination of the ion source, particularly in the area around the filament utilized to emit electrons where the high temperature causes pyrolysis of the reagent gas and contamination. High pressure also limits the choice of reagent gases able to be utilized and thus also limits the choice of chemical properties and reaction pathways available for CI. High pressure also limits the CI yield. Because ions are not trapped in a high-pressure ion source, the time in which the sample can interact and react with the reagent ions is limited by the volume of the ion source and the total gas flow rate. The gas flow rate in a high-pressure ion source is high, and thus the residence time of sample molecules in the ionization region where the reagent ions reside is low.
As an alternative to external ion sources, a 3D ion trap itself may be utilized to effect CI. In this case, the reagent ions are formed directly in the interior region defined by the electrodes of the 3D ion trap and the sample is subsequently introduced into the same interior region. In this case, the sample is ionized in this interior region and the resulting sample ions are subsequently scanned from the same interior region to produce a mass spectrum. Internal ionization is advantageous because it is performed at the low operating pressure of the ion trap. However internal ionization is disadvantageous because, unlike external ionization, it is necessary to heat the entire electrode assembly of the ion trap to prevent the sample from the GC from condensing on the electrodes. Operating the mass analyzer at elevated temperatures is disadvantageous in that it requires heating equipment and may produce inaccurate spectral data due to sample adsorption on the large surface area of the electrodes. Moreover, the electrode assembly must be fabricated by special techniques designed to enable the electrode assembly to reliably withstand repeated high-temperature operation.
In view of the foregoing, there is a need for providing apparatus and methods for implementing low-pressure EI and CI in which the sample is ionized in an ion processing device that is external to an ion trap utilized for mass analysis. |
Mediterranean Salad With Creamy Herb Dressing
Autumn is the perfect time to try new pasta recipes and make some all-time favorites, using authentic, great-tasting Dreamfields Pasta, available in seven delicious varieties. With Dreamfields you get the healthy advantage with only 5g of digestible carbs and 5g of fiber per 1-cup serving, making it the perfect pasta for all pasta lovers.
Directions
In medium bowl combine mayonnaise, herbs and vinegar. Stir to combine. Add to pasta mixture; toss until well coated (add extra vinegar if necessary for consistency). Season with salt and pepper as desired.
Refrigerate, covered, 4 hours or overnight to chill completely.
Tips & Techniques
*If traditional pasta is used in this recipe there is a total of 58g carbohydrate. For more information go to www.DreamfieldsFoods.com. |
Hamad Mansor
Hamad Mansor (Arabic:حمد منصور) (born 13 August 1994) is a Qatari footballer. He currently plays for Qatar on loan from Al-Sadd .
External links
References
Category:Qatari footballers
Category:1994 births
Category:Living people
Category:Al Sadd SC players
Category:Muaither SC players
Category:Al-Khor SC players
Category:Qatar SC players
Category:Qatar Stars League players
Category:Association football midfielders |
Q:
Rails url_for and namespaced models
In Rails, is it possible to namespace models in modules and still get correct behavior from url_for?
For instance, here, url_for works as expected:
# app/models/user.rb
class User < ActiveRecord::Base
end
# config/routes.rb
resources :users
# app/views/users/index.html.haml
= url_for(@user) # /users/1
Whereas after putting the User model into a module, url_for complains about an undefined method m_user_path:
# app/models/m/user.rb
module M
class User < ActiveRecord::Base
end
end
# config/routes.rb
resources :users
# app/views/users/index.html.haml
= url_for(@user) # undefined method 'm_users_path'
Is it possible to have url_for ignore the module in M::User and return user_path for url_for(@user) instead of m_user_path?
UPDATE
So, after almost 5 years, here's the solution, thanks to esad. This has been tested in Rails 4.2.
# app/models/m/user.rb
module M
class User < ActiveRecord::Base
end
end
# app/models/m.rb
module M
def self.use_relative_model_naming?
true
end
def self.table_name_prefix
'm_'
end
end
# config/routes.rb
resources :users
# app/views/users/index.html.haml
= url_for(@user) # /users/1
Note: when generating model, view and controller with bin/rails g scaffold m/user, the views and the controller will be namespaced, too. You need to move app/views/m/users to app/views/users and app/controllers/m/users_controller.rb to app/controllers/users_controller.rb; you also need to remove references to the module M everywhere except in the model M::User.
Finally, the goal here was to namespace models but not views and controllers. With esads solution, the module M (containing User) is explicitly told to not appear in routes. Thus, effectifely, the M is stripped of and only User remains.
The user model can now reside in app/views/models/m/user.rb, the users controller lives in app/views/controllers/users_controller.rb and the views can be found in app/views/users.
A:
Just define use_relative_model_naming? in the containing module to avoid prefixing the generated route names:
module M
def self.use_relative_model_naming?
true
end
end
A:
Use
namespace "blah" do
resources :thing
end
Then routes will be named appropiately.
rake routes
To view all routes
A:
Specify the module on the route
resources :users, :module => "m"
or use scope to do it
scope :module => "m" do
resources :users
end
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##VERSION: 2.0
build.gradle||GHIDRA||||END|
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Cardboard packers are a prefabricated boxes usually use for packaging and protecting goods. These boxes are light weighted and easy to handle while transporting from one place to another. These are qu...mehr
Cardboard packers are a prefabricated boxes usually use for packaging and protecting goods. These boxes are light weighted and easy to handle while transporting from one place to another. These are qu...mehr |
Miami Northwestern has been one of the most dominant high school football programs in the America for most of the last decade, producing college prospects by the truckload. The latest superstar to emerge from Northwestern is junior quarterback Teddy Bridgewater, a 6-foot-3, 170-pound dual-threat prospect that has quickly captured the attention of top programs from around the country.
"I've got offers now from Florida, Miami, Tennessee, LSU, Rutgers and Florida International," Bridgewater said. "I'm not really concerned about my offers at this point. I don't want to make any decisions based on who has offered me now because I think there will be some more coming."
"I'm hearing a lot from Notre Dame, Indiana, Florida State and Alabama too. Alabama is one that I'm really excited about. I like what I've seen of them this year. They play a lot of young guys at pretty much every position and their quarterback is playing really good right now. That defense is one of the best I've ever seen."
Bridgewater showed last week why some recruiting analysts are calling him one of the best quarterback prospects of the 2011 class by setting a Dade County record with seven touchdown passes in a single game for Northwestern.
"We can definitely still improve as a team and I'm sure there are some things I can do to improve as a quarterback. I think we have a pretty special team this year but we have to keep pushing ourselves and trying to constantly get better. If we do that, we could go pretty far."
While Bridgewater says that most college coaches say they want him as a quarterback, he feels confident that if called upon he could succeed at a number of positions at the college level.
"So far, everybody is recruiting me as a quarterback. I've played quarterback pretty much my whole life but I'd be willing to change to another position if it helps me get on the field. I feel like I have the athletic ability to play something like wide receiver or running back."
When it comes to recruiting, Bridgewater finds himself with plenty of options to consider and the junior star says that while he doesn't have any favorites at this point, he has already set a date to announce his eventual decision.
"I made a decision a while ago that I would make my commitment on my birthday during my senior year, which is November 10. Right now, that's the plan so hopefully I can take some visits during the summer and know enough to make my decision by then." |
Follow the following bedroom decorating ideas to make. If you use a high-quality cotton duvet cover, which protects your comforter, you can ditch the top sheet for a cozy night’s sleep and also a no-fuss morning when creating your bed.” If it is easy for them to perform, they may sleep better even though you should not expect guests to create their bed. Give a lamp and chair and a desk.
Having cushions on the bed completes the appearance of the bedroom. Hanging the lights behind a sheet or thin curtain helps make your space feel less like a Christmas screen and more . Spray the amount in blankets and your pillow; this can help you be more comfortable, if you prefer the scent.
Studies have found that wearing socks to bed helps you sleep, although there’s no solid explanation for this. The Westin Hotel is known for its “Heavenly Bed” concept (pictured) made to give guests the ultimate in sleeping luxury. Your mattress sags, you wake up with stiffness or pain, you may need a new mattress if your mattress is 5 to 7 years old or has lumps, or you discover whenever you aren’t in your bed, that you get better sleep.
We enjoy shooting off the sheet because it adds time to making the bed and generally becomes jumbled mess at the foot of the bed every morning every morning. Here, a strong canopy creates a snug cocoon for Sunday mornings. Mattresses help give your spine the right quantity. Start looking for an easy-to-wash duvet cover made from soft, natural fiber (flannel, cotton, linen, etc).
Fantastic Housekeeping has recommendations for the finest. For a twin bed, stick with 2 standard pillows and a single Euro cushion. Our sheets breathe, keeping the skin dry and comfortable even on nights top tips to make bed comfortable. Try sheets made or ones of easy cotton. Whether you’re currently trying to cozy up your mattress or just have difficulty we’ve found the 10 items that the internet is buzzing going to make sleeping a dream come true.
When deciding of a duvet remember that the you go, the mattress linens will cost to cover it. Should you prefer sleeping without the sheet but enjoy the look of the perfectly made bed, the Nova Duvet Cover might be for you. The Westin Hotel “Heavenly Bed” feather and down king-size cushions cost $85 each.
To attain thread counts greater bedding makers often times utilize creative weaving and counting. Having said that, if you are allergic to down (or just how much it costs), there are still plenty of feather-free and budget-friendly alternatives that will give you the BIG, FLUFFY FEELING you’re looking for. |
import { FORWARD, LEFT } from '@warriorjs/geography';
import shootCreator from './shoot';
describe('shoot', () => {
let shoot;
let unit;
beforeEach(() => {
unit = {
damage: jest.fn(),
log: jest.fn(),
};
shoot = shootCreator({ power: 3, range: 3 })(unit);
});
test('is an action', () => {
expect(shoot.action).toBe(true);
});
test('has a description', () => {
expect(shoot.description).toBe(
`Shoots the bow & arrow in the given direction (\`'${FORWARD}'\` by default), dealing 3 HP of damage to the first unit in a range of 3 spaces.`,
);
});
describe('performing', () => {
test('shoots forward by default', () => {
unit.getSpaceAt = jest.fn(() => ({ getUnit: () => null }));
shoot.perform();
expect(unit.getSpaceAt).toHaveBeenCalledWith(FORWARD, 1);
expect(unit.getSpaceAt).toHaveBeenCalledWith(FORWARD, 2);
expect(unit.getSpaceAt).toHaveBeenCalledWith(FORWARD, 3);
});
test('allows to specify direction', () => {
unit.getSpaceAt = jest.fn(() => ({ getUnit: () => null }));
shoot.perform(LEFT);
expect(unit.getSpaceAt).toHaveBeenCalledWith(LEFT, 1);
expect(unit.getSpaceAt).toHaveBeenCalledWith(LEFT, 2);
expect(unit.getSpaceAt).toHaveBeenCalledWith(LEFT, 3);
});
test('misses if no receiver', () => {
unit.getSpaceAt = jest
.fn()
.mockReturnValueOnce({ getUnit: () => null })
.mockReturnValueOnce({ getUnit: () => null })
.mockReturnValueOnce({ getUnit: () => null })
.mockReturnValueOnce({ getUnit: () => 'anotherUnit' });
shoot.perform();
expect(unit.log).toHaveBeenCalledWith(
`shoots ${FORWARD} and hits nothing`,
);
expect(unit.damage).not.toHaveBeenCalled();
});
describe('with receiver', () => {
beforeEach(() => {
unit.getSpaceAt = jest
.fn()
.mockReturnValueOnce({ getUnit: () => null })
.mockReturnValueOnce({ getUnit: () => 'receiver' })
.mockReturnValueOnce({ getUnit: () => 'anotherUnit' });
});
test('damages receiver', () => {
shoot.perform();
expect(unit.log).toHaveBeenCalledWith(
`shoots ${FORWARD} and hits receiver`,
);
expect(unit.damage).toHaveBeenCalledWith('receiver', 3);
});
test('shoots only first unit', () => {
shoot.perform();
expect(unit.damage).not.toHaveBeenCalledWith('anotherUnit', 3);
});
});
});
});
|
Smart meter rollout in the UK: towards a low carbon future?
On 19 November consumer rights group Which? published a report warning energy suppliers that they need to triple the current rate of smart meter installation to meet the target of replacing existing meters in every UK home by 2020. This would mean installing 30 smart meters per minute every day for the next two years to replace the existing 46 million meters, which is a challenge to say the least. Back in August 2018, Citizens Advice was already calling for the deadline to be extended for another three years.
The same day the Which? report came out, the Centre on Innovation and Energy Demand (CIED) hosted an event bringing together key stakeholders to discuss the progress of the smart meter rollout, its challenges and opportunities and how the technology can contribute to the low carbon transition.
A panel of speakers from Citizens Advice, Smart Energy GB, the Energy Saving Trust and energy supplier Npower all provided their perspective on the Smart Meter Implementation Programme, the largest government-run information technology project in history.
Image by Nora Blascsok
A key topic of discussion at the event was how the rollout is communicated in mainstream media. Despite its challenges, participants agreed that the rollout can be regarded a success, however, stakeholders needed to come together and create a positive narrative, a message to consumers they all can get behind. This message should emphasize the fact that smart meters are a step towards a better future and a technology that will empower consumers to participate in the new energy world.
Another point highlighted was the way smart meters are a means to an end. Alongside the direct benefits of helping consumers understand their energy use better, minimise energy waste, access energy when most affordable and least carbon intensive; they should also lead to people installing more energy efficiency technologies. They can not only help link data to the provision of energy advice, but also, help drive action to achieving energy efficiency standards – the Minimum Energy Efficiency Standards set by the UK government (all homes to be EPC band C or higher by 2025, fuel poor homes by 2030).
The panellists also focused on the importance of not leaving anyone behind. Smart meters can not only make prepay more affordable –a metering method mostly used by low-income consumers – they will help vulnerable consumers keep track of their energy use, lead to accurate bills and put an end to back-billing. However, as an audience member pointed out, there are questions around whether it is the consumer who is making the significant saving or the energy supplier.
Another take-away message was the importance of consumer choice and the problematic issue of the 2020 deadline. There have been calls to make the installation of smart meters mandatory, however, this would eliminate the incentive to make a pitch and sell the benefits allowing consumers to have a choice in the matter. Furthermore, having to prioritise speed due to the deadline will likely go to the detriment of quality or cost, as one panel member pointed out.
Regarding issues around trust, it was highlighted by an audience member that there needs to be more transparency around how suppliers use consumers’ data. Setting tariffs based on energy use profiles could affect vulnerable consumers, as their flexibility around when they use energy is limited.
The discussion provided a lot of food for thought and linked well into the research done at CIED around smart homes, the media discourse around the rollout and the issues around vulnerable consumers and their response to the rollout.
The SMIP is certainly not without its challenges but if all stakeholders work together, the technology will enable a faster transition to a low carbon future. |
1. Field of the Invention
The present invention generally relates to semiconductor laser elements and typically to a semiconductor laser element of a high output power and low power consumption for use in an optical disk apparatus, an optical transmission system or the like, and a manufacturing method thereof.
The present invention also relates to an optical disk apparatus and an optical transmission system having such a semiconductor laser element.
2. Description of the Related Art
Among various semiconductor laser elements, a semiconductor laser element of buried ridge structure is able to reduce the parasitic current that does not contribute to laser oscillation and to separate a crystal regrowth interface from the active layer in the element manufacturing stage because of its own structure and is therefore known as having a structure capable of achieving high temperature operation and transverse mode stability while establishing compatibility between high reliability and low power consumption (low threshold current).
The buried ridge structure, of which the portion to be exposed to the atmosphere during the manufacturing process is located apart from the active region at the time of laser oscillation, is therefore able to restrain excess photoabsorption to a low level and secure reliability.
Moreover, providing a current block layer of a small refractive index outside the ridge portion achieves the optical confinement in the horizontal direction by only current constriction and real refractive indices. Since a so-called real refractive index guide structure does not use the photoabsorption for the optical confinement in the horizontal direction, the waveguide loss at the time of laser oscillation is small, and the power consumption can be restrained to a low level.
In fabricating the buried ridge structure semiconductor laser element, it is a general practice to subject a semiconductor substrate having stacked crystal layers including an active layer to two more crystal (re)growth processes.
However, carrying out two crystal regrowth processes is a very large cost increase factor in forming a semiconductor laser element. Accordingly, there is an idea of simplifying the manufacturing process by providing an electrode that forms a Schottky junction with a cladding layer and an ohmic junction with a contact layer (refer to, for example, JP 04-111375 A).
FIG. 34 shows a cross-sectional structure of a semiconductor laser element described in JP 04-111375 A. This semiconductor laser element is manufactured as follows. First, an n-InGaP cladding layer 702, an InGaAs/GaAs strained quantum well active layer 703, a p-InGaP cladding layer 704 and a p-InGaAs contact layer 705 are successively formed on an n-GaAs substrate 701 by the MOCVD (metal-organic chemical vapor deposition) method. Etching is performed until a middle of the p-InGaP cladding layer 704 is reached by the technique of photolithography or the like to form a ridge portion 708 of a forward mesa shape. Thereafter, Ti/Pt/Au and Au—Ge—Ni/Au are deposited as a p-side electrode 706 and an n-side electrode 707, respectively. As a result, a Schottky junction portion is formed between the p-side electrode 706 and the p-InGaP cladding layer 704, while an ohmic junction portion is formed between the p-side electrode 706 and the p-InGaAs contact layer 705. If a current is applied between the p-side electrode 706 and the n-side electrode 707 of the thus fabricated element, then a current flows through only the ohmic junction portion. In this way, current constriction is effected.
With the above-mentioned structure, the crystal growth process, which has been carried out three times in total in an ordinary buried ridge structure, is allowed to be carried out only once, and the manufacturing process steps can consequently be largely reduced.
However, the semiconductor laser element of JP 04-111375 A, in which the current constriction is effected by using the Schottky junction, has been unable to carry out operation with a low threshold current (e.g., 30 mA or less) and a high output power (e.g., output power exceeding 150 mW) as achieved in the conventional semiconductor laser elements of ordinary buried ridge structure. Moreover, it has been difficult to provide an element design matched to the optical characteristic specifications demanded for various applications. Furthermore, the Schottky junction portion has had poor reliability, and long-term reliability has not been able to be achieved.
Reasons for the failure in achieving the operation of a low threshold current and a high power may include the fact that the current constriction property at the Schottky junction portion was so insufficient that the leakage current particularly in a fine stripe-shaped structure could not be sufficiently reduced.
Moreover, a construction capable of establishing compatibility between a low device resistance and the current constriction property is not disclosed. As a result, the device resistance was increased. This has also been a factor that disturbs the high power operation.
Moreover, in order to secure the current constriction property, it is required to use, for the cladding layer, materials such as InGaP having a great energy discontinuity (barrier) ΔEv in the valence band (so as to be lattice matched to GaAs) and AlGaAs having a high Al mole fraction, and there has been little scope for changing the refractive index of the cladding layer. Therefore, the degree of freedom in designing the optical characteristics has been limited. Furthermore, no Schottky junction structure having resistance to breakdown has been disclosed, and so far there have been only structures that lack long-term reliability.
In addition, the semiconductor laser element and the manufacturing method disclosed in JP 04-111375 A has had a problem that the optical characteristics of the laser element are largely influenced by the variation in the layer thickness of the remaining p-InGaP cladding layer after etching, occasionally causing variation in the far-field pattern (FFP) in the horizontal direction and a deterioration in the stability of the transverse mode. Furthermore, due to the variation in the InGaP layer thickness, the characteristic of the Schottky junction formed on that layer may also vary, and this may lead to an insufficient current constriction. Moreover, since the refractive index of the InGaP layer that is a lattice matching condition is uniquely determined, adjustment thereof is only achievable by changing the InGaP film thickness when making an optical design. Thus, the degree of freedom of design is small. There is a further problem that use of InGaP for the p-cladding layer tends to increase ΔEv, which may result in a limit to the injection efficiency of holes into the active layer. |
Q:
WPF: Button which is a path
I have a bunch of Buttons in my application which look almost the same. The only difference between them is the path data that they use.
This is the style I have for the "minimize button" of my application:
<Style x:Key="MinimizeButton" TargetType="{x:Type Button}">
<Setter Property="Template">
<Setter.Value>
<ControlTemplate TargetType="{x:Type Button}">
<!-- The border is here to make the entire "block" clickable.
Without it, only the actual path is clickable. -->
<Border Background="Transparent">
<Path Name="ThePath" Data="{StaticResource MinimizeIconPath}" Style="{StaticResource WindowButtonPath}"/>
</Border>
</ControlTemplate>
</Setter.Value>
</Setter>
</Style>
As you can see, I'm basically just changing the control template to use a path instead of the default button stuff. But this declaration is only for the minimize button – I have a few other buttons, such as "maximize", "restore" and "close" to deal with now. The problem is that the styles for those buttons will be identical, with the only difference being the Data attribute of their Paths.
What would you recommend me to do to use as little code as possible?
A:
User control?
http://www.codeguru.com/cpp/data/data-misc/xml/article.php/c12521/
http://www.codeproject.com/KB/WPF/XSButton.aspx
|
// Copyright 2018 The Go Authors. All rights reserved.
// Use of this source code is governed by a BSD-style
// license that can be found in the LICENSE file.
package lockedfile
import (
"fmt"
"os"
"sync"
)
// A Mutex provides mutual exclusion within and across processes by locking a
// well-known file. Such a file generally guards some other part of the
// filesystem: for example, a Mutex file in a directory might guard access to
// the entire tree rooted in that directory.
//
// Mutex does not implement sync.Locker: unlike a sync.Mutex, a lockedfile.Mutex
// can fail to lock (e.g. if there is a permission error in the filesystem).
//
// Like a sync.Mutex, a Mutex may be included as a field of a larger struct but
// must not be copied after first use. The Path field must be set before first
// use and must not be change thereafter.
type Mutex struct {
Path string // The path to the well-known lock file. Must be non-empty.
mu sync.Mutex // A redundant mutex. The race detector doesn't know about file locking, so in tests we may need to lock something that it understands.
}
// MutexAt returns a new Mutex with Path set to the given non-empty path.
func MutexAt(path string) *Mutex {
if path == "" {
panic("lockedfile.MutexAt: path must be non-empty")
}
return &Mutex{Path: path}
}
func (mu *Mutex) String() string {
return fmt.Sprintf("lockedfile.Mutex(%s)", mu.Path)
}
// Lock attempts to lock the Mutex.
//
// If successful, Lock returns a non-nil unlock function: it is provided as a
// return-value instead of a separate method to remind the caller to check the
// accompanying error. (See https://golang.org/issue/20803.)
func (mu *Mutex) Lock() (unlock func(), err error) {
if mu.Path == "" {
panic("lockedfile.Mutex: missing Path during Lock")
}
// We could use either O_RDWR or O_WRONLY here. If we choose O_RDWR and the
// file at mu.Path is write-only, the call to OpenFile will fail with a
// permission error. That's actually what we want: if we add an RLock method
// in the future, it should call OpenFile with O_RDONLY and will require the
// files must be readable, so we should not let the caller make any
// assumptions about Mutex working with write-only files.
f, err := OpenFile(mu.Path, os.O_RDWR|os.O_CREATE, 0666)
if err != nil {
return nil, err
}
mu.mu.Lock()
return func() {
mu.mu.Unlock()
f.Close()
}, nil
}
|
-- predictability
SET synchronous_commit = on;
-- fail because we're creating a slot while in an xact with xid
BEGIN;
SELECT txid_current() = 0;
?column?
----------
f
(1 row)
SELECT 'init' FROM pg_create_logical_replication_slot('regression_slot', 'test_decoding');
ERROR: cannot create logical replication slot in transaction that has performed writes
ROLLBACK;
-- fail because we're creating a slot while in a subxact whose topxact has an xid
BEGIN;
SELECT txid_current() = 0;
?column?
----------
f
(1 row)
SAVEPOINT barf;
SELECT 'init' FROM pg_create_logical_replication_slot('regression_slot', 'test_decoding');
ERROR: cannot create logical replication slot in transaction that has performed writes
ROLLBACK TO SAVEPOINT barf;
ROLLBACK;
-- succeed, outside tx.
SELECT 'init' FROM pg_create_logical_replication_slot('regression_slot', 'test_decoding');
?column?
----------
init
(1 row)
SELECT 'stop' FROM pg_drop_replication_slot('regression_slot');
?column?
----------
stop
(1 row)
-- succeed, in tx without xid.
BEGIN;
SELECT 'init' FROM pg_create_logical_replication_slot('regression_slot', 'test_decoding');
?column?
----------
init
(1 row)
COMMIT;
CREATE TABLE nobarf(id serial primary key, data text);
INSERT INTO nobarf(data) VALUES('1');
-- decoding works in transaction with xid
BEGIN;
SELECT txid_current() = 0;
?column?
----------
f
(1 row)
-- don't show yet, haven't committed
INSERT INTO nobarf(data) VALUES('2');
SELECT data FROM pg_logical_slot_get_changes('regression_slot', NULL, NULL, 'include-xids', '0', 'skip-empty-xacts', '1');
data
-----------------------------------------------------------
BEGIN
table public.nobarf: INSERT: id[integer]:1 data[text]:'1'
COMMIT
(3 rows)
COMMIT;
INSERT INTO nobarf(data) VALUES('3');
SELECT data FROM pg_logical_slot_get_changes('regression_slot', NULL, NULL, 'include-xids', '0', 'skip-empty-xacts', '1');
data
-----------------------------------------------------------
BEGIN
table public.nobarf: INSERT: id[integer]:2 data[text]:'2'
COMMIT
BEGIN
table public.nobarf: INSERT: id[integer]:3 data[text]:'3'
COMMIT
(6 rows)
SELECT 'stop' FROM pg_drop_replication_slot('regression_slot');
?column?
----------
stop
(1 row)
|
bird on a wire
It was as if he continued to fall. Even after the explosion of pain from when his head smacked into the roof of the car. Falling, falling through a darkness that watched him. He could feel something vile looking into his mind, its evil tenacity prying past his eyes and into his brain relentlessly crushing his will, peeking into his body and violating him. A sensation of spiders covered in oily hair that were crawling inside of his skin and skull. Gnawing, scratching, nibbling, tickling. Laughter erupted from the greasy arachnids, their mandibles quivering with devilish glee and dripping with saliva. He fell into the black screaming, crying and clawing at his eyes scratchi —
A solitary heartbeat, thumping in the darkness.
Glasses clinked. A toast. Familiar music fuzzily found its way to his ears, that haunting tune:
“Stopped into a churchI passed along the wayWell, I got down on my knees (got down on my kneeees)And I pretend to praaay…”
Robert’s eyelids were heavy, and he blinked away the sleep. He sat up and looked at a darkened bar, men leaning against a wooden wall talking. A dart thumped into a board amidst mixed cheers and groans, and R.J. wiped at his eyes. He felt like he was drugged, or still drunk from the night before. A nausea twinged in his gut as he smelled himself. He looked down at the ragged suit he was wearing, tattered and stained. He really smelled like something that had died and sat in the sun for too long. Robert mused about the witty comments Linda would have made if she could see him now. The faint smile disappeared from his face as his past experiences came rushing back to him. The bloody mess in the forest, the vacuum of space, the post-apocalyptic hell all filled his mind’s eye.
Where is this? Seems… Normal enough.
There was an empty glass on the table in front of him, sitting on top of a newspaper that read the date. Robert’s heart skipped a beat and he stood up with a start, looking for the bartender. He noticed that his cheeks itched as he strode across the dark wooden floor. A thick beard has found its home where his clean-shaven face used to be. Odd.
“Say, barkeep…” Robert said with a wave of his hand as he sat at the counter. The man turned and hesitated before walking over. Picking up a glass to clean, he looked at R.J. and nodded.
“What city is this?” Robert asked, barely able to hide his hope.
Laughter swelled in the tavern as the bartender told him, and Robert’s eyes lit up with joy. He was home! Well, almost home.
“Pour me a cold one, friend.” Putting a bill on the counter he smiled at the fellow, and the fellow could not help but smile back at Robert’s happy face. R.J. had one of those types of smiles: rare, but when they came you HAD to smile back. Perfect for a travelling salesman. He had closed a few big deals with this talent before. The man poured R.J. a nice cold beer with a modest head, and he took a sip. It was impossibly refreshing to Robert, and he felt he could cry he was so happy. Finally he was safe. Suddenly, the bartender’s smile became stretched almost… like a caricature. His eyes narrowed and changed somehow.
Did it just get darker in here? Or…
“You… do not belong.” The barkeep said with an ominous monotone. The voice was distorted and had undertones of static. Invisible ice crept over Robert’s shoulders and shot down his legs as the man leaned toward him threateningly. Now Robert could see that darkness was emanating from the space around the being. The shadows were pulsing, bubbling out from nowhere, and R.J. felt like he was making eye contact with it. As an elongated tongue curled from the being’s mouth Robert leaned back from the demon barkeeper in awkward horror, and he looked to his left at the man sitting two chairs away. The jovial fellow did not seem to notice this petrifying thing pouring drinks! Robert felt as if he was freezing solid, and leaping from his bar stool he made his way to the entrance, clumsily weaving past people as they enjoyed their night. Passing back frenzied glances at a thing of madness.
“YOU CANNOT ESCAPE.” A roar blew from the darkened space swirling behind the bar, framing the shadowy humanoid. Robert noticed how unnaturally tall it was, and that everyone around him seemed to not notice anything. Talking and laughing, blissfully unaware that something had consumed the…barkeeper? With the blink of an eye the devil vanished from existence, the darkness resting for now. Glasses clinked again and conversations blended into a chorus of humanity. Maybe he was seeing things.
A bell chimed as he left the bar, twinkling in the doorway with warmth.
So much for not escaping.
Considering where he woke up the last few times, Robert felt at ease despite what he saw. He looked at this old city, one he had grown to hate, and saw a paradise. It’s amazing what peering into hell can do to your world-view. It was a nice day out, and Robert walked briskly through the crowd with ease. Folks were avoiding him due to his odor and appearance, and a light laugh came from his chest. There was a homeless man in the window pane next to him, smiling back and wearing the same tattered rags. He couldn’t let his beautiful wife see him like this! She would make him sleep on the couch and bathe in tomato soup for a WEEK! Her smile, with those ridiculously perfect teeth, filled his head with feelings of longing. The beard could wait, but he needed new clothes. Luckily his wallet was still hanging in the clump of threads that used to be his back pocket. A storekeepers eyes changed from suspicious to thrilled quickly with some clean green bills.
As he walked out, buttoning his black suit coat, he could feel the owner watching him. Casting a glance over his left shoulder as he strode away, he caught shadows of darkness surrounding a figure wearing the smile of a Cheshire. Robert’s blood ran cold in his veins despite the sun shining onto the city streets, and he picked up his pace. He felt his paranoia was getting the better of him. Thinking back as he walked, he thought that this really must have all been some strange drug induced amnesia. Robert felt certain that all that had happened had been just dreams, and he felt braver because of this. Finally he was on his way home, to his wife who must have had every cop in the state looking for him. He turned down a block and he could see the park that he went to each morning and —
My car!! Yes!!
He produced car keys from his coat pocket and trotted to his sedan. Amazed that it could still be here after what seemed like forever, he stood and looked around at this day. It really was a lovely one. Birds sat lazily on a wire, watching people going about their lives.
“Hey, R.J.,! Is that you?” A voice shouted out over the hustle and bustle of the city. Robert turned quickly to look at an old friend. The doctor he met once during a sales call, and R.J. tried to get him to buy several vacuums. Several!! At the time, Dr. Charley was incredulous to the point of hilarity. He instantly had a soft spot for this bold salesman, rattling on about how useful it would be to have several vacuums — one for the house, the practice, and back-ups just in case the others broke! Ridiculous! Yet there was logic in his rhetoric. And the only reason Robert did this was so that he could negotiate down to just the two. Start high, they always tell you, set a high benchmark to set the tone of a negotiation. Robert smiled at him as he walked over through the crowd.
“Barely recognized you with that beard, R.J. Lowman! What are you doing with one? Found a job that let you keep it?”
“No… Just…” Robert paused and looked up to the sky. A crow was flying against the wind, struggling and getting nowhere. But it was beautiful, he supposed. Sighing, he looked back to his friend:
“I’ve taken some time off, I guess. From the search. I’ve been meaning to ask you about those sleeping pills you gave me, are you sure that they were OK?”
“What do you mean?” Dr. Charley tilted his head and crossed his arms. He looked as if he was still in the office, wearing his lab coat and stethoscope.
“I had some very strange… dreams, and I don’t remember the last…” Robert sheepishly looked to the ground, “… I don’t KNOW how long.” A moment of silence passed.
“That’s just too bad.” Dr. Charley replied flatly. Robert looked at him in surprise.
“What do you mean, ‘that’s too bad’?! You’re a doctor! Sort it out!” The doctor burst into loud laughter at him, gregariously throwing his head back. After a moment he calmed down and caught his breath, wiping the tears from his eyes.
“Robert, do you still think that I am Dr. Charley?” He looked at the smiling doctor, who watched him with the gleeful curiosity of a child. A bitter cold spread itself through R.J., tracing its path down his back to his feet. He felt weak.
“We are everywhere, Robert. You cannot escape us!” At this, Robert backed up, reaching behind him for the door handle — eyes locked onto his friend.
“What are you talking about? You’re Doc Charles!” The doctor stared back at him in disbelief. He chuckled and shook his head, looking down at the ground as if remembering some punchline to some joke. Robert was horrified. He knew now that what he looked at was not his friend. There was no doubt. He felt the tingling of fear again, surprised he was not desensitized to the feeling. In one swift motion he swung himself into the car and shut the door, turning the engine on. Dr. Cha — something leaned casually onto his car and stared into the window, looking right into Robert’s eyes with a knowing smile. Like a friend would.
“Try as you might, but the cycle must continue, Robert.”
Robert slammed on the accelerator and peeled into traffic. Glancing into the rearview mirror he could see the fake doctor, standing there waving at him with one hand while the other was tucked into his lab coat. Robert whirled his car around the corner, tires squealing over the black asphalt. Pedestrians threw themselves out of his way as he raced out of the city. He had to get home. He had to get to his wife before they did.
” We are everywhere.”
The city finally began to grow smaller in his rear-view mirror, and R.J. breathed a sigh of relief. The radio quietly comforted him as did the dull roar of his engine. He tapped his fingers on the steering wheel over-zealously to the rhythm of the song, the same evocative tune from the bar:
He drove for a while, and stopped for gas. As he pumped he noticed a man sitting by his motorcycle in all black, watching him. Dark sunglasses hid his gaze, but Robert could feel eyes on him. R.J. cut the pump early, and got back into his car to leave. The man kicked on his bike and sat on it as it rumbled, glancing at his watch. Robert carefully turned back onto the main road, and got up to speed as fast as he could. He felt uneasy after what happened in the city, despite attempts to calm himself down. The rear-view mirror held no dark motorcyclist. He breathed a sigh of relief, but still his heart pounded in his chest. Thumping against his ribs.
The reflection off of the motorcycle’s chrome flashed in his mirrors. The man in black was coming up on him. Robert accelerated, pushing the gas down and shifting into gear. There was no way that he could out run the bike, he knew, but maybe somehow he could cause him to wreck. The man was barreling up the road, coming closer and closer. Robert felt his heart in his throat beating mercilessly. They were on a straightaway now, and the man in black flew up behind him, and passed him without effort. Then he kept going. Apparently, the only thing that man was looking for was the open road. R.J. felt like a nervous fool.
But then the motorcycle stopped off in the distance, and turned around. It looked like the exhaust was pumping out black smoke but he was surrounded by that darkness. The same thing that consumed the others. The motorcycle roared toward Robert, some kind of demon flying toward him with the throttle pulled back. Robert pushed further on the accelerator as he wiped the sweat from his palms. |
Duration:
Long Enough and Just So Long
I’d never wanted to go to Earth until the doctor told me I couldn’t, that my bones were too brittle. After that, it wasn’t an obsession, just an edge to my days.
Otherwise, my life’s good.
I run a courier ship between Earth, Luna, the space stations, Mars, and the Inner Gate. You need as little mass as possible to run a snipship, and due to what that doctor called my defects, I’m one of the smallest, fastest. Good pay, and most of the time I’m low-g, which is easiest on me.
Freetime I slum around Luna, where my best girlfriend Pippi lives. Or she and I go prospecting out in the shadow of the Gate, like the dozens of other crazies, hoping to stumble on an alien artifact, make us all rich. Not too impossible a dream, though. It’s happened before.
I had a permanent cradle walker left at Luna, that’s how much time I spent there. Pippi worked as a sportscaster for the biggest Moon channel, MBSA. Her name’s not really Pippi, but she had orange braids and long legs and freckles everywhere, so what else could everyone call her?
I’m used to my name getting distorted. My parents named me Podkayne after a girl in an old story about Mars. It becomes Poddy and Special K, usually Kayne.
In college, though, they called me the Gimp. Most of the time it was affectionate. Pippi was my roommate, there from day one. She had eight siblings, ranging from twelve years to three months. A roomie with lower limb reduction syndrome didn’t faze her. I’d come in with a chip pre-loaded on my shoulder, but I relaxed after a couple of weeks.
Pippi was borderline Aspie, called it like it was, which caused her enough troubles on her own. You had to explain to her why you were angry or sad or whatever, but once she knew what was going on, she knew what sounds to make.
The Aspiness makes her an excellent sportscaster. She knows every sports score for the last half century, and a lot of pre-Net stuff too. You can’t come up with a trivia question that’s lunar sports-related that she can’t answer. That was the only thing she really got passionate about, and in a way that charmed the camera.
We never hooked up. Both of us were wired straight. Pippi had a regular friend named Trevor who was usually away on business trips. I paid for it or went virtual every once in a while, and left things at that.
We were both enjoying sunlight at our favorite park, two blocks away from Pippi’s apartment complex. Sitting beside a sculpture there I’ve always loved, spindly rails of color tumbling taller than me like animation lines, edges glinting pink and blue and purple. The smell of tomato and basil and sage filled the air.
Pippi had her face turned up to the light, soaking in the warmth. She had been indulging in tanners again. Her orange shirt and shorts were vibrant against the expanse of her brown skin.
I was more cautious. I don’t want skin tumors later on, so I keep a gauzy over-shirt and hat about me. Silvery sleeves to deflect the light were set over my arms, strapped into the walker’s maneuvering legs. Underneath the sleeves, mercurial light played over my skin.
We both saw him when he entered the park: Tourist-new, still dressed in arrival shorts and paper shirt with “Be nice, I’m a newbie” printed on the back, which guaranteed him a 10% discount at any participating business.
Pippi squinted over. “Is that…”
I followed her gaze. Dark glasses gave me the advantage. “Yep. It’s an AI.”
It was just after what the newsies were calling the Sexbot Scandal, when that Senator was caught traveling with an AI and had used the momentary notoriety to call for AI rights. Now the Senator’s ‘droid and several others of its kind had bought themselves free. I’d seen an interview with one while trapped in line picking up Chinese takeout the night before. Its plans for the next year were to travel with its friend, another of the bots. Wink wink, nudge nudge.
The oldest human urge: Curiosity about who or what each other was fucking.
He had the white plastic skin most AIs were affecting that year. On his head a slouched wool hat like a noir detective’s.
He looked up and saw us looking at him. He froze, like a car grinding gears to a stop. Then he moved again, almost impatient, flinging an arm up as though against us, although I realized a second later that it shielded his eyes from the dazzle of sunlight off the sculpture. Trapezoids of colored light danced over his tunic, glittered on the lenses that were his eyes.
Pippi waved.
He stepped backwards, ducked into the tunnel.
Of course we went in pursuit.
#
He took the West tunnel. Moving fast, dodging between walkers moving between stations, grabbing handholds to hurl himself along. It wasn’t hard to follow him—I’m small, and mostly muscular in the chest and shoulders, so I can rocket along as far as anyone from handhold to handhold. Pippi slowed me down, kept hissing at me to wait up for her.
We emerged in the most touristy of plazas, the complex of malls near the big hotels, the public gardens. I thought I’d seen the flicker of his tunic, his hat’s crumpled feather, as he ducked into the Thai garden.
The dome overhead admitted unadulterated sunlight. There were parrot flowers and bua pood, a waterfall, and a grove full of gibbons, safely behind mesh. Trails led off to discreet clothing and lifestyle boutiques, a restaurant, and a walkway to the next mall. I saw his hat bob through its glass confines and elbowed Pippi, pointing.
She said, “He could be going anywhere from there. There’s a tube stop in the middle of the mall.”
“Where would a sexbot go?”
“Do you think he’s for hire?” she said.
The interview had said only a few sexbots had chosen to keep their professions. Most of the others had made enough to fund other careers. Most had become solo-miners or explorer pilots.
“It can’t be the first time he’s been asked the question,” Pippi said.
I hesitated. I could talk her into asking. Could machines feel embarrassment? What was the etiquette of communication? Was a sexbot, like a human, capable of being flattered by a flirtatious or even directly admiring question?
Gibbons hooted overhead. A long-billed bird clung upside down to the other side of the mesh. If we stayed here much longer, we’d have a park fee added to our monthly taxes. Two parks in a single day was way too extravagant.
We went home.
#
I had a run to the Gate the next morning, so I got up early, let myself out. Took the West tunnel to the tube stop. Grabbed a mushroom roll on the way and ate it on the platform, peering into shop windows at orange and blue scarves and fake ferns and a whole window wall’s worth of animate Muffs, the latest wearable animals. The sign said they lived off air impurities. They had no eyes, which to some people made them cute, I guess, but to me just looked sad.
This time I followed at a distance. Got in the train car at the opposite end, but kept an eye on him. Luckily for me he was getting out at the port. I don’t know what I would have done if it’d looked as though he was going further.
Maybe followed him.
Why? I don’t know. There was something charming about the way he held himself. And I was curious—who wouldn’t be?—about the experience of someone made for sex, someone for whom sex was his entire rationale for existence. What would it have been like for him (it?) awakening to that?
The port platform straddled the Dundee cliffs, overlooking the Sea of Tranquility. He was there at that flickering curtain of energy and I remembered what it did to constructs—shorted them out, wiped them clean. He had his hand outstretched, and I’m the last to deny anyone their choices, but even so I shouted, “Hey.”
He turned, his hand dropping.
I caught up to him. I was in the cradle walker because I was being lazy that day. I could see him taking it in, the metal spidering my lower body, the bulge where my flesh ended, where legs might have been on someone else, the nubs of my left hand—two but as useful as three of your fingers, I swear.
I said, “Want to get a cup of tea and talk about it?”
So cliché, like something you might have seen in a cheap-D. But he said, “Okay,” and his voice sounded as sincere as a mechanical voice can.
The café was half-deserted, just a couple of kids drinking coffee near the main window. We were between main shifts, and I was late for my pick-up, but I thumbed a don’t-bother-me code, knowing I was one of the most reliable usually. They’d curse me but let it slide.
It’s weird, talking to a mechanical. Half the time your mind’s supplying all the little body movements, so you feel like you’re talking to a person. Then half the time you’ve got a self-conscious feeling, like you were talking to your toaster in front of your grandmother.
Maybe it was just as strange for him. There’s a lot of Gimps up here—lower gravity has its advantages, and in a lot of spaces, like my rig, the less you mass the better. Plus times are lean—less elective surgery. Here he was in the land of the unbeautiful, the people who didn’t care as much about their appearance. Strange, when he was beautiful in every single inch, every graceful, economical move.
We didn’t say a word about any of that.
I told him the best places to sightsee, and where he could take tours. I thought maybe he had some advantages—did he need to breathe, after all? Could he walk Outside just as he was?
The big casinos are worth seeing, particularly Atlantis and Spin City. I sketched out a map on my cell and shot it to him.
“Where do you like to go?” he said.
I’m not much for shopping, and I said so. I liked to take the mega-rail between Luna and the Cluster—cheap and you could stare out the window at the landscape.
“Let’s do that,” he said.
#
The Cluster used to be a fundamentalist-founded station that ended up selling its space to private concerns in order to fund itself. The remnants of the church were there. They ran the greenhouses that grew food for Luna, where most of the water got processed too. The stuff at the market there was always fresh and good and cheaper than in stores.
A jazz club had bought space, and a tiny government office matched its grander counterpart in Luna. And there was Xanadu, which was a co-op of five wealthy families. Along with a scattering of individuals who dealt in rare or hand-crafted goods.
There was always music there, and it had enough reputation for being dangerous that all but a few tourists steered clear.
His name was Star. He would be all right with me. I knew enough to keep him safe.
We ate berries and sat beside rippling water. He told me about Earth—never about the people, but the landscape. Trees, pines and sycamores and madrona, maples and honey locusts and cedar. He talked about cliffs that were bound with color: Yellows and reds and deep browns. Everything grew there, it seemed.
He talked about rain, about slow gray clouds and tearing nor’easters. Rain drumming on a tin roof versus its sound on slate. Fine spring mist and the hot rain that fell during drought, coin-sized and evaporating too quickly. Rain on sand, echoed by waves. Thunderheads, gathering themselves over the ocean. He had lived beside the sea for a few years, he said.
I wondered who he had lived with.
So much was unsaid. It was like a cloud in the room. We relaxed despite it.
He didn’t know where he was staying. He had no luggage. I approved of that. I stick to plas-wear and carry no souvenirs other than the rooms inside my head. Even my ship, where I spend more time than anywhere else, is unpersonalized. I liked it that way.
I was staying with Pippi. Star had money, or so he said, and asked where a clean hotel was. I steered him to Blizz, which caters to the Gate regulars, and went back to Pippi’s.
She was surprised to see me. I hadn’t felt like going out on a trip, I said, and offered to take her out to dinner.
All the time we were eating sweet potato fries and tempeh steaks, I tried to figure out how to tell her about Star.
I don’t know what kept me from just blurting it out. That was usually the level we communicated at. Straightforward and without pretense.
I felt like a shit keeping quiet. Eventually it would come out and the longer it took, the worse it would be.
Her place is tiny. Three of us made it feel crowded. We stood around the table, bumping it with our hips.
“How much do you cost?” Pippi asked Star.
He looked at me. “I don’t do that anymore.”
“Then why are you here?”
“I came to see Podkayne.”
Pippi was unembarrassed. She shrugged and said, “Okay.”
He wanted advice about buying into the colony, where to pick a spot. I made him buy me lunch in return for my advice, and we took Pippi along since she knew better than I where the good deals were.
“Over there in Cluster, someone told me a month or two ago,” she said. “He was saying the Church is going to sell off more space, and it’s going to get gentrified. It’s a long ways off though, over an hour by tram.” She licked barbecue sauce off her fingers. Star pushed a wipe across the table towards her.
#
“I don’t think he likes me much,” she said to me, later.
“I don’t think he likes humans much,” I said. “He makes allowances, but I think he’d be just as happy dealing with mechanicals only.”
“Not many mechs up here,” she said.
“Why?” I said. “You’d think it would be ideal for them. No rust. Less dirt. Fewer pollutants in the air.”
“It would make sense,” she said. “What does it say about us, we’re so crazy we pick a place even mechanicals don’t want to live?”
Maybe ten thousand on the face of the moon. The space stations ranged in size from a few hundred to a few thousand. Twenty thousand on the surface of Mars. I didn’t go back there much, even though it was where I had grown up, after my parents died in a crash. Maybe two or three thousand existing around the bounty of the Gate, another hundred pilots and vagabonds and Parasite-ridden.
The few, the proud, the crazy.
Why had Star chosen to come up here?
#
I asked.
He said, “There’s too many living things on the planet.”
“Why not Mars? It’s enough people to qualify as civilization.”
“They’re spread out and it’s dusty. Here it’s clean.”
“You like the sterility up here,” I said. “Then why think about living over in the Cluster? It’s the most organic spot on the moon.”
His face never smiled, just tilted from one degree to another. “It’s a controlled organic.”
“But what do you want to do?”
“Live,” he said. “By myself, with a few friends,” he nodded towards me, “according to my own devices.”
“What about sex?” I blurted out.
He froze like a stuck strut’s shadow. “I beg your pardon?”
“I’m sorry,” I said. “I didn’t mean to intrude. It’s just that I was somewhat interested, but only if you were.”
He shook his head, mere centimeters of rejection. “I’m afraid not.”
Words I’d heard before. Including what he said next. “We can be friends, though.”
#
“He’s not interested,” I told Pippi.
“Screw him,” she said. “Let’s go play Sex Rangers.”
We climbed into the virtual suits and tapped in. I found someone interested in fooling around on a rocky shore, underneath fuzzy pines. The suit’s as good as sex, any day—releases all the tensions you need released, in my opinion—and a lot cleaner.
Afterwards we logged out and ate pizza and watched a deck about boxing. Pippi said the guy had an 87 percent chance of winning (he did), 54 percent chance by knock-out (he did not).
“I asked Star to come mining with us,” I said to her when we were getting ready for bed. I took the couch; she had a fold-down bunk.
“You did what?”
“He’ll be an extra pair of eyes. Not like he’ll take up oxygen.”
She paused. “Fair enough.”
#
He was good enough at spotting. He learned the difference between ice and metal fast enough to satisfy impatient Pippi, who hated explaining things. I focused on getting us close to the debris that swirled in and out of the Gate. You never knew what you might find. One guy picked up a device that fueled a company in food replication and yielded over forty patents. One pilot found a singing harp. Another the greasy lump that ended up becoming snipship fuel.
You never knew.
Pippi and I had a routine. Star didn’t intrude on it much, went to the secondary display and focused on looking for mineral spikes.
Usually we chatted back and forth. There, Star was an intrusive, if silent, presence. Pippi ended up thumbing on the usual newschat channel. Nothing much. An outbreak on Mars, but small and well-contained. An ambassador stricken but rallying in order to continue his mission through the Gate. How much he looked forward to being the fifth human through the interstellar passage that allowed us access to the wild and varied universe. How much he looked forward to opening new trade channels.
Who knew what he might find out there?
“What’s this?” Star said.
From afar just a glitter. Then, closer, a silver-sided chest, the size of a foot locker but covered with golden triangles. An odd, glittery powder encrusted the hinges and catch as it spun in space.
Nothing hissed out. A glass sphere inside, clouded with bubbles and occlusions. As Pippi slipped it out of the gray material surrounding it, we could see oily liquid filling it.
“Could be useless,” Pippi said, her voice unhappy. “Plenty of stories like that before.”
“Could be beaucoup bucks,” I pointed out.
“Of course,” Pippi said, her voice loud and angry, “it’s the time you bring someone along, to split it three ways, that we actually hit a lode.”
“I don’t want any claim,” Star said.
Flummoxed, I stared at him. What must it be like, to have enough to not need more, to have just that one extra layer against yourself and poverty? My parents had left me enough to buy my snipship, but all my capital was tied up in that rig.
“I just wanted the company,” he said. “I thought it would be interesting.”
“Fucking tourist,” Pippi said. “Want to watch the monkeys dance? We’ll kiss for another five grand.”
He backed up, raising his hands. His feet clattered on the deck. Before he had moved quietly. Did he choose to make that sound to remind us he was a machine?
“Thought we’d just love to take the walking vibrator on tour?” Pippi said. When he remained silent, she turned on me. “See, it doesn’t have anything to say to that.”
“He,” he said.
“He? What makes you a he, that you’ve got a sticky-out bit? I bet you’ve got a sticky-in bit or two as well.” She laughed. Meanness skewed her face.
“Enough,” I said. “Let’s tag the find, in our names, Pippi.”
She dropped back. I clung to the rigging, started to thumb in figures. She pushed forward, “Let me, it’s faster.” Fingers clicking, she muttered under her breath, “Get us all home faster that way.”
I took over after she’d tagged the spot and put the coordinates in. I was trying not to be angry. Hope mellowed out some of the harsh emotion. It could be a significant find. It was nice of Star to give up his claim.
#
Back in the ship bay, the lights laddered his face till he looked like a decoration. Pippi was strapping our find into a jitney.
“Why not a place where there’s rain?” I said.
“That could only be Earth,” he said. “Do you know the worst thing about rain there?”
“What?”
Pippi tied a rope into place, tested it with a quick tug, glanced over her shoulder at us.
“Rain there has gotten so acidic that if I stand out in it I have to come in and shower after a few minutes. It damages my outer skin.”
I tried to picture the cold, then acid burn. Luna was better.
“I’m sorry about Pippi.”
She honked the horn.
“Go ahead. I’m taking the tram over to the Cluster,” he said.
I hesitated. “Meet me later?”
“I’ll call you.”
He didn’t, of course. We cashed in the case—a lump sum from a company’s R&D division that doubled our incomes and then some.
I texted him, “Come celebrate with us, we’re dockside and buying dumplings.” But he didn’t reply until three days later. “Sorry, things got busy. Bought house. Come out and see it.”
“When?”
“Tomorrow morning. I’ll make you breakfast.”
I left in the morning before Pippi was awake.
#
His place was swank, built into a cliff-side, with a spectacular view of the endless white plains below. He made me waffles with real maple syrup. He was an amazing cook. I said so.
“I was programmed that way,” he said, and made a sound that was sort of a laugh.
The sexbots—all of the AIs struggling for emancipation lately—had had to demonstrate empathy and creativity. I wondered what that had been like.
He was standing uncomfortably close. I leaned forward to make it even closer, thinking he’d draw back.
He didn’t.
“I’m programmed a certain way,” he said.
“How is that?”
“I want to please you. But at the same time I know it’s just the way I’m programmed.”
“It can’t be something more than that?” My arm was pressed against his surface. It was warm and yielding as flesh. I couldn’t have told the difference.
He pulled away. I bit my lip in frustration, but I liked him enough to be civilized.
I drank the last of my coffee. Real Blue Mountain blend. He kept his kitchen well stocked for human visitors—who did he hope would stop in?
#
As it turns out, Pippi. Next time I came through on a quick flight (I might be rich, but who was I to turn down fast and easy money?), she told me how he’d fed her.
“Pasta,” she said, rolling the words out. “And wine, and little fish, from Earth. And afterwards something sweet to drink.”
She said they’d fucked. I believed her. It wouldn’t be her style to lie. It would never occur to her.
So I did and said I’d fucked him too. She didn’t respond, not right off the bat, but I caught her looking at me oddly by the time I said toodle-oo and went off to sleep in my ship.
It wasn’t the first time I’d slept in there, not by a long shot.
I wished them both happiness, I supposed.
#
Still, two weeks later, I came in response to Pippi’s panicked call. He was going back to Earth, she said.
We both showed up at the farewell hall. He was standing with a tall blonde woman, Earth-fat. Star slipped away from us, came over with a bearing jaunty and happy, his polished face expressionless as always.
“Who is that?” Pippi said.
“A journalist. She’s going to help me tell my story, back on Earth.”
“I see,” Pippi said. She and I both surveyed the woman, who pretended not to notice us. Her manicured hand waved a porter over to take her luggage aboard, the hard-shelled cases the same color as her belt.
Pippi said, “Is this because you don’t want to fuck me any longer? You said you liked it, making me feel good. We don’t have to do that. We can do whatever you like, as long as you stay.”
He averted his face, looking at the ship. “That’s not it.”
“Then what?”
“I want to go back to the rain.”
“Earth’s acid rain?” I said. “The rain that will destroy you?”
Now he was looking at neither of us.
“What about your place?” Pippi said.
“You can have it,” he said. “It never felt like home.”
“Will anyplace?” I asked. “Anywhere?”
“When I’m telling my story, it feels like home,” he said. “I see myself on the camera and I belong in the world. That’s what I need to do.”
“Good luck,” I said. What else could I say?
#
Pippi and I walked away through the terminal. There were tourists all around us, going home, after they’d played exotic for a few days, experienced zero-grav and sky-diving and painted their faces in order to play glide-ball and eaten our food and drunk our wine and now were going home to the rain.
We didn’t look at each other. I didn’t know how long Star’s shadow would lie between us. Maybe years. Maybe just long enough for sunlight to glint on forgotten metal, out there in the sky. Maybe long enough and just so long.
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Cat Rambo
Cat Rambo writes in the Pacific Northwest. Her collection, Eyes Like Sky and Coal and Moonlight, appeared from Paper Golem Press in 2009, following her collaboration with Jeff VanderMeer, The Surgeon’s Tale and Other Stories in 2007. Among the places her work has appeared are Asimov’s, Weird Tales, and Clarkesworld. |
Peter & Sally Cant
Peter & Sally Cant
Making a difference with stem cell research.
Peter and Sally Cant spent years searching for a breakthrough in autism research. But it wasn’t until they toured U of T’s molecular genetics labs—and looked through a microscope at a tiny cluster of stem cells—that they might have found what they were searching for.
Their son David, who died at 45 in 2008, had lived with autism since childhood. “Looking down the microscope was our ‘hallelujah’ moment,” recalls Peter. “Sally and I looked at each other and we knew this was it.” Sally accepts her grieving will never end, “but then I thought that giving to stem cell research would make a reason for David’s life.”
After touring U of T’s molecular genetics lab, the Cants decided to fund a graduate award in stem cell research. They have also revised their wills to support neurological research at U of T.
“It has been an absolute delight and pleasure to be involved with the University, including becoming members of the Presidents’ Circle,” says Peter. “It is our hope that a cornucopia of opportunity will come from this endowment in David’s name. We hope this gift will truly make a difference.” |
Talamantes-Becerra, B., Georges, A. Carling, J., Kennedy, K., and Gahan, M.2019.Identification of bacterial isolates from a public hospital in Australia using complexity-reduced genotyping.Journal of Microbiological Methods, in review.
Alam, S.M.L., Georges, A., Gleeson, D., Sarre, S.D. and Ezaz, T.2018.Sex-linked markers in the Australian grassland earless dragon Tympanocryptis pinguicolla.Combined Meeting of the Australian Society of Herpetologists and the Society for Research on Amphibians and Reptiles in New Zealand. November 15-18, 2018. Kindilan, Queensland
Berman, D., Unmack, P., Georges, A. and Gruber, B.2018.River structure and scale as determinants of population structure in the eastern basins of Australia.Annual Conference of the Genetics Society of Australasia, University of Canberra, July 1-4, 2018
Caprero, A., Georges, A. and Waters, P.2018.Waking the sleeping dragon: epigenetic mechanisms that govern hibernation in the central bearded dragon.Annual Conference of the Genetics Society of Australasia, University of Canberra, July 1-4, 2018
Castelli, M., Cherryh, C., Georges, A. and Holleley, C.E.2018.Sex reversal is a geographically and temporally widespread phenomenon in the central bearded dragon.Annual Conference of the Genetics Society of Australasia, University of Canberra, July 1-4, 2018
Dissanayake, D., Hill., L, Holleley, C., Deakin, J., and Georges, A.2018.How sex can go wrong in the cold: Sex reversal of wild alpine skinks Bassiana duperreyi (Eastern Three-lines Skink).Combined Meeting of the Australian Society of Herpetologists and the Society for Research on Amphibians and Reptiles in New Zealand. November 15-18, 2018. Kindilan, Queensland.
Georges, A. and Patel, H.2018.Using gene synteny to define putative superscaffolds as an aid to physical mapping.Annual Conference of the Genetics Society of Australasia, University of Canberra, July 1-4, 2018
Georges, A., Spencer, R.J., Kilian, A., Welsh, M. and Zhang, X.2018.Assault from all sides: Hybridization and introgression threaten the already critically endangered Myuchelys georgesi.Combined Meeting of the Australian Society of Herpetologists and the Society for Research on Amphibians and Reptiles in New Zealand. November 15-18, 2018. Kindilan, Queensland
Ferronato, B.O., Roe, J.H. and Georges, A.2017.Responses of an Australian freshwater turtle to drought-flood cycles along a natural to urban gradientAustral Ecology 42:442–455
[pdf]
Georges, A.2017.The dragon genome -- sequence, annotation, physical mapping -- what we have learned an what questions have been raised.Cytogenetics in the Genomics Era. February 2-3, 2017, Canberra, Australia
Georges, A.2017.Genetics, conservation and climate change: Lessons from the dragon.Genomics and Animal Conservation Symposium in Honour of Prof. Jennifer Graves and her receipt of the PM Prize for Science. LaTrobe University, Melbourne. September 29, 2017.
Ellis, R. and Georges, A.2015.An annotated type catalogue of the turtles (Testudines: Pleurodira: Chelidae) in the collection of the Western Australian Museum.Records of the Western Australian Museum 30:52-60
[pdf]
Georges A. and Spencer, R.J.2015.Bellinger River Turtles: Assessment of genetic diversity and hybridization in a species under threat.Report prepared for the Ecosystems and Threatened Species Unit, Office of Environment and Heritage, PO Box A290, Sydney South, NSW 1232. 25-Aug-2015.
Rhodin, A.G.J., Kaiser, H., van Dijk, P.P., Wüster, W., O’Shea and Thomson, S. with 74 other authors, including Georges, A.2015.Comment on Spracklandus Hoser, 2009 (Reptilia, Serpentes, ELAPIDAE): request for confirmation of the availability of the generic name and for the nomenclatural validation of the journal in which it was published (Case 3601; BZN 70:234-237; 71:30-38; 133-1Bulletin of Zoological Nomenclature 72(1):65-78.
[pdf]
2014
Adams, M., Raadik, T., Burridge, C. and Georges, A.2014.Global biodiversity assessment and hyper-cryptic species complexes: more than one species of elephant in the room?Systematic Biology 63:518-533.
[pdf]
Eisemberg, C. and Berra, T.2014.Notes on the Sale of the Nurseryfish (Kurtus gulliveri) in the Kikori Market, Papua New GuineaJoint Meeting of Ichthyologists and Herpetologists, Chattanooga, Tennessee, 30 July – 3 August, 2014
Ferronato, B., Roe, J.H. and Georges, A.2014.The other side of the fence: Reptile bycatch in a predator-proof fence?50th Anniversary Meeting of The Australian Society of Herpetologists, Greenhills Conference Centre, Canberra, ACT. January 29-31, 2014.
Georges, A.2014.Missionaries, misfits and manna from heaven -- Challenges of turtle conservation in Papua New Guinea12th Annual Symposium on the Conservation and Biology of Tortoises and Freshwater Turtles. Keynote Address.
Georges, A.2014.Wetlands. What frehwater turtles can tell us about their management.Yass Area Network (YAN) AGM (11 Sept 2014), Dinner Address.
Hodges, K., Georges, A. and Donnellan, S.2014.The biogeography of hybridisation in east Australian long necked turtles. International Biogeography Society Early Career Conference, Canberra ACT, Jan 7-10, 2014.
Todd, E.V., Blair, D., Georges, A., Lukoschek, V. and Jerry, D.R.2014.A biogeographical history and timeline for the evolution of Australian snapping turtles (Elseya: Chelidae) in Australia and New Guinea.Journal of Biogeography 41:905-918.
[pdf]
Unmack, P., Gruber, B. and Georges, A.2014.Comparative phylogeography of four aquatic species from the Murray-Darling BasinJoint Meeting of Ichthyologists and Herpetologists, Chattanooga, Tennessee, 30 July – 3 August, 2014
Zhang, X. and Georges, A.2014.Complete mitochondrial genome sequence for the Australian turtle, <em>Chelodina longicollis</em>, with reassessment of the light strand replication origin (OL) and frameshift mutations in the <em>Chelodina</em>50th Anniversary Meeting of The Australian Society of Herpetologists, Greenhills Conference Centre, Canberra, ACT. January 29-31, 2014.
Eisemberg, C. and Georges, A.2013.A pibotal temperature for the pig-nosed turtle (Carettochelys insculpta) in the Kikori River, Papua New Guinea.37th Meeting of the Australian Society of Herpetologists, Point Wolstoncroft, Lake Macquarie, New South Wales, Jan 29 to Feb 1, 2013. Pp 60.
Georges, A.2013.Commentary: For reptiles with temperature-dependent sex determination, thermal variability may be as important as thermal averages.Animal Conservation 16, 493-494.
[pdf]
Georges, A.2013.Life in the slow lane. Spatial dynamics and persistence of turtles in the Cooper.Lake Eyre Basin Under the Spotlight. The Future of the World's Last Great Desert River System. Symposium, Longreach, Feb 25-28, 2013.
Georges, A.2013.Announcing the first genome sequence for an Australian reptile.37th Meeting of the Australian Society of Herpetologists, Point Wolstoncroft, Lake Macquarie, New South Wales, Jan 29 to Feb 1, 2013.
Georges, A.2013.Stories of PNG.Dinner presentation at the Science Educators Association of the ACT, Pod Food, Pialligo ACT, November 8.
Georges, A.2013.The Kikori Project: Science underpinning community-led conservation and environmental education in the Kikori Delta, PNG.Research School of Biological Sciences, Australian National University, May 16, 2013.
Georges, A.2013.Finding a Needle in a Haystack - subtracting male and female dragon genomes.Seminar presented to the Techniques in Computational Genomics Group, Australian National University, March 1, 2013.
Georges, A.2013.Sex in dragons: Finding a needle in a haystack.School of Biologicals Sciences, Florida State University, Florida USA. May 1, 2013.
Georges, A.2013.Sex in dragons.Department of Genetics, University of Texas in Arlington, Texas USA. May 10, 2013.
Georges, A.2013.Genomics for Dummies - A step by step introduction to the full genome sequence for bearded dragon.ACT Herpetological Association, Belconnen Way Soccer Club, June 18, 2013.
Hodges, K., Georges, A. and Donnellan, S.2013.The biogeography of itrogressive hybridisation in eas Australian long necked turtles.37th Meeting of the Australian Society of Herpetologists, Point Wolstoncroft, Lake Macquarie, New South Wales, Jan 29 to Feb 1, 2013:16 [Awarded prize for best PhD presentation]
Holleley, C.E.2013.Crowd Funding in Universities: The Sugar Glider Genetics Project Case Study. Workshop on Is Crowd Funding an Option in a University Setting? University of Canberra, Australia. [Plenary Speaker]
Holleley, C.E.2013.Sex-reversed dragons can reproduce: a naturally occurring transition from genotypic to temperature dependent sex determination.Seminar presented to the Institute for Applied Ecology, University of Canberra, Australia.
Todd, E.V., Blair, D., Georges, A., Zhang, X., Alacs, E. and Jerry, D.2013.Biogeographic history and timeline for the evolution of Australian snapping turtles (genus Elseya) in Australia and New Guinea.37th Meeting of the Australian Society of Herpetologists, Point Wolstoncroft, Lake Macquarie, New South Wales, Jan 29 to Feb 1, 2013:47.
Fielder, D., Vernes, K., Alacs, E. and Georges, A.2012.Mitochondrial variation among Australian freshwater turtles (genus Myuchelys), with special reference to the Endangered M. bellii.Endangered Species Research 17:63-71.
2011
Bower, D.2011.Conservation biology of freshwater turtles in the lower Murray River of Australia.PhD Thesis, Applied Ecology, University of Canberra
Eisemberg, C.C.2011.Nesting ecology, harvest and conservation of the pig-nosed turtle (Carettochelys insculpta) in the Kikori Region, Papua New Guinea.PhD Thesis, Applied Ecology, University of Canberra
Fielder, D.P.2010.Population ecology, ecophysiology, phylogenetics and taxonomy of the threatened western sawshelled turtle, Myuchelys bellii, from the Murray-Darling Basin of Australia. PhD Thesis, Applied Ecology, University of Canberra
Georges, A. and Thomson, S.2010.Diversity of Australasian freshwater turtles, with an annotated synonymy and keys to species.Zootaxa 2496:1-37.
[pdf]
Kennard, M., Pusey, B., Boyden, J., Burrows, D., Leigh, C., Perna, C., Bayliss, P. and Georges, A.2010.Compilation of species distribution datasets for use as biodiversity surrogates.Pp. 48-56 in Kennard, M. (2010). Identifying high conservation aquatic ecosystems in northern Australia. Final report for the Department of Environment, Water, Heritage and the Arts and the National Water Commission prepared by Charles Darwin University.
Wilson-Wilde, L., Norman, J., Robertson, J., Sarre, S.D. and Georges, A.2010.Current issues in species identification for forensic science and the validity of using the cytochrome oxidase I (COI) gene.Forensic Science, Medicine and Pathology 6:233-241.
Baggiano, O., Hughes, J., Schmidt, D. and Georges, A.2009.The importance of flood for the connectivity of freshwater turtle populations in the Moonie catchment in the upper Murray Darling Basin, Queensland, and its effect on their genetic structure.Minisymposium on the Biology and Conservation of Australasian Freshwater Turtles, Marine Turtle Symposium, Brisbane, Feb 14-19, 2009.
Bower, D.2009.Water saving strategies conflict with turtle conservation in the Lower-Murray River.Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Bower, D.2009.Symposium: Herpetological conservation in wetlands: Wetter and better or dryer and dire?Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Corey, B., Fordham, D.A., Georges, A. and Brook, B.2009.Predation by feral pigs threatens the indigenous harvest and local persistence of snake-necked turtles in northern Australia.Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Eisemberg, C.C., Rose, M., Silva, R.F. and Georges, A.2009.The decline of the Piku empire: Harvest and conservation of the pignosed turtle on the Kikori Delta, Papua New Guinea.Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Ezaz, T., Moritz, B., Waters, P.D., Graves, J.A.M., Georges, A. and Sarre, S.D.2009.The ZW sex microchromosomes of an Australian dragon lizard share no homology with those of other reptiles or birds.Chromosome Research 17:965-973
[pdf]
Fielder, D., Vernes, K., Alacs, E. and Georges, A.2009.A phylogeny of Australias threatened sawshelled turtles using mtDNA.Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Georges, A.2009.Temperature dependent sex determination in reptiles: fact or furphy?Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Georges, A.2009.Kangaroo Cull Tag Allocations (Licence to Cull) for 2009. Assessment of the formula used to determine the number of tags allocated.Advice to the ACT Office of the Commissioner for Sustainability and the Environment prepared by the Institute for Applied Ecology, University of Canberra, June 2009.
Georges, A.2009.End we might not like.Canberra Times, Times 2, 13-Apr-09
Georges, A. and Thomson, S.2009.Diversity of Australasian freshwater turtles, with an annotated synonymy, keys to species and a web-based database of distributional data.Minisymposium on the Biology and Conservation of Australasian Freshwater Turtles, Marine Turtle Symposium, Brisbane, Feb 14-19, 2009.
Georges, A., Sarre, S.D. and Ezaz, T.2009.Reptiles with TSD: Are they inherently resistent to climate change?Fifth International Symposium on the Biology of Sex Determination, April 20-24, 2009, Kona, Hawaii:87-88.
Hare, K.M., Cree, A. and Georges, A.2009.Symposium: How ecophysiology can support conservation of reptiles.Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Hodges, K., Georges, A. and Donnellan, S.2009.Integrating genetic data and ecological niche models to test alternative phylogeographic hypotheses.Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Hodges, K., Georges, A. and Donnellan, S.2009.Maps and molecules - comparative phylogeography of Chelodina expansa and Chelodina longicollis.Joint Meeting of the Society for Research on Amphibians and Reptiles in New Zealand and the Australian Society of Herpetologists, Aukland February 20-23, 2009.
Corey, B. and Fordham, D.2008.Collaborative research demonstrates that turtle harvest in the Manigrida region is sustainable.Kantri Laif: News for North Australian Land and Sea Managers. Issue 4, August 2008, pp 34.
Eisemberg, C.2008.Piku in Trouble: Indigenous communities in PNG work with Oil Search to conserve the pig-nosed turtle.Kantri Laif: News for North Australian Land and Sea Managers. Issue 4, August 2008, pp 7.
Eisemberg, C.C., Georges, A. and Rose, M.2008.Piku in trouble: Conservation of the pig-nosed turtle in the Kikori Delta, Papua New Guinea.Sixth World Congress for Herpetology, Manaus, Brasil. August 17-22, 2008.
Georges, A.2008.Overview of turtle research and conservation projects conducted through the University of Canberra.Seminar delivered to IEPA (Institute for Science and Technological Research), Macapa, Brasil. August 14, 2008.
Martinez, P. A., Ezaz, T., Valenzuela, N., Georges, A. and Graves, J.A.M.2008.An XX/XY heteromorphic sex chromosome system in the Australian chelid turtle Emydura macquarii : A new piece in the puzzle of sex chromosome evolution in turtles.Chromosome Research 16:815-825.
Martinez, P.A. Ezaz, T. Valenzuela, N., Georges, A. and Graves, J.A.M.2008.A novel sex chromosome system in the Australian chelid turtle Emydura macquarii, a new piece in the ever increasing complexity of sex chromosome evolution.Joint Meeting of the Society for the Study of Amphibians and Reptiles and the Herpetologists League, Montreal, Canada. July 23-28, 2008.
Martinez, P.A., Ezaz, T., Valenzuela, N., Georges, A. and Graves, J.A.M.2008.Sex chromosomes in the Australian chelid turtle Emydura macquarii.Annual Joint Meeting of the Society for the Study of Evolution, and the Society of Systematic Biologists, Minneapolis, Minnesota. June 20-24, 2008.
Bower, D.2007.Take them with a pinch of salt: Comparative tolerance of freshwater turtles to increase in ambient salinity.33rd Meeting of the Australian Society of Herpetologists, Albany 2007, Camp Quaranup, WA.
Corey, B., Fordham, D. A. and Georges, A.2007.Optimal conditions for egg storage, incubation and post-hatching growth for the freshwater turtle, Chelodina rugosa: Science in support of an indigenous enterprise.33rd Meeting of the Australian Society of Herpetologists, Albany 2007, Camp Quaranup, WA.
Corey, B., Fordham, D. and Georges, A.2007.Optimal conditions for rearing Chelodina rugosa Chelodina rugosa eggs and hatchlings: Science in support of an indigenous enterprise.Proceedings of the Australian Society for Herpetologists Conference, Albany WA, 4-7 December 2007.
Fordham, D., Georges, A. and Brook, B.W.2007.Indigenous harvest, exotic pig predation and local persistence of a long-lived vertebrate.Fenner Conference on the Environment: Wildlife Population Dynamics and Management. Academy of Science, Canberra. December 2-5, 2007.
Fordham, D., Georges, A. and Corey, B.2007.Optimal conditions for egg storage, incubation and post-hatching growth for the freshwater turtle, Chelodina rugosa: Science in support of an indigenous enterprise.Aquaculture 270:105-114.
Georges, A.2007.From molecules to landscapes: genealogical concordance and defining bioregions.Fenner Conference on the Environment: Wildlife Population Dynamics and Management. Academy of Science, Canberra. December 2-5, 2007.
Georges, A. and Alacs, E.A.2007.The Pig-nosed Turtle in the Kikori: Options for Research to Support Conservation and Management.Report to Oilsearch Pty Ltd, Sydney, by the Institute for Applied Ecology, University of Canberra. February 2007.
Georges, A., Alacs, E.A. and Kinginapi, F.2007.Freshwater turtles of the Kikori (with special reference to the pig-nosed turtle).Report to Oilsearch Pty Ltd, Sydney and World Wide Fund for Nature (WWF), by the Institute for Applied Ecology, University of Canberra. January 2007.
Georges, A., Walsh, R., Spencer, R.J., Welsh, M. and Shaffer, HB.2007.The Bellinger Emydura.Challenges for Management. Report to NSW National Parks and Wildlife Service, Sydney, by the Institute for Applied Ecology, University of Canberra. July 2007.
Hodges, K.2007.Comparative phylogeography of two long-necked turtle species in the Murray-Darling basin: How genetics can answer ecological questions.54th Annual Conference of the Genetics Society of Australasia, Sydney, Australia. 27th-29th June 2007.
Spencer, R-J., Georges, A., and Welsh, M.2007.The Bellinger Emydura. Ecology, Population Status and Management.Report to NSW National Parks and Wildlife Service, Sydney, by the Institute for Applied Ecology, University of Canberra. July 2007.
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2006
Abramyan, J., Shaffer, H.B. and Georges, A.2006.Phylogeography and genetic variation in the genus Emydura.Proceedings of the Australian Society of Herpetologists Conference, Healsville, Vic. 18-21 April, 2006.
Bower, D.2006.Chelodina expansa - Rare and endangered or cryptic and secure?Proceedings of the Australian Society of Herpetologists Conference, Healsville, Vic. 18-21 April, 2006.
Corey, B. and Doody, J.S.2006.Ecology and conservation of carpet pythons (Morelia spilota) in semi-arid rural landscapes of western NSW.Proceedings of the Australian Society of Herpetologists Conference, Healsville, Vic. 18-21 April, 2006.
Ezaz, T., Quinn, A. Sarre,S., Georges, A., Graves, J.A.M.2006.Sex in dragons: where boys can be girls.Molecular Genetics and Evolution Seminar series, Research School of Biological Sciences, The Australian National University, October 18, 2006.
Georges, A., Fordham, D. and Alacs, E.2006.Science in support of Aboriginal harvest of snake-necked turtles as part of an indigenous industry.Powdermill Freshwater Turtle Meeting, Southwestern Research Station, Portal, Arizona. 1-3 September 2006.
Pezaro, N., Doody, J.S. and Georges, A.2006.Testing for sex allocation in the water dragon, Physignathus lesueurii, a species with temperature-dependent sex determination.Proceedings of the Australian Society of Herpetologists Conference, Healsville, Vic. 18-21 April, 2006.
Roe, J.H., Brinton, A.C. and Georges,A.2006.The secret terrestrial life of an Australian freshwater turtle, Chelodina longicollis.Proceedings of the Australian Society of Herpetologists Conference, Healsville, Vic. 18-21 April, 2006.
Steer, D. and Doody, J.S.2006.Living on the Edge: the foraging games played out between agile wallabies (Macropus agilis) and saltwater crocodiles (Crocodylus porosus)Proceedings of the Australian Society of Herpetologists Conference, Healsville, Vic. 18-21 April, 2006.
2005
Alacs, E.2005.Monitoring trade in the northern long-necked turtle, Chelodina rugosa: a phylogenetic approach.Joint meeting of the Australian Society of Herpetologists, the Society for Research on Reptiles and Amphibians in New Zealand and the Fijian Society of Herpetologists, Springsbrook, Queensland 7-11 February, 2005:10.
Davies, C., Doody, J.S., Green, B., Georges, A. and Steer, D.2005.Basking at night during the cold tropical winter: Thermoregulation is important for pig-nosed turtles.Joint meeting of the Australian Society of Herpetologists, the Society for Research on Reptiles and Amphibians in New Zealand and the Fijian Society of Herpetologists, Springsbrook, Queensland 7-11 February, 2005:17.
Doody, J.E. and Welsh, M.2005.First glimpses into the ecology of the red-faced turtle, Emydura victoriae, in tropical Australia.Herpetofauna 35:11-14
Doody, J.S., Guarino, F. and Georges, A.2005.Testing for among-generation adjustment to offspring sex ratios: Clinal variation in sex-determining attributes in the water dragon Physignathus lesuerii.Joint meeting of the Australian Society of Herpetologists, the Society for Research on Reptiles and Amphibians in New Zealand and the Fijian Society of Herpetologists, Springsbrook, Queensland 7-11 February, 2005:19.
Georges, A., Guarino, F. and Bito, B.2005.Freshwater Turtles of the TransFlyReport to the Suki/Aramba Wildlife Management Committee, Serki, Western Province, Papua New Guinea by the Institute for Applied Ecology, University of Canberra. October 2005. 31 pp. plus 2 attachments.
Georges, A., Sarre, S., Quinn, A., Guarino, F. and Ezaz, T.2005.Are reptiles predisposed to temperature-dependent sex determination?Joint meeting of the Australian Society of Herpetologists, the Society for Research on Reptiles and Amphibians in New Zealand and the Fijian Society of Herpetologists Pp. 21.
Holloway, R. H. P.2005.Ecology of the River Terrapin in Sre Ambel, CambodiaRegional Workshop on the Research and Conservation of the River Terrapin, September 2004, Terengganu, Malaysia
Raadik, T.A.2005.Dorrigo Plateau: A biodiversity hot-spot for galaxiids.Fishes of Sahul. Journal of the Australia New Guinea Fishes Association 19:98-107.
Roe, J.H.2005.Are roads taking their toll on aquatic reptiles?Joint meeting of the Australian Society of Herpetologists, the Society for Research on Reptiles and Amphibians in New Zealand and the Fijian Society of Herpetologists, Springsbrook, Queensland 7-11 February p. 45.
Sarre, S.D., Georges, A., Quinn, A., Ezaz, T. and Guarino, E.2005.Girls will be boys and boys will be girls: Investigating the TSD-GSD continuum in reptiles.5th World Congress for Herpetology, Stellenboch, South Africa 19-24 June 2005. Pp 90.
Trembath, D. F. and Freier, D.2005.Accidental ingestion of barbecue scraps leads to death of a varanid in the northern territory.Herpetofauna 35: 48-49.
Trembath, D.F.2005.Demographic consequences of superabundance of Emydura krefftii (Testudines: Chelidae) in the Ross River, Townsville.Joint meeting of the Australian Society of Herpetologists, the Society for Research on Reptiles and Amphibians in New Zealand and the Fijian Society of Herpetologists, Springsbrook, Queensland 7-11 February, 2005:52.
Trembath, D.F.2005.The comparative ecology of Kreffts River Turtle Emydura krefftii in Tropical North Queensland.Masters Thesis, University of Canberra, August 1991.
2004
Doody, J.S., Georges, A. and Young J.E.2004.Determinants of reproductive success and offpring sex in a turtle with environmental sex determination.Biological Journal of the Linnean Society, London, 80:1-16.
Georges, A., Doody, J.S., Beggs, K. and Young, J.E.2004.TSD in reptiles: how the physical and physiological can conspire to defeat global climate change.Pp. 44 in Scientific Program and Abstracts, Third International Conference of Comparative Physiology and Biochemistry, KwaZulu-Natal, South Africa.
Holloway, R. H. P.2004.Conservation Biology of the River Terrapin in CambodiaInternational Congress of Zoology, Beijing, China
Holloway, R. H. P. and Heng Sovannara2004.Conservation of the River Terrapin, Batagur baska (Gray, 1831) in Cambodia.p276 in Proceedings of the XIXth International Congress of Zoology. August 23-27, 2004.
Doody, J.S. and W. Osborne.2003.Animal ways in rice bays: a year spent with vertebrates on rice farms.IREC Farmers Newsletter Rice Research and Development Special. 162:55-57
Doody, J.S., Georges, A. and Young, J.E.2003.Twice every second year reproduction in the pig-nosed turtle, Carettochelys insculpta, in the wet-dry tropics of Australia.Journal of Zoology, London 259:179-188.
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Georges, A.2003.Delights of Tiputini, EcuadorSeminar presented to the ACT Herpetological Association, March 2003.
Georges, A. et al.2003.The January 2003 ACT Fires: Priorities for Research to Inform Management.Outcomes of a Workshop held at the University of Canberra, Friday October 31, 2003, on behalf of the ACT Natural Resource Management Committee.
Georges, A., Guarino, E., Webster, I., Jolly, P. and Thoms, M.2003.Modelling the impact of flow regulation on a flagship species, the pig-nosed turtle.Joint Meeting of the American Society of Ichthyologists and Herpetologists American Elasmobranch Society, Herpetologists League, and the Society for the Study of Amphibians and Reptiles, Manaus, Brazil. 26 June to 2 July, 2003.
2002
Doody, J.S.2002.Backbones in flooded zones: on-farm biodiversity in the rice-growing Riverina.IREC Farmers Newsletter Rice Research and Development Special. 159:57-58.
Doody, J.S.2002.The ecology and sex determination of the pig-nosed turtle, Carettochelys insculpta, in the wet-dry tropics of Australia.PhD Thesis, Applied Ecology, University of Canberra
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Doody, J.S., Young, J.E. and Georges, A.2002.Sex differences in activity and movements in the Pig-Nosed turtle, Carettochelys insculpta, in the wet-dry tropics of Australia.Copeia. 2002:93-103
Doody, S. Simms, R.A. and Georges, A.2002.Use of thermal springs for aquatic basking by the Pig-nosed turtle, Carettochelys insculpta.29th Annual Meeting of the Australian Society of Herpetologists. Birragai, ACT, 11-15 July 2002.
Doody, S. and Sims, R.2002.Patterns of predation in pig-nosed turtles.29th Annual Meeting of the Australian Society of Herpetologists. Birragai, ACT, 11-15 July 2002.
Georges, A. and Cottingham, P.2002.Biodiversity in Inland Waters: Priorities for its Protection and Management. Recommendations from the 2001 Fenner Conference on the Environment.CRC for Freshwater Ecology, March 2002. Technical Report 1/2002.
Georges, A., Adams, M. and McCord, W.P.2002.Electrophoretic delineation of species boundaries within the genus Chelodina (Testudines: Chelidae) of Australia, New Guinea and Indonesia.Zoological Journal of the Linnean Society 134:401-21.
Georges, A., Beggs, K., Doody, S. and Young, J.2002.How the physical and the phyiological can conspire to defeat global climate change.29th Annual Meeting of the Australian Society of Herpetologists. Birragai, ACT, 11-15 July 2002.
Goodsell, T.2002.Freshwater turtles in wet and dry waterholes: Tracking metapopulation patterns of the Cooper Creek turtle with microstatellites.29th Annual Meeting of the Australian Society of Herpetologists. Birragai, ACT, 11-15 July 2002.
Goodsell, T.L.2002.Gene flow in highly variable environments: Population structure of an Australian freshwater turtle, Emydura macquarii>.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
2001
Cann, J. and Georges, A.2001.A new look at the freshwater tortoises of Australia.Pp. 38-46 in Ettling, J. and Becker W. (Eds). Proceedings of the 24th International Herpetological Symposium on Captive Propogation and Husbandry. Audubon Park and Zoological Gardens, New Orleans. July 19-22, 2000.
Doody, J.S.2000.Do development equivalents produce phenotypic equivalents? A test of the comparative influences of constant and fluctuating incubation temperatures on phenotypes of hatchling turtles.Chelonian Conservation and Biology, 3:529-531
Doody, J.S., Georges, A. and Young, J.E.2000.Monitoring plan for the pig-nosed turtle in the Daly River, Northern TerritoryUnpublished report to the Parks and Wildlife Commission of the Noerthern Territory, Darwin. June 2000.
Thomson S.A.2000.On the identification of the holotype of Chelodina oblonga (Testudinata: Chelidae) with a discussion of the taxonomic implications.Chelonian Conservation and Biology 3:745-749.
Thomson, S., Kennett, R. and Georges, A.2000.A new species of long necked turtle (Chelidae:Chelodina) from the sandstone plateau of Arnhem Land, Northern Australia.Chelonian Conservation and Biology 3:675-685
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Thomson, S.A.2000.A revision of the fossil chelid turtles (Pleurodira) described by C.W. De Vis (1897).Memoirs of the Queensland Museum 45:593-598
Young, J.E., Georges, A., Doody J.S. and West, P.2000.Management plan for the Pig-nosed Turtle, Carettochelys insculpta, in the Northern TerritoryUnpublished report to the Parks and Wildlife Commission of the Noerthern Territory, Darwin. June 2000.
Doody, J.S. and Georges, A.1999.The ecology of sex determination of pignose turtles, Carettochelys insculpta, in tropical Australia.Pp. 92-93 in Joint Meeting of the American Society of Ichthyologists and Herpetologists American Elasmobranch Society, Herpetologists League, and the Society for the Study of Amphibians and Reptiles, Pennsylvania State University.
Doody, J.S., Georges, A. and Young, J.E.1999.Reproductive biology of the pignose turtle, Carettochelys insculpta, in Australia: Why lay two clutches every other year rather than one clutch each year?Pp. 93 in Joint Meeting of the American Society of Ichthyologists and Herpetologists American Elasmobranch Society, Herpetologists League, and the Society for the Study of Amphibians and Reptiles, Pennsylvania State University.
Fortescue M.1999.The marine and terrestrial ecology of a northern population of the Little Penguin, Eudyptula minor, from Bowen Island, Jervis Bay.PhD Thesis, Applied Ecology, University of Canberra
Guarino, F.1999.Ecology and ecophysiology of the lace monitor (Varanus varius) in a temperate environment.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
Shaffer, H.B., Storz, B.L., Georges, A. and Starkey, D.1999.Pleistocene glaciation, population substructure, and genetic differentiation in long-generatio time vertebrates: a case study in eastern Australian turtles.Evolution 99, Madison, Wisconsin, USA. Society for the Study of Evolution, the American Society of Naturalists and the Society of Systematic Biologists, 22-26 June 1999.
Thomson S.A. and Mackness B.S.1999.Fossil turtles from the early pliocene bluff downs local fauna, with a description of a new species of ElseyaTransactions of the Royal Society of South Australia 123:101-105
Welsh M.1999.Resource partitioning among the freshwater turtles of the Daly river, Northern Territory.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
West P.B.1999.Variation in the distribution and habitat utilisation of rabbits, Oryctolagus cuniculus, in central-western NEW South Wales.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
Young, J.E., West, P.B., Georges, A., Doody, J.S. and Alderman, R.L.1999.Thermosensitive period of Carettochelys insculpta from the Northern Territory, Australia.Pp. 239 in Joint Meeting of the American Society of Ichthyologists and Herpetologists, American Elasmobranch Society, Herpetologists League, and the Society for the Study of Amphibians and Reptiles, Pennsylvania State University. State College, Pennsylva
Beggs, K.E.1998.Embryonic development of the pig-nosed turtle, Carettochelys insculpta, under constant and fluctuating temperature regimes.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
Georges, A.1997.Modelling incubation period and constant temperature equivalence for reptile embryos incubated under fluctuating temperature regimes.Proceedings of the 77th Annual Meeting of American Society of Ichthyologists and Herpetologists, University of Washington, Seattle. 26 June-2 July. Pp:139.
Georges, A.1997.Population dynamics of Australian freshwater turtles: Past, present and future research.Proceedings of the 77th Annual Meeting of the American Society of Ichthyologists and Herpetologists, University of Washington, Seattle. 26 June-2 July. Pp:138.
Jones, S.1997.Resource partitioning of rocky habitat within a grassland reptile community.Proceedings of the 24th Conference of the Australian Society of Herpetologists, Wellington Mills, WA, 22-25 September, 1996. Pp:97.
Robinson, W.1997.Ant communities in the grasslands of the Australian Capital Territory and the role of ants in the ecology of the pink-tailed legless lizard, Aprasia parapulchella.Masters Thesis, University of Canberra, August 1991.
Thomson, S., Adams, M., Sedden, J. and Georges, A.1997.The western Australian turtle Chelodina oblonga (Testudines: Chelidae) and its phylogenetic placement within the genus Chelodina.Proceedings of the 77th Annual Meeting of American Society of Ichthyologists and Herpetologists, University of Washington, Seattle. 26 June-2 July. Pp:290.
Thomson, S., White, A. and Georges, A.1997.Re-evaluation of Emydura lavarackorum: Identification of a living fossil.Memoirs of the Queensland Museum 42:327-336.
Thomson, S., White, A., Adams, M. and Georges, A.1997.A synthesis of diverse disciplines: How Elseya lavarackorum, the Gulf Snapping Turtle (Testudines: Chelidae), was identified as a living fossil.Proceedings of the 24th Conference of the Australian Society of Herpetologists, Wellington Mills, WA, 22-25 September, 1996. Pp:112.
1996
Doody, J.S.1996.The effect of incubation temperature on performance of hatchling turtles: phenotypic response to fluctuatng vs. constant temperatures.Proceedings of the 76th Annual Meeting of the American Society of Ichthyologists and Herpetologists, University of New Orleans, 13-19 June 1996, p 126
Doody, J.S.1996.Summers with softshells.Bulletin of the Chicago Herpetological Society. 31:132-133.
Fortescue, M.E.1996.Breeding success and foraging patterns of the Little Penguin Eudyptula minor on the east coast of Australia.Pp. 16-17 proceedings of the Third Interantional Penguin Conference, Capetown, South Africa, 2-6 September 1996. African Seabird Group
Fortescue, M.E.1996.Temporal and spatial variation in breeding success of the Little Penguin Eudyptula minor on the east coast of Australia.Presented at the Annual Conference of the Royal Australian Ornitholoical Union, Wollongong University, 25 May 1996
Georges, A.1996.Modelling incubation period and constant temperature equivalence for reptile embryos incubated under fluctuating temperature regimes.Proceedings of the 9th Meeting of the Australasian Wildlife Management Society, University of Canberra, 3-5 December 1996, pp:26-27.
Georges, A. and Adams, M.1996.Electrophoretic delineation of species boundaries within the short-necked chelid turtles of Australia.Zoological Journal of the Linnean Society, London 118:241-260.
Jones, S.1996.Resource partitioning of rocky habitat within a grassland reptile community.Proceedings of the 9th Meeting of the Australasian Wildlife Management Society, University of Canberra, 3-5 December 1996, pp:29.
Jones, S.1996.When is absence an absence? Using probability of non-detection to determine true absences of the threatened pink-tailed legless lizard, Aprasia parapulchella.Proceedings of the 9th Meeting of the Australasian Wildlife Management Society, University of Canberra, 3-5 December, pp 35
Kennett, R.1996.Nhaltjan nguli miwatj yolngu djaka miyapunuwu: Sea turtle conservation and the Yolngu people of north east Arnhem Land, Australia.Proceedings of the 9th Meeting of the Australasian Wildlife Management Society, University of Canberra, 3-5 December, pp 42
Kennett, R.M.1996.Sea turtle research and management and the Yolngu people of north east Arnhem Land.16th Annual Symposium on Sea Turtle Conservation and Biology, Hilton Head, South Carolina, USA, February 1996
Kennett, R.M.1996.Science turns to Aboriginal knowledge to save endangered turtles.On the Brink! The Newsletter of the Endangered Species Progran, Australian Nature Conservation Agency, Canberra
Kennett, R.M., Munungurritj, N. and Yunupingu, D.1996.An overview of the Dhimurru Land Management Aboriginal Corporation and the Miyapunu Project.Indigenous Land Corporation, Land Management Workshop, Mirrambeena Resort, Darwin, August 13-14, 1996
Podreka, S., Georges, A. and Maher, W.1996.The environmental contaminant DDE fails to influence the outcome of sexual differentiation in the marine turtle Chelonia mydas.Proceedings of the 9th Meeting of the Australasian Wildlife Management Society, University of Canberra, 3-5 December, pp:65.
Thomson, S.A.1996.Field keys to the turtles of the Northern Territory.Applied Ecology Research Group, University of Canberra. pp 24
Thomson, S.A., Littlejohn, M.J., Robinson, W.A. and Osborne, W.S.1996.Taxonomy of the Litoria aurea comples: A re-evaluation of the Southern Tableland populations of the ACT and NSW.In: Pyke, G.H. and Osborne, W.S. (eds). Biology and Conservation of the Green and Golden Bell Frog (Litoria aurea). Australian Zoologist 30:158-169.
1995
Barratt, D.1995.Ecological impacts of domestic cats in the ACT suggested from predation and movement data.Pp. 182-187 in Proceedings of the 10th Australian Vertebrate Pest Control Conference, Hobart, Tasmania, 29 May-2 June 1995
Barratt, D.G.1995.Predation and movement by house-based domestic cats (Felis catus L.) in suburban and rural habitats: preliminary findings.Pp:181-187 in Bennett, A., Backhouse, G. and Clark, T. (eds). People and Nature Conservation. Perspectives on private land use and endangered species recovery. Beatty and Sons, Sydney.
Barratt, D.G.1995.Movement patterns and prey habits of house cats <i>Felis catus</i> (L.) in Canberra, Australia.Master of Applied Science Thesis, University of Canberra.
Kilgour, S.1995.Water balance and energy metabolism of the eastern long-necked turtle (Chelodina longicollis) during terrestrial migration and aestivation.23rd Meeting of the Australian Society of Herpetologists, Laurel Hill, NSW. February 9-12, 1995.
Nunan, D.1995.Trophic ecology of the nationally vulnerable striped legless lizard Delma impar within the Australian Capital Territory.23rd Meeting of the Australian Society of Herpetologists, Laurel Hill, NSW. February 9-12, 1995.
Nunan, D.1995.Diet and feeding ecology of the striped legless lizard Delma impar (Fischer, 1882) within the Australian Capital Territory.Unpublished report to the ACT Parks and Conservation Service, Canberra, July 1995.
Nunan, D.1995.Trophic ecology of the striped legless lizard Delmar impar (Fischer, 1882) within the Australian Capital Territory.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
Robinson, W.1995.Dietary preferences of Aprasia parapulchella in laboratory feeding experiments.23rd Meeting of the Australian Society of Herpetologists, Laurel Hill, NSW. February 9-12, 1995.
Robinson, W.1995.Choosing the right home: an importANT decision.Ecological Society of Australia. Open Forum and Symposium Conference, Hobart, 27-29 September. Pp:64.
Thomson, S. and Georges, A.1995.Neural bones in Australian turtles.Proc. 23rd Meeting of the Australian Society of Herpetologists, Laurel Hill, NSW. February 9-12, 1995.
Thomson, S. and Georges, A.1995.A new genus of Australian chelid turtle and the relationships of the short-necked taxa.Proceedings of the 5th Conference on Australian Vertebrate Evolution, Paleontology and Systematics. National Science and Technology Centre, Canberra. April 18-20
Williams, D.G., Osborne, W., Georges, A. and Holloway, S.1995.Principles and strategic options for the conservation of native grasslands and their threatened fauna in Gungahlin, A.C.T.Report to the ACT Planning Authority, Canberra. June 1995. 88 pp.
1994
Baverstock, P., Johnson, A., Georges, A. and Donnellan, S.1994.Evolutionary divergence in the 12S ribosomal RNA gene in four Gondwanic groups: A test of the molecular clock hypothesis.Second World Congress for Herpetology, Adelaide, January 1994.
Fortescue, M.E.1994.Biology of the Little Penguin Eudyptula minor on Bowen Island and at other Australian colonies: a case for scientific reference reserves.In: Penguins of the World. Surrey Beatty and Sons, Chipping North, NSW.
Georges, A. and Adams, M.1994.Electrophoretic determination of species boundaries among the shortnecked chelid turtles of Australia.Joint Meeting of the Herpetologists League and the Society for the study of Reptiles and Amphibians, University of Georgia, September 1994
Georges, A., Limpus, C.J. and Stoutjesdijk, R.1994.Proportion of development at a temperature, not daily duration of exposure, determines sex in the marine turtle Caretta caretta.Journal of Experimental Zoology 270:432-444.
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Nunan, D.L.1994.Trophic ecology of the nationally vulnerable striped legless lizard Delma impar within the Australian Capital Territory.Workshop on Temperate Grasslands Research in the ACT: Current Research and Future Priorities 30 August 1994.
1993
Barratt, D. and Hall-Martin, A.J.1993.Impact of elephants (Loxodonta africana) on sub-tropical thicket vegetation in south-east South Africa.Poster presentation for the Sixth International Theriological Congress, University of New South Wales, Sydney, 4-10 July 1993.
Druhan, J.1993.Evaluation of the Frequency-of-Capture, Petersen and three non-parametric techniques for estimating population size from capture-recapture data.Statistics in Ecology and Environmental Monitoring Conference, Dunedin, New Zealand, 13th-17th December 1993.
Druhan, J.1993.Evaluation of the Frequency-of-Capture, Petersen and three non-parametric techniques for estimating population size from capture-recapture data.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
Georges, A.1993.Jervis Bay: So many plans, so where is the action? Opening talk, Jervis Bay: Conservation, Management and the Future.HMAS Creswell, Jervis Bay, February 11-14, 1993.
Georges, A. and Robinson, G.1992.An introduction to statistical computing using SAS.A three day short-course for the NSW Water Resources Commission, July 1992.
Georges, A. and Robinson, G.1992.An Introduction to Statistical Computing using SAS.A three-day workshop organized for staff of the New South Wales Department of Water Resources. Parramatta, NSW, June 17-19, 1992
Georges, A. and Thomas, K.1992.Statistical computing using SAS.A three day short-course for the Conservation Commission of the Northern Territory, July 1992.
Jones, S.1992.Habitat relationships, diet and abundance of the endangered pygopodid, Aprasia parapulchella in the Australian Capital Territory and surrounding New South Wales.Australian Society of Herpetologists Conference, Sydney 6-9 th December, 1992.
Jones, S.1992.Abundance and habitat relationships of the legless lizard Aprasia parapulchella.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
Morton, C. M.1992.Diets of three species of tree-rat, Mesembriomys gouldii (Gray), M. macrurus (Peters) and Conilurus penicillatus (Gould) from the Mitchell Plateau, Western Australia.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
1991
Barratt, D.G. and Hall-Martin, A.J.1991.The effect of indigenous browsers on valley bushveld in the Addo Elephant National Park.Proceedings of the First Valley Bushveld/Subtropical Thicket Symposium: Special Publication of the Grassland Society of Southern Africa.
Beynon, F.1991.Comparisons of oxygen consumption and developmental rates in the eggs of Chelodina rugosa and Chelodina longicollis.21st Annual Meeting of the Australian Society of Herpetologists, Armidale, November, 1991
Beynon, F.1991.Comparative rates of oxygen consumption and development in the eggs of two species of turtle in the genus Chelodina.4th Australasian Wildlife Management Society Conference, Dubbo, 27-29 November, 1991.
Beynon, F.1991.Comparative rates of oxygen consumption and development in two species of turtle.Honours Thesis, Applied Ecology, University of Canberra, January 1992.
Foulkes, J.N. and Kerle, J.A.1991.Feasibility study for the reintroduction of the brushtail possum to Uluru National Park.Unpublished report to the Australian National Parks and Wildlife Service, Canberra. December, 1991
Georges, A.1991.A management plan for the sustainable harvest and export of the edible jellyfish Catostylus mosaicus.Prepared for Taiga Food Pty Ltd for submission to the Australian National Parks and Wildlife Service
Georges, A.1991.A management programme for the sustainable harvest and export of the edible jellyfish Catostylus mosaicus.Report to Australian National Parks and Wildlife Service on behalf of Taiga Food, Sydney. June, 1991.
Georges, A., Limpus, C.J. and Stoutjesdijk, R.1991.Variance in temperature affects sex determination independently of the mean in the marine turtle Caretta caretta.21st Annual General Meeting of the Australian Society of Herpetologists, Armidale, November, 1991.
Georges, A., Williams, D. and Richards, G.1991.An Introduction to Q & A for urban park managers.A one-day workshop for the ACT Parks and Conservation Bureau. University of Canberra, March 1991
Jones, S.1991.Abundance, habitat use and diet of the endangered Pygopodid Aprasia parapulchella in the ACT and surrounding NSW.21st Annual General Meeting of the Australian Society of Herpetologists, Armidale, November, 1991.
Norris, R.H. and Georges, A.1991.A review of the Deepwater Ocean Outfalls Environmental Monitoring Program (Pre-Commissioning Report).Report commissioned by the State Pollution Control Commission (NSW)
1990
Beynon, F.1990.The comparative rates of development and oxygen consumption in eggs of tropical and temperate zone turtles in the genus Chelodina: An honours proposal.20th Annual Meeting of the Australian Society of Herpetologists, Gemini Downs, September 1990.
Georges, A. and Thomas, K.1990.Statistical Computing using SAS.A three-day workshop organized for staff of the Conservation Commission of the Northern Territory. Palmerston, NT, July 21-23, 1992
Georges, A. and Williams, D.1990.An Introduction to Statistical Analysis using SAS.A three-day workshop organized for staff of the ACT Parks and Conservation Service. University of Canberra, February 1990
Georges, A. and Williams, D.G.1990.An introduction to statistics using SAS.Workshop for ACT Parks and Conservation Bureau, Canberra, February 1990.
Georges, A., Williams, D. and Richards, G.1990.An Introduction to Q & A for data entry and retrieval.A one-day workshop organized at the request of the ACT Parks and Conservation Service. University of Canberra, April 1990
Kennett, R., Georges, A. and Palmer-Allen, M.1990.Great mysteries of the north: Do northern long-necked turtles nest under water?20th Annual Meeting of the Australian Society of Herpetologists, Gemini Downs, September 1990.
Palmer-Allen, M. and Beynon, F.1990.Hatchling sex ratios are independent of temperature in field nests of the long-necked turtle Chelodina longicollis.20th Annual Meeting of the Australian Society of Herpetologists, Gemini Downs, September 1990.
Georges, A.1989.Female turtles from hot nests: Is it amount of development or duration of incubation at high temperatures that matters?Oecologia (Berlin) 81:323-328.
Georges, A.1989.Conservation and biology of the pig-nosed turtle Carettochelys insculpta in northern Australia.Invited paper, First World Congress of Herpetology, University of Kent, Canterbury, September, 1989
Georges, A.1989.The pig-nosed turtle Carettochelys insculpta in Kakadu National Park: A response to the Final Environmental Impact Statement on the Coronation Hill Gold, Platinum and Paladium Project.Unpublished report to DASETT, September, 1989.
Georges, A. and Adams, M.1989.Biochemical systematics of Australian chelid turtles.First World Congress of Herpetology, University of Kent, Canterbury, September, 1989
Georges, A. and Kennett, R.1989.Life in ephemeral habitats: How Australian turtles cope with the cycles of wet and dry.First World Congress of Herpetology, University of Kent, Canterbury, September, 1989.
Georges, A.1988.The Warradjan Carettochelys insculpta: A literature review and annotated bibliography.Report to the Australian National Parks and Wildlife Service, February 1988. 38 pp
Georges, A. and Kennett, R.1988.Dry-season distribution and ecology of the Warradjan (Carettochelys insculpta Ramsay) in Kakadu National Park.Report to Australian National Parks and Wildlife Service, February 1988. 62 pp
[pdf] |
What to watch at the Sochi Olympics
The Sochi Winter Olympics will be about more than skiing, skating, and sledding. Every Olympics can promote peace by putting a spotlight on the host country. Russia has already found the Games can stir change for the better, despite what Putin expects.
AP Photo
President Vladimir Putin speaks while visiting the Coastal Cluster Olympic Village ahead of the 2014 Winter Olympics Feb. 5 in Sochi, Russia.
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February 5, 2014
By the Monitor's Editorial Board
An original purpose of the modern Olympics has been to promote peace – not only between nations but often inside a host country. With the opening of the Olympic Winter Games in Sochi this weekend, the focus will be mainly on skating, skiing, and sledding. But what will the Games do for peace, especially within Russia?
The country has yet to end terrorist threats from the nearby Caucasus region. Russian democracy is broken. Many dissidents remain in jail. With the world’s eyes on Russia for two weeks of elite winter sports, these Olympics might well be a catalyst for change.
To some degree, they already are.
Under pressure from the International Olympic Committee (IOC), President Vladimir Putin had to back down from a ban on protests during the two weeks of the Olympics. He conveniently released a few prominent people from prison, such as former opponent and oil tycoon Mikhail Khodorkovsky, feminist punk rockers Pussy Riot, and Greenpeace protesters. And he was on the defensive after a foreign backlash against a new Russian law against gays.
Mr. Putin had sought to use the Olympics to “buck up” his people and unite them behind his strong rule, especially if Russian athletes collect more than a few gold medals. The Games were meant to project an image of Russia as a great power and spur a slowing economy. He also wanted to develop the area around the Black Sea resort town of Sochi.
Instead, Russians are upset at the estimated $51 billion spent to prepare for the Games – an outsized amount in the history of the Olympics. Worries over a possible terrorist attack during the Games – countered by the presence of 37,000 security forces – have served as a reminder of Russia’s weaknesses within its own border. And local residents are upset at the ecological damage in their area.
One possible effect of the Games: Russian political activists have found renewed energy in reports of corruption and overruns in spending on Sochi. In both India and Brazil since 2011, massive protests have erupted in response to suspect spending on international megasports events. The demonstrations have pushed clean politics to the fore in those two nations.
Even more than corruption, Russians are watching to see if Putin has set the stage for a successful Olympics. If he hasn’t, people ask, how can he manage the economy. For more than a century, the IOC has chosen host nations based in part on political reasons. The 1936 Berlin Games ended up puncturing Hitler’s claim to Aryan superiority with the performance of black running star Jesse Owens. The 1964 Olympics restored Japan’s role in the world. The 1980 Games further isolated the Soviet Union because of a Western boycott. The 1988 Games in Seoul, helped bring democracy to South Korea.
Since the 2008 Summer Olympics in Beijing, in contrast, China’s rulers have only tightened their grip on power.
The Olympics, either by design or circumstance, put a global spotlight on a host country’s strengths and weaknesses. Such scrutiny can stir change, usually for the better. Humanity expects much in terms of universal values at these giant events. The Sochi contests could help reverse Putin’s descent into dictatorship and raise prospects for peace. If so, the Olympics themselves deserve a medal.
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1. Overview of the DNA Damage Response {#sec1}
======================================
Genomic instability is one of the most common characteristics of tumor cells and is probably due to the combined effect of DNA damage, tumor-specific DNA repair defects, and a failure to arrest the cell cycle before the damaged DNA is passed on to daughter cells. Genomic instability is recognized as a characteristic of most solid tumors and adult-onset leukaemias and is manifested as alterations in chromosome number and structure (chromosomal instability) and as changes to the structure of DNA, such as nucleotide substitutions, insertions, and deletions. To maintain genomic stability and to counteract DNA damage, cells have evolved a complex cellular response, called DNA damage response (DDR), which is coordinated by the DNA damage checkpoints \[[@B1], [@B2]\].
Somatic mutations in DDR genes have been found in several cancer types \[[@B3]\]. Indeed, on one hand, inactivation of the DDR favors the accumulation of mutations in proto-oncogenes increasing the risk of tumor development. On the other, since the anticancer activity of most chemotherapeutic drugs relies on the induction of DNA damage, alterations in the DDR also affect the tumor\'s sensitivity to chemotherapy \[[@B4]\].
Conceptually the molecules that orchestrate the DDR can be functionally organized in sensors, mediators, transducers and effectors. Recognition of DNA damage is the first step in the activation of the signaling cascade that controls the DNA damage checkpoints. DNA lesions are recognized by various sensor proteins: the MRN (MRE11-RAD50-NBS1) complex that signals double-strand DNA breaks (DBSs), and by RPA that binds single-strand DNA at sites of DNA damage. Subsequently, recruitment and activation of the highly conserved apical DDR kinases ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and RAD3-related (ATR) occur. In both yeast and mammals Tel1/ATM recognizes DSBs, while Mec1/ATR is activated in the presence of long single-stranded DNA tracts. Once activated, ATR and ATM transduce the DDR signal by promoting the phosphorylation/activation of downstream kinases, such as CHK1 and CHK2, which in turn regulate downstream checkpoint effectors. Checkpoint activation elicits a multifaceted cellular response that coordinates cell cycle progression with DNA repair activity, thus allowing cells to block the cell cycle until the damage is repaired. In addition, ATM phosphorylates the histone 2A variant *γ*H2AX that marks chromatin regions flanking DSBs. These phosphorylation events promote the recruitment of several mediator proteins that facilitate ATM/ATR signaling (see \[[@B5]\]).
Recently, a novel layer of complexity in the cellular response to DNA damage has emerged with the involvement of RNA metabolism. A first link between mRNA biogenesis and genome stability has been provided by the observation that transcription is inhibited in response to DNA damage, both generally and locally at DNA damage sites \[[@B6], [@B7]\]. Changes in the pre-mRNA splicing pattern of crucial genes in the DDR have long been observed (for review see \[[@B8]\]), and splicing factors have been observed to change their intracellular distribution following genotoxic damage. Finally, DNA damage is known to affect mRNA stability both positively and negatively (for review see \[[@B9]\]). This review is focused on the interplay between AS and DDR. We initially discuss how activation of the DDR signaling cascade can influence the activity of splicing factors and how this can affect cell fate. Then, we examine how, in turn, alternative splicing factors can contribute to the DDR. Finally, we will discuss how alternative splicing regulators can represent novel targets for cancer therapies.
2. How DDR Can Affect Alternative Pre-mRNA Splicing (AS) {#sec2}
========================================================
In recent years several reports have uncovered how DNA damage can induce splicing changes that give rise to mRNA variants encoding different protein isoforms with the potential to affect the cellular response and the cell fate. A first indication that the DDR can alter AS came from the observation that *Drosophila* S2 cells treated with camptothecin, a topoisomerase I inhibitor that leads to replication forks arrest, or exposed to ionizing radiation (IR) express specific alternative splicing variants of the transcription factor TAF1 that was shown to control the G2/M transition \[[@B10]\]. Alternative splicing of TAF1 in response to DNA damage was shown to depend on ATM/CHK2 and ATR/CHK1 signaling and to induce degradation of the splicing factor Tra2 \[[@B10], [@B11]\]. Another example is provided by the effect of UV irradiation and cisplatin on the splicing of MDM2 and MDM4 transcripts \[[@B12]\]. The MDM2 gene encodes an E3 ubiquitin ligase that targets p53 for proteasome-mediated degradation. MDM2 expression is positively controlled by p53 at the transcription level, generating a feedback loop. The proteins encoded by the mRNA variants induced by DNA damage lack the p53 interaction domain so that they may favor p53 activation \[[@B12]\]. A detailed description of the regulation of the alternative splicing of the MDM2 transcript in response to various genotoxic treatments can be found in \[[@B8]\].
How genotoxic stress can influence the activity of the splicing machinery is to date largely unknown. Two major mechanisms are known to control the activity of splicing factors in response to external and internal stimuli: (i) changes in expression level and (ii) posttranslational modifications. In addition, it has emerged in recent years that pre-mRNA splicing occurs largely cotranscriptionally and that also the processivity of RNA polymerase II (RNAPII) can influence the recognition of alternative exons (\[[@B13], [@B14]\], [Figure 1](#fig1){ref-type="fig"}).
2.1. The Expression Level of Splicing Factors Changes in Response to DNA Damage {#sec2.1}
-------------------------------------------------------------------------------
The simplest way by which DNA damage can affect the splicing machinery is by modifying the expression level of specific splicing factors. SR proteins and hnRNP proteins were the first splicing factors identified \[[@B15], [@B16]\]. These proteins are components of the basal splicing machinery. However, since their concentration can influence splice site selection, they contribute to alternative splicing. Indeed, deregulation of several members of the SR protein and hnRNP families of splicing regulators has been observed following various genotoxic treatments \[[@B17], [@B18]\]. One additional recent example is provided by the upregulation of SC35 by E2F1, a transcription factor that has a key function during S phase progression and apoptosis in response to DNA-damaging agents \[[@B19]\].
2.2. Posttranslational Modification of Splicing Factors {#sec2.2}
-------------------------------------------------------
As mentioned above the presence of phosphorylated *γ*H2AX is a hallmark of sites of DSBs. However, phosphorylation of H2AX is only the tip of the iceberg: a host of posttranslational modifications of both the histones and components of the DDR machinery have been reported at sites of DNA damage, including acetylation, ubiquitination and SUMOylation, and arginine methylation (for review see \[[@B20]\]). These modifications play a central role in coordinating cell cycle progression and DNA repair \[[@B21]\]. Therefore, it is not surprising that activation of the damage signaling cascade can lead to the post-translational modification of splicing factors that can modify their intracellular localization and/or activity. Here, we will discuss examples of posttranslational modifications of splicing regulatory proteins in response to DNA damage that have recently been reported.
### 2.2.1. Phosphorylation {#sec2.2.1}
The activity and the intracellular distribution of the serine/arginine (SR) proteins are tightly regulated by phosphorylation. SR proteins are RNA-binding proteins that are characterized by at least one domain enriched in RS dipeptides. This region, often located at the C-terminus of the protein, is termed RS domain and is subjected to reversible phosphorylation. The assembly of the splicing complex and the catalysis of the splicing reaction require dephosphorylation/phosphorylation cycles. While phosphorylated SR proteins favor spliceosome assembly, intron removal is associated with dephosphorylation of SR proteins. After splicing, a subset of SR proteins remains associated with the mature mRNA and is exported to the cytoplasm. Here, their reimport into the nucleus requires phosphorylation by SRPK1 and SRPK2, two SR protein kinases that are predominantly localized in the cytoplasm. It was recently shown that genotoxic stress can induce the phosphorylation and the relocalization of these kinases to the nucleus where they in turn hyperphosphorylate SR proteins leading to changes in pre-mRNA splicing \[[@B22], [@B23]\]. Moreover, chronic replication-dependent DNA damage was shown to induce the hyperphosphorylation of ASF/SF2 (SRSF1) \[[@B24]\].
Several studies underline the importance for catalysis of dephosphorylation within the assembled spliceosome \[[@B25]--[@B27]\]. Interestingly, the protein phosphatase PPM1G, which promotes pre-mRNA splicing, is phosphorylated in response to DNA damage and is rapidly recruited to DNA damage sites \[[@B28]\].
### 2.2.2. Acetylation {#sec2.2.2}
The first evidence of the role of lysine acetylation in the DDR came with the observation that overexpression of a dominant-negative allele of the acetyltransferase TIP60 reduced the efficiency of DSB repair \[[@B29]\]. Now, we know several TIP60 targets among crucial DDR factors, including H2AX, H4, and ATM \[[@B30]\]. A recent quantitative mass spectrometry analysis revealed that splicing factors, including SR proteins and hnRNPs, have numerous acetylation sites \[[@B31]\], often located within their RNA-binding domain. Since acetylation of lysine neutralizes the positive charge of the amino acid, this reversible modification could contribute to the regulation of their activity in splicing. Consistent with this report, Edmond and colleagues recently identified the SR protein ASF/SF2 as a novel substrate of TIP60 acetyltransferase activity in response to genotoxic treatments \[[@B23]\]. In the case of ASF/SF2, however, acetylation does not influence the splicing activity but rather controls its protein turnover by promoting degradation. Although these observations clearly demonstrate that acetylation can contribute to the regulation of the DDR, a recent genome-wide characterization of the DDR-regulated acetylome revealed a rather weak overall increase in site-specific acetylation compared to phosphorylation suggesting that acetylation may be a more selective/subtle modification \[[@B28]\].
### 2.2.3. Ubiquitination/SUMOylation {#sec2.2.3}
In addition to targeting proteins to proteasome-mediated degradation, ubiquitination has emerged as an important regulatory signal that functions in many cellular processes. Ubiquitination involves the covalent attachment of a 76 amino acid ubiquitin chain to a lysine of the modified protein by an E3 ubiquitin ligase. Multiple lysines can be ubiquitinated in the target protein, and following addition of the first ubiquitin additional ubiquitin molecules can be added, yielding a polyubiquitin chain.
Many different E3 ubiquitin ligases participate in the DDR. For example, RNF2 catalyzes the monoubiquitination of H2AX contributing to ATM recruitment \[[@B34], [@B35]\], while depletion of RNF4 impairs RAP80, BRCA1, and RAD51 recruitment to sites of DNA damage \[[@B36]--[@B38]\].
Indirect evidence suggests that ubiquitination of splicing factors may also be involved in the DDR. Ubiquitin regulates spliceosome assembly \[[@B39]\]. Moreover, the essential yeast splicing factor Prp19 and its human ortholog have E3 ligase activity *in vitro* \[[@B40]\]. A role of PRP19 in the mammalian DDR first emerged with the report that it was strongly upregulated by DNA damage in human cells and that its depletion by siRNA resulted in an accumulation of DSBs and apoptosis and reduced survival after exposure to ionizing radiation \[[@B41]\]. Prp19 is part of a large complex that contains \>30 proteins \[[@B42]\]. PRP19/Pso4 itself is modified by ubiquitination in response to DNA damage, and this modification reduces its affinity for other members of the Pso4 complex \[[@B43]\].
Besides ubiquitin, vertebrate cells encode several other ubiquitin-like proteins that are structurally related to ubiquitin. The best characterized one is small ubiquitin modifier (SUMO). Similar to ubiquitination, SUMOylation is a reversible posttranslational modification that plays a crucial role in the control of the DDR and in DNA repair pathways by modulating protein:protein interactions (for review see \[[@B44]\]).
ASF/SF2, the best characterized member of the SR protein family of splicing factors, can act as a cofactor stimulating SUMO conjugation by the SUMO E2 conjugating enzyme Ubc9 \[[@B45]\]. ASF/SF2 also interacts with PIAS1 and regulates its SUMO E3 ligase activity in response to DNA damage \[[@B45]\].
HnRNP K, as several other hnRNPs, is SUMOylated \[[@B46]\]. HnRNP K is a multifunctional protein involved in many steps of mRNA biogenesis \[[@B47]\]. Various genotoxic treatments stimulate SUMOylation of hnRNP K by the Polycomb E3 ligase Pc2 that in turn is activated by DNA damage via phosphorylation by HIPK2 kinase \[[@B48]\]. HnRNP K cooperates with p53 in the transcriptional activation of cell cycle arrest genes such as 14-3-3*σ*, GADD45, and p21 in response to DNA damage \[[@B49]\], and its SUMOylation stimulates p53 transcriptional activity \[[@B48]\].
2.3. Transcriptional Effects on AS {#sec2.3}
----------------------------------
Many chemotherapeutic drugs are potent inducers of DNA damage that interferes with transcription. Platinum derivatives such as cisplatin and carboplatin induce DNA adducts and intra- and interstrands cross-links between purine bases. Platinated adducts distort the DNA helix impairing replication and transcription elongation, which in turn can lead to the formation of DSBs \[[@B50]\]. Camptothecin (CPT) is a topoisomerase I inhibitor that leads to a block of transcription elongation and DNA replication. Doxorubicin is an inhibitor of DNA topoisomerase II that induces structural alterations in promoter DNA \[[@B51]\].
Although pre-mRNA splicing can occur independently of transcription, many different studies have provided evidence that AS, as other RNA processing events required for the synthesis of the mature mRNA, is mostly cotranscriptional (for review see \[[@B52]\]). Several RNA processing factors are recruited on the C-terminal domain of RNAPII and are deposited on the nascent pre-mRNA molecule during transcription elongation. Moreover, AS is influenced by the rate of transcription elongation; slowing down the polymerase may favour the use of weak splice sites by delaying the synthesis of downstream splice sites, thus facilitating the recognition of suboptimal exons (for review see \[[@B53]\]). In the light of these considerations it is not surprising that genotoxic treatments have been reported to induce transcription-dependent splicing alterations.
Recent studies have uncovered how the processivity of RNAPII can influence the recognition of alternative exons \[[@B54]\]. Muñoz and colleagues showed that DNA damage induced by UV can directly modulate the activity of RNAPII during transcript elongation thereby affecting the selection of alternative exons \[[@B55]\]. The effect of UV and cisplatin treatment on AS was initially characterized on the EDI alternative cassette exon of the fibronectin gene. Both genotoxic treatments despite eliciting quite different types of DNA damage strongly stimulated the inclusion of the EDI exon. The effects induced by UV were independent of p53 since they were observed also in Hep3B cells that are considered to be p53 null. Moreover, they were not due to changes in the expression or intracellular localization of splicing factors known to regulate this splicing event. Interestingly, however, both UV and cisplatin induced the hyperphosphorylation of RNAPII\'s CTD leading to the inhibition of transcription elongation. Therefore, in this case genotoxic damage affects the kinetic coupling between transcription and splicing, thereby affecting cotranscriptional AS. Interestingly, UV does not generally affect the level of either gene expression or AS, but its effects are restricted to a subset of responsive genes. In addition, the effects on AS induced by DNA damage may depend on the specific type of damage-inducing treatment. Indeed, when doxorubicin was used in this same study it did not induce the same splicing changes observed upon UV treatment \[[@B55]\].
Several splicing-sensitive microarray studies have examined the effect of CPT on cotranscriptional AS \[[@B56]--[@B58]\]. CPT appears to reduce RNA polymerase elongation rate promoting predominantly exon inclusion \[[@B56], [@B57]\]. Interestingly, many AS events leading to exon inclusion result in the production of mRNAs containing premature stop codons (PTCs) that will undergo nonsense-mediated decay. Gene Ontology analysis of the functional categories associated with the AS indicated that CPT treatment appeared to affect transcription and splicing of RNA-binding proteins \[[@B56], [@B57]\]. These observations suggest that this may represent a mechanism that allows the cell to respond to genotoxic damage by rapidly adjusting the level of RNA processing factors to the level of transcription.
A specific example of AS event that is affected by DNA damage is provided by the MDM2 gene. As mentioned previously, the MDM2 gene produces several different mRNA variants due to AS. The biological significance of these variants is presently unknown since only few of them are translated into proteins. The best characterized alternative MDM2 mRNA isoform is the ALT1 transcript that lacks 8 of 12 exons due to exon skipping. It has been reported that the ALT1 variant is upregulated by UV treatment \[[@B12], [@B59]\]. Dutertre and colleagues demonstrated that production of ALT1 and other variants due to exon skipping is regulated cotranscriptionally \[[@B58]\]. Specifically, different genotoxic treatments such as camptothecin, doxorubicin, and cisplatin induce MDM2 exon skipping by disrupting the interaction between EWS, a transcriptional coregulator \[[@B60]\], and the splicing factor YB-1 \[[@B58]\]. These results suggest that DNA damage may interfere with the coupling between transcription and splicing leading to the production of alternative or aberrant mRNA variants.
Recent work that examined the response to IR using exon sensitive microarrays in lymphoblastoid cells and in fibroblasts confirms the genome-wide effects on transcription and splicing induced by genotoxic stress. Exon-level analysis revealed a general increase in internal exon skipping in response to radiation. The affected genes are involved in cell cycle regulation, chromatin dynamics, p53 regulation, and cell growth. In addition this study revealed an increase in the use of alternative promoters. These promoters have p53 binding elements at or near the start site suggesting that the protein isoforms encoded by these mRNA variants may have an active role in regulating the response to IR \[[@B140]\].
Collectively, these reports suggest that genotoxic damage can interfere with RNA polymerase II activity and may influence cotranscriptional AS by distinct mechanisms. However, considering that the functional categories associated with the affected genes are related to crucial cellular programs one can speculate that these changes might be functionally relevant for the response to DNA damage.
3. How AS Can Affect the DDR {#sec3}
============================
Regulation of AS depends, on one hand, on the presence of specific sequence elements in the pre-mRNA and, on the other hand, on *trans*-acting protein factors. In this section we first report the most recent findings on how mutations in genes involved in the DDR affect splicing of the transcript thereby altering protein function. Then, we review how alteration of the expression of splicing factors can contribute to genomic instability and cancer ([Figure 2](#fig2){ref-type="fig"}).
3.1. Mutations in Genes Involved in the DDR Can Disrupt Pre-mRNA Splicing {#sec3.1}
-------------------------------------------------------------------------
The splicing machinery assembles on conserved sequence elements that define the intron-exon junctions, the so-called splice sites, and on the branch point sequence (BPS), a poorly conserved sequence located near the 3′ end of the intron. In addition to these core splicing signals, splicing is influenced by other regulatory elements \[[@B61]\]. These elements are conventionally classified as exonic splicing enhancers (ESEs) or silencers (ESSs) depending whether they function to promote or inhibit inclusion of the exon they reside in and as intronic splicing enhancers (ISEs) or silencers (ISSs) if they enhance or inhibit usage of adjacent splice sites or exons from an intronic location. These regulatory elements function by recruiting factors that activate or inhibit splice site recognition or spliceosome assembly.
Considering that about the 95% of our genes produce at least two isoforms \[[@B62]\] there is a high probability that mutations affecting these *cis-*acting elements could induce aberrant splicing with deleterious consequences. Accordingly, it has widely been suggested that most of the unclassified mutations (missense or silent) could affect splicing \[[@B63]--[@B66]\]. Furthermore, synonymous mutations, that do not change the protein sequence and therefore have traditionally been considered innocuous polymorphisms, could induce splicing aberrations by modifying splice sites (either canonical or cryptic) or splicing regulatory sequences \[[@B64], [@B65]\]. Splicing affecting mutations could lead to transcript instability by nonsense mediate decay (NMD) or to the synthesis of truncated or dysfunctional protein products. Despite their potential functional relevance, characterization of splicing-affecting mutations, in general, and in DDR genes, in particular, is not extensive. This may be in part due to historical reasons and also to technical issues related to the experimental validation of the functional consequences of a specific mutation. Hereafter, we describe examples of these types of mutations affecting critical genes in the DDR (see also [Table 1](#tab1){ref-type="table"}). First, we will discuss mutations in the well-characterized BRCA1 and BRCA2 genes. Second, we will describe an example of intronic mutation affecting AS of the ATM gene. Then, we will illustrate how different AS isoforms of key DDR regulator can have different functions. Finally, we will review how the loss of splicing factors can cause DNA damage.
### 3.1.1. BRCA1 and BRCA2: The Reclassification Issue {#sec3.1.1}
BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185), the two most important breast cancer susceptibility genes \[[@B67]\], are key players in DDR. They are involved in homologous recombination (HR) and DNA repair (or review see \[[@B1]\]).
More than 3500 mutations have been reported that affect the BRCA1/2 genes, about one-third of which are unclassified variants (UVs) (Breast Cancer Information Core Database (BIC), French UMD-BRCA1/2 mutation database: <http://www.umd.be/BRCA2/>, <http://www.umd.be/BRCA1/>) that may induce aberrant splicing. In 1998 Mazoyer et al. described a missense mutation within the exon 18 of the BRCA1 gene, which leads to exon skipping and consequently to the disruption of the BRCT domain, producing a nonfunctional protein \[[@B63]\]. Using an *in vitro* splicing system Liu and colleagues subsequently showed that exon skipping resulted from the disruption of a splicing enhancer in exon 18 \[[@B68]\]. Subsequently, Fackenthal et al. detected a missense mutation in BRCA2 affecting an ESE that caused the skipping of exon 18 and the out-of-frame fusion of exons 17 and 19 \[[@B69]\]. Later on, several other mutations that affect splicing were described in BRCA1 and BRCA2 contributing to a better understanding of the mechanism underlying the role of these proteins in tumorigenesis \[[@B66], [@B70]\]. More recently, Gaildrat et al. used a BRCA2 minigene reporter system to study the effect of predicted splice-site mutation and some unclassified mutations occurring at a distance from the splicing sites. The study identified a group of mutations that induced the aberrant splicing of exon 7 \[[@B71]\].
In recent years, several groups have proposed a combined approach to study the potential effects of BRCA1/2 genetic variants on splicing efficiency \[[@B65], [@B72]\]. These strategies exploit splicing prediction programs to detect potential splicing alterations followed by functional assays to analyze BRCA1/2 unclassified mutations. Using this approach Sanz and colleagues identified 57 putative splicing-affecting mutations. However, an effect on splicing could be confirmed by functional analysis only for half of the tested mutations \[[@B65]\]. This low rate of correlation could be explained by the high false positive outputs of the algorithm predicting ESE and ESS mutations. Therefore, the results of this study and of other similar reports underlie the importance of a functional validation of bioinformatic predictions \[[@B65], [@B72]\]. Nevertheless, these studies support the reclassification of many UVs as splicing affecting mutations.
### 3.1.2. ATM: The Intronic Case {#sec3.1.2}
The ataxia-telangiectasia mutated (ATM) (OMIM 607585) gene, a key player in the DDR, is mutated in the autosomal recessive disorder ataxia-telangiectasia (AT, OMIN number 208900). The gene was identified in 1995 by positional cloning; it encodes 66 exons spanning \~160 kb of genomic DNA \[[@B73]\]. It soon became clear that a significant proportion of the known mutations in the ATM gene causes splicing defects \[[@B73]--[@B75]\]. Here, we will focus our attention on a specific aberrant splicing event that illustrates an additional layer of complexity in splicing regulation.
The aberrant inclusion of a pseudoexon of 65 nt located in intron 20 (termed here exon 20A) was reported more than 10 years ago in a patient affected by AT \[[@B76]\]. The inclusion of exon 20A was associated with a deletion of four nucleotides (GTAA) in intron 20. The authors identified a novel regulatory element within intron 20, termed "intron-splicing processing element" (ISPE), which acts as an intronic silencer of a cryptic 3′ splice site \[[@B77]--[@B79]\]. The ISPE is recognized by the U1 snRNP, a core component of the splicing apparatus that normally binds the 5′ splice site. Sequestration of U1snRNP by the ISPE prevents inclusion of exon 20A. However, in the AT patient the four nucleotides deletion impairs U1 snRNP binding to the regulatory element, causing the inclusion of the cryptic exon \[[@B78]\].
3.2. Same Gene, Two Isoforms with Quite Different Functions {#sec3.2}
-----------------------------------------------------------
Cyclin D1 was first characterized as a cell cycle regulator. Cyclin D1 associates with CDK4/6 promoting the phosphorylation of Rb; this last event leads to the derepression of E2F, a transcription factor that controls the expression of DNA replication genes, allowing cell cycle progression (for review see \[[@B80]\]). In addition, cyclin D1 has a transcriptional regulatory activity that is independent of its association with CDK4/6 \[[@B80]\].
Two AS variants of cyclin D1 have been described, called D1a and D1b \[[@B81]\]. The respective protein isoforms display different intracellular localization: while D1a shuttles between the nucleus and the cytoplasm, D1b is constitutively nuclear \[[@B82]\]. Both transcript variants are expressed in normal tissues but D1b often appears upregulated in several forms of cancer, including breast cancer \[[@B83], [@B84]\]. The oncogenic properties of D1b isoform have been extensively described \[[@B82], [@B85]\]. Instead, cyclin D1a has directly been related to the DDR \[[@B86]\]. Recruitment of cyclin D1a, but not of D1b, to chromatin is sufficient to activate the DDR promoting H2AX phosphorylation. Moreover, after genotoxic stress D1a enhances the recruitment of repair factors contributing to checkpoint activation and G2/M arrest \[[@B86]\]. A recent report by Myklebust and colleagues identifies high expression of cyclin D1a protein as a positive predictive factor for the benefit of adjuvant chemotherapy with levamisole and 5-fluorouracil (5-FU), which leads to replication stress, due to the depletion of the intracellular deoxythymidine triphosphate (dTTP) pool in colorectal cancer \[[@B87]\]. This last report underlies the importance to link expression patterns of splicing isoforms with therapeutic approaches.
Two recent reports show that the splicing factors Sam68 and ASF/SF2 regulate cyclin D1 splicing favoring the D1b isoform \[[@B88], [@B89]\]. In addition, transcriptional regulation also affects the cyclin D1a/D1b ratio. Sanchez and colleagues reported that the EWS-FL11 fusion protein, a well characterized transcription factor, is able to favor the expression of the D1b isoform by decreasing the rate of polymerase II elongation \[[@B90]\]. Additionally, a polymorphism at the 3′ splice site of intron 4 may influence splice site choice favoring the production of the D1b isoform \[[@B81]\]. All these mechanisms show that alternative splicing of cyclin D1 is under a tight control making it a very interesting target for the development of new therapeutic strategies.
Another quite interesting example of AS variants was recently described by Pabla and colleagues \[[@B91]\]. CHK1-S is a novel splice variant of CHK1, which functions as endogenous regulator of CHK1 in both physiological conditions and after DNA damage. The authors demonstrated that in normal conditions CHK1-S is able to interact with CHK1 inhibiting its activity and promoting the S to G2/M transition. Upon DDR activation, CHK1 becomes phosphorylated and the CHK1-S/CHK1 interaction is disrupted, so that CHK1 can induce cell cycle arrest facilitating DNA repair. Interestingly, the authors reported a deregulated expression of CHK1-S in testicular and ovarian cancer. How the CHK1-S expression is regulated remains to be explored.
3.3. Loss of Splicing Factors and Genomic Instability {#sec3.3}
-----------------------------------------------------
A growing body of evidence suggests that the depletion of splicing factors may induce genomic instability. It is by now well established that transcription and RNA processing are tightly coupled processes \[[@B13], [@B14]\]. This, on one hand, favors accurate and efficient mRNA processing, and on one hand, protects the genome from the likely disastrous effects of the nascent transcripts themselves \[[@B92]\]. Accordingly, in *S. cerevisiae*, when genes involved in mRNA processing are mutated, defects occur in the packaging of nascent mRNAs \[[@B93]\]; as a result the nascent pre-mRNA hybridizes with the transcribed strand generating an RNA-DNA duplex, known as R-loop, causing genomic instability. Analogous effects have been observed in chicken DT40 cells upon silencing of the splicing factor ASF/SF2 \[[@B92], [@B94]\]. Similarly, in mouse embryonic fibroblasts, loss of SC35 (SFSR2) resulted in G2/M cell-cycle arrest and genomic instability \[[@B95]\]. Indeed, not only the siRNA-mediated silencing of splicing factors but also the genetic depletion of a transcriptional coactivator, such as SKIIP, besides affecting splicing, induced genomic instability \[[@B96]\]. Interestingly, overexpression of RNAse H, which cleaves the RNA molecule in a RNA-DNA hybrid, reduced the formation of H2AX foci \[[@B96]\]. Although the precise mechanism through which the formation of R-loops results in genomic instability is still unclear, these structures have recently been demonstrated to impair the replication fork progression \[[@B97]\].
Finally, in the last few years numerous mutations affecting components of the splicing machinery have been reported in several types of cancer \[[@B98]--[@B103]\]. The functional consequences of these mutations, and how they contribute to tumorigenesis not fully understood. However considering the genotoxic consequences of splicing factor depletion, discussed previously, it is possible that at least some of these mutations may result in the functional inactivation of the splicing factors thus affecting genomic stability.
3.4. Splicing-Related Proteins as Novel Factors in the DDR {#sec3.4}
----------------------------------------------------------
The results of several recent genome-wide studies strongly suggest an overlap between the pathways leading to mRNA biogenesis and the cellular response to DNA damage. Some years ago, Matsuoka and colleagues performed a large-scale proteomic analysis that identified more than 700 proteins phosphorylated by ATM and ATR in response to DNA damage. Interestingly, RNA processing was among the enriched Gene Ontology categories that had not been previously linked to the DDR \[[@B104]\]. The involvement of RNA maturation in the cellular response to DNA damage has been subsequently supported by various siRNA-based screens to identify genes involved in DNA damage sensitivity and genome stabilization \[[@B96], [@B105]\]. In particular, two genome-wide shRNA screens identified the Ewing sarcoma (EWS) protein, a member of the TET (TLS/EWS/TAF15) family of RNA- and DNA-binding proteins, as necessary for resistance to camptothecin \[[@B106]\] and IR \[[@B105]\]. Consistent with a role in the DDR, EWS knock-out mice show hypersensitivity to ionizing radiation \[[@B107]\]. Recently, Paronetto et al. showed that the Ewing sarcoma (EWS) protein, a member of the TET (TLS/EWS/TAF15) family of RNA- and DNA-binding proteins, regulates AS in response to DNA damage \[[@B108]\].
Using a similar approach to identify novel genes contributing to telomeres protection Lackner and colleagues observed that silencing of several splicing-related genes (including SKIIP and SF3A1) induced a general activation of the DDR, possibly because of R-loop formation while having a weak effect on telomere stability \[[@B109]\]. More recently, RBMX, an hnRNP that associates with the spliceosome and influences alternative splicing \[[@B110]\], was found in a genome-wide siRNA-based screen to detect regulators of homologous recombination (HR) regulators. RBMX regulates HR in a positive manner, accumulates at sites of DNA damage in a poly(ADP-ribose) polymerase 1- (PARP1-) dependent manner and promotes resistance to several DNA damaging agents \[[@B111]\]. As discussed in [Section 2.2](#sec2.2){ref-type="sec"}, Beli and colleagues identified the protein phosphatase PPM1G, that regulates spliceosome activity, using high resolution mass spectrometry combined with stable isotope labeling with amino acids in cell culture (SILAC) to quantify regulated changes in phosphorylation and acetylation induced by different DNA-damaging agents \[[@B28]\]. In addition, they detected the phosphorylation of THRAP3, a protein involved in RNA processing and stability \[[@B112], [@B113]\]. Interestingly, while many DDR factors are recruited to H2AX foci, THRAP3 is excluded from sites of DNA damage in a manner that parallels transcription inhibition. Two THRAP3-binding proteins BCLAF1 and PNN \[[@B114]\], that have also been implicated in mRNA metabolism, behaved in a similar manner suggesting that they are part of a novel protein complex, which may link RNA splicing to the DDR. A comprehensive review, that appeared while this paper was under revision, describes in detail some of these novel RNA-binding proteins involved in DDR \[[@B9]\].
4. Summary and Future Perspectives {#sec4}
==================================
Genomic instability is a hallmark of cancer cells, and the understanding of the mechanisms able to limit or counteract it can positively impact on the therapy of tumors. Genome-wide approaches have revealed that genes involved in RNA processing are often deregulated in response to genotoxic treatments. Since many chemotherapeutic compounds are DNA-damaging agents, AS can be an important determinant of how tumor cells respond to therapy.
Pre-mRNA splicing is a crucial step in the control of gene expression. The activity of splicing factors must therefore be tightly regulated since both their depletion and their upregulation can have harmful consequences. On one hand, deregulation of splicing factors may affect AS leading to the generation of cancer driving transcripts \[[@B115], [@B116]\]. On the other hand, depletion of splicing factors may induce aberrant splicing of critical DDR effectors altering indirectly the cellular response to DNA damage \[[@B117]\]. Moreover, splicing factors\' depletion may slow down intron removal favoring the formation of DNA/RNA hybrid thereby leading to the collapse of replication forks and to the generation of DSBs \[[@B92], [@B96], [@B97]\]. Finally, the activation of the DDR can promote the posttranslational modification of splicing factors altering their intracellular localization and/or their activity \[[@B22], [@B23], [@B43], [@B48], [@B118]\]. Although some aspects of the relationship between DDR and mRNA processing have been clarified, certain observations are still to be explained. Why are some splicing factors recruited to sites of DNA damage? What are their functions there? Moreover, when splicing changes are considered, a causal link between splicing alteration and disease occurrence can only be established if the stability of the mRNA variant and the function of the encoded protein have been determined. Finally, the possibility should be considered that an involvement of splicing factors in the DDR may not necessarily imply an involvement of splicing regulation in the DDR but may reflect other functions of these proteins. Nevertheless, considering that AS plays a major role in the regulation of the apoptotic response (see C. Sette\'s review in this issue) and that AS variants have been demonstrated to regulate chemoresistance \[[@B87]\], it is reasonable that splicing modulation has been proposed as an appealing therapeutic target \[[@B119], [@B120]\]. Strategies to modulate AS by antisense oligonucleotides are already in advanced clinical trial phases for some neuromuscular disorders, such as Duchenne muscular dystrophy or spinal muscular atrophy, and oligonucleotides are being developed to target specific mRNA variants to enhance the efficacy of conventional chemotherapy \[[@B121]\]. In addition, in recent years several bacterial compounds, and other small molecules have been identified that target spliceosomal components \[[@B122]\]. Interestingly, some of these compounds display strong cytostatic effects and significant antitumor activity in animal models. Further understanding of how AS regulation and the DDR are interconnected and linked to different signal transduction pathways should help us to better understand tumor progression and provide a basis for innovative splicing-targeted cancer therapies.
{#fig1}
{#fig2}
######
List of DDR-related genes found to be aberrantly spliced in several cancer types.
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Gene Function Cancer References
--------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------ -----------------------------
BRCA1 An E3 ubiquitin ligase contained in several cellular complexes, involved in DNA repair, genome stability maintenance, and cell cycle checkpoint control. Breast and ovarian cancer \[[@B63], [@B65]--[@B72]\]
BRCA2 Involved in HR, it associates with RAD51 Breast and ovarian cancer\ \[[@B65], [@B72]--[@B123]\]
Familial pancreatic cancer
ATM Apical kinase of DDR response, mainly involved in HR Ataxia-telangiectasia\*\ \[[@B124]--[@B127]\]
Hereditary breast and ovarian cancer\
Mantle cell lymphoma\
Colon tumor derived cell lines\
Leukemia-lymphoma-derived cell lines
MRE11 Component of DNA damage sensor complex MRN Mismatch repair deficient colorectal cancer\ \[[@B126], [@B128]\]
Leukemia-lymphoma and colorectal cancer-derived cell lines
ATR Apical kinase of DDR response, mainly involved in HR Seckel syndrome\*\ \[[@B129]--[@B132]\]
Hodgkin\'s lymphoma\
Breast and ovarian cancer
XPA Nucleotide excision repair Xeroderma pigmentosum\* \[[@B133]\]
DNAPK Apical kinase of DDR response, mainly involved in NHEJ Xeroderma pigmentosum\* \[[@B134]\]
MSH2 and MLH1 Mismatch repair Hereditary nonpolyposis colorectal cancer \[[@B135]--[@B137]\]
CHEK2 DNA damage checkpoint kinase Breast cancer\ \[[@B138], [@B139]\]
Li-Fraumeni syndrome
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
HR: homologous recombination; NHEJ: nonhomologous end joining. \*ataxia-telangiectasia, xeroderma pigmentosum, and Seckel syndrome were included because they display strong predisposition to malignancies.
[^1]: Academic Editor: Claudio Sette
|
445 P.2d 254 (1968)
MISSOURI-KANSAS-TEXAS RAILROAD COMPANY, a corporation, and H.S. Whitlock, an individual, Plaintiffs in Error,
v.
Kim HAYES, by and through John Hayes, her father and next friend, Defendant in Error.
No. 42107.
Supreme Court of Oklahoma.
July 16, 1968.
Rehearing Denied September 24, 1968.
E.J. Doerner and Harry D. Moreland, of Doerner, Stuart, Moreland, Saunders & Daniel, Tulsa, for plaintiffs in error.
Baker & Baker, Tulsa, for defendant in error.
*256 BLACKBIRD, Justice.
This is a companion case to Cause No. 41767, styled "Missouri-Kansas-Texas Railroad Company, a corporation, and H.S. Whitlock, an individual, Plaintiffs in Error, vs. Laurie Hayes, by and through John Hayes, her father and next friend, Defendant in Error", Okl., 445 P.2d 779.
In the present action, the same father, as plaintiff, sued the same plaintiffs in error, as defendants, for damages on account of personal injuries another of his minor children, Kim Hayes, suffered in the same auto-train collision. Kim, like her sister, Laurie, on whose behalf the cited action was instituted, was riding in the back seat of the 1959 Chevrolet Sedan driven by Jimmy Geren, when it collided with the Railroad Company's train, at the same crossing near Broken Arrow.
This case was tried before a jury approximately six months after the trial of Cause No. 41767, supra, and, as did that trial, resulted in a verdict and judgment for the plaintiff.
The first three propositions, and the arguments thereunder, advanced for reversal by plaintiffs in error, hereinafter *257 referred to as "defendants", are in no material respect different from those they presented in Cause No. 41767, supra. There were a few differences in the evidence introduced in the two cases, one of them being that, in this case, no member of the defendant railroad company's train crew directly, or unequivocally, admitted that the crossing involved was a very dangerous one, or more hazardous than many others. However, since there was no material difference in the evidence of the two cases concerning the physical characteristics of the crossing, and the facts surrounding the collision, and we think the evidence here was ample to support the jury's evident conclusion that the crossing was an extra hazardous one, we find none of defendants' arguments under their first three propositions sufficient to justify reversal of the trial court's judgment, for the same reasons that we found substantially the same arguments insufficient in Cause No. 41767, supra. Therefore, in view of the foregoing, we find it unnecessary to repeat, or add to, what we there said, either, by way of describing the evidence, or of discussing the rules and/or law applicable thereto. Accordingly, we hereby adopt what was therein said and held, as controlling here on defendants' first three propositions, and the arguments advanced thereunder.
In a fourth proposition presented in this appeal, defendants urge that the verdict in this case "was clearly excessive and clearly indicates that it is the result of passion or prejudice."
In plaintiff's petition, he alleged, among other things, in substance, that Kim, who, at the time of the collision was approximately 6 1/2 years old, received severe head injuries, which rendered her unconscious "for many days" and resulted in permanent brain damage; that she also received injuries to the chest, arms, legs and in the area of her abdomen, which tore and ruptured her liver and made abdominal surgery necessary, resulting in permanent damage to that organ; and, that she will be permanently disabled and required to undergo future medical treatment. In his amended petition, plaintiff prayed for damages in the total sum of $87,450.00 for these injuries. The jury's verdict awarded him the lump sum of $32,500.00 and his costs in the action.
The evidence introduced at the trial failed to unequivocally show that Kim will be permanently disabled, or will certainly require future medical treatment. Her mother testified, in substance, that, when Kim was taken to the hospital, on the day of the accident, she was bruised, and her face was so swollen that "you couldn't even tell where her nose, eyes or anything was * * * her mouth was full of blood and * * * (she) had blood in her hair * * * (and) all the hide burned off of her forehead up into her hair line." Mrs. Hayes further testified that Kim was in an intensive care unit of the Hospital "about a week"; that abdominal surgery was performed on her by Dr. S. which left scars on her abdomen; that she had "I.V. cutdowns" on her arms and ankles, because her veins weren't large enough to accommodate the needle through which she was fed intravenously; that Kim had some kind of a medicine for pain every day for about the first two weeks of the continuous three-week and one-day period that she was hospitalized.
Dr. S, a specialist in general chest and vascular surgery, testified that on the day of the accident he was asked, by the orthopedic consultant for Kim's sister, Laurie, to go to the hospital's intensive care unit and see Kim; that, when he did so, he found her blood pressure was dropping, she was beginning to go into shock, and her abdomen was beginning to distend. Dr. S further testified, in part, as follows:
"* * * We performed a four quadrant tap, put a needle in all four quadrants of the abdomen, and got back blood which didn't clot, which is indicative of a rupture of one of the organs. So we immediately prepared her for surgery and took her down to the operating *258 room, where we found that somewhere between a fifth and a fourth of the right lobe of the liver had been knocked off of the liver, had been what we called avulsed, and we had to continue this removal with our surgical technique and control the bleeding and take out this part of the liver that had been crushed or had been damaged in the accident. * * *."
Dr. S further testified that, since part of the liver had already been "knocked loose" and "cracked open", the surgical technique was to completely remove that fragment because "it could never have been sutured back on and wouldn't have stayed alive * * *". He further testified that the portion of Kim's liver that was removed, was about a quarter of its right lobe, which "roughly makes up about two-thirds of the liver." Dr. S further testified that Kim was in the hospital's intensive care unit, where there were "special nurses around the clock * * *" for several days; that she had the "usual amount of pain" of "any patient undergoing * * * major surgery, but after a period of three or four days, she was not in too much pain and recovered uneventfully." Dr. S further testified that, from the hemorrhage in Kim's abdomen, she lost two quarts of blood, but this was replaced by transfusion at the time of the surgery; that if she had not been operated and her internal hemorrhage have been stopped, she would have bled to death. Dr. S further testified that with one-sixth of her liver removed, Kim still had enough liver left to sustain life "in its fullest sense"; and, on cross examination, he referred to this organ's injury as an "avulsion", and testified to the effect that a normal human being has a larger liver than is necessary to meet the usual needs of the body, and that the body will rejuvenate it, like some other organs, but to only a minimal extent. Dr. S. further testified that Kim's recovery from the operation was not accompanied by some of the complications which sometimes occur, and that, since the recovery, it had not been necessary "to give her any further treatments with respect to her liver."
Kim's neurosurgeon testified that "We" started watching her in regard to her head injuries, when she was brought to the hospital, and she "gradually became conscious and was able to talk, and to respond at the end of about a week." He further testified that Kim's "brain wave has been abnormal * * *" and evidenced some "short circuiting", as shown by encephalograms that have been taken. He called this condition "dysthymia", and further testified that "she had headaches for a considerable period of time", but he indicated that this had ceased and that, at the time of the trial, she was taking no medication for her head condition. He further testified that no focal scarring of the brain was indicated. When interrogated as to her prognosis, Dr. S testified, in substance, that no further medication should be necessary unless "the brain wave becomes abnormal." He further testified, in substance, that though Kim's "brain wave forms remain abnormal," he could not predict whether this condition would improve, or not, and stated that: "We have no real knowledge that there will be any marked abnormality showing up." He further testified, in substance, that there was a "fifty-fifty" chance that the brain wave may get better, or may get worse, in the passage of time. He indicated that he had taken the last encephalograms the day before the trial, and recommended that Kim's "brain wave should be taken every six months to a year, to see if medication would be needed." He further testified, in substance, that if Kim should ever receive another head injury, her brain would be "a little bit more sensitive" to it, than if she had not received the subject one.
In support of their "Proposition IV", defendants first say, in substance, that, at the hearing on their motions for new trial, the trial judge listened to their arguments that the verdict was excessive. Then they quote his oral comments upon that issue, as showing that he failed to make a determination as to it. They contend that, for that reason, there is no "ruling *259 to which this (appellate) court can attach customary * * * weight." We do not agree. Unlike the cases in which we have refused to consider oral remarks trial judges have made from the bench in connection with their rulings on motions for new trials, the ones in question were incorporated in the journal entry of the court's judgment. As to this and related matters, see Moree v. Moree, Okl., 371 P.2d 719, and the cases therein cited. But, even if these remarks were regarded as "findings", or as the reasons why the motion for new trial was overruled, they are not inconsistent with, or contradictory to the decretal part of the court's order. They, like the other expressions in the order, clearly show that the trial judge was unable to conclude, after an apparently thorough, adversary, hearing on the matter, that the verdict was excessive; and those expressions cannot be held to impeach his ruling or to diminish the weight of its presumptive correctness, nor to relieve defendants of their burden of demonstrating that the verdict was excessive, or was the result of passion and/or prejudice. We reject, as unfounded, the defendants' charge that, by this order, the trial court failed to rule on their motions for a new trial, and "left the matter to the Supreme Court."
The record fails to show anything related to Kim's injuries that could conceivably have incited sympathy, or passion, on the part of the jury, unless such might have been induced by the vivid detail with which those injuries, and the steps taken to alleviate them, were described. But no complaint is made of that. Instead, defendants content themselves with making general statements to the effect that plaintiff's counsel "exaggerated" the "weight and value of medical testimony", the jury reached its verdict "under the influence of said counsel's argument", and "adroit and generalized argument has lead the jury to adopt an exaggerated belief of a continuing brain injury of a critical nature." We have no way of considering counsel's arguments before the jury, as they do not appear in the casemade. See Kansas City Southern Ry. Co. v. Martin, Okl., 293 P.2d 600, and Westgate Oil Co. v. McAbee, 181 Okl. 487, 74 P.2d 1150.
We recognize that the evidence in this case does not unequivocally show that Kim will ever definitely and certainly suffer any future pain, permanent disability, or other ill effect, either from the brain injury or her liver injury, but the testimony of the expert witnesses, as to such future damages, if any, seems to have been "as complete, exact and unequivocal as the nature of * * * (her) condition would permit." St. Louis San Francisco Ry. Co. v. McBride, Okl., 376 P.2d 214, 219. And, as indicated in the cited case, the difficulty of proving such damages should not have the effect of lessening the monetary burden of defendants' legal responsibility for Kim's condition, or for the pain and suffering she has already undergone. As to the latter, there is no fixed standard of measurement. (Chicago, Rock Island & Pac. R. Co. v. Hawes, Okl., 424 P.2d 6, 15, and Bready v. Tipton, Okl., 407 P.2d 194, 206, quoting Collins v. McPherson, 91 Ga. App. 347, 85 S.E.2d 552, 555). This being true, and the lump-sum verdict furnishing no clue to how much of its amount was awarded for any particular item of plaintiff's damages, we are without any sound basis, in view of the foregoing, for a determination that the verdict and judgment were excessive. See Kansas City Southern Ry. Co. v. Johnston, Okl., 429 P.2d 720, 731, cert. den., 389 U.S. 895, 88 S.Ct. 481, 19 L.Ed.2d 471.
In accord with the foregoing, the judgment of the trial court is affirmed.
All the Justices concur.
|
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