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Vulnerability and resiliency to suicidal behaviours in young people.
We aimed to examine factors that influence vulnerability/resiliency of depressed young people to suicidal ideation and suicide attempt. Data were gathered during a 21-year longitudinal study of a birth cohort of 1,265 New Zealand young people. Measures included: suicide attempt; suicidal ideation; major depression; childhood, family, individual and peer factors. Young people who developed major depression had increased rates of suicidal ideation (OR = 54: 95% CI 4.5-6.6) and suicide attempt (OR = 12.1; 95% CI 7.9-18.5). However, the majority of depressed young people did not develop suicidal ideation or make suicide attempts, suggesting that additional factors influence vulnerability or resiliency to suicidal responses. Factors influencing resiliency/vulnerability to suicidal responses included: family history of suicide; childhood sexual abuse; neuroticism; novelty seeking; self-esteem; peer affiliations; and school achievement. These factors operated in the same way to influence vulnerability/resiliency among those depressed and those not depressed. Vulnerability/resiliency to suicidal responses among those depressed (and those not depressed) is influenced by an accumulation of factors including: family history of suicide, childhood sexual abuse, personality factors, peer affiliations and school success. Positive configurations of these factors confer increased resiliency, whereas negative configurations increase vulnerability.
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Q:
Sharing sockets over separate nodeJS instances
I'm making chat application with multiple chat servers(nodeJS) and one redis server which should help grouping all nodeJS instances. Well I have this:
var io = require('socket.io').listen(3000);
// Create a Redis client
var redis = require('redis');
client = redis.createClient(6379, '54.154.149.***', {});
// Check if redis is running
var redisIsReady = false;
client.on('error', function(err) {
redisIsReady = false;
console.log('redis is not running');
console.log(err);
});
client.on('ready', function() {
redisIsReady = true;
console.log('redis is running');
});
io.sockets.on('connection', function(socket){
socket.on('new user', function(data){
client.set('user:' + data, socket.id);
})
socket.on('send message', function(data){
client.get('user:' + data['id'], function (err, socketId) {
io.sockets.connected[socketId].emit('new message',data['msg']);
});
})
socket.on('disconnect', function(data){
});
});
The redis is working perfectly and if I have two users on the same server they can chat. But if they are on different servers they cannot because the sockets aren't shared between the servers. How can I resolve this? I checked about pub/sub on redis and I'm sure that's the answer but I didn't manage to implement it.
A:
The Socket.IO docs have a section about doing sticky sessions and distributing messages. The quick answer though is to use a module like socket.io-redis.
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Michael Hirsh was national editor for Politico Magazine from 2014–2016.
There is a photograph of Maj. Gen. John Batiste, eyes brimming with tears after the memorial ceremony for Capt. Humayun Khan in Baqubah, Iraq, on June 11, 2004, three days following Khan’s death. Standing next to Batiste—who was the commanding general of the 1st Infantry Division in which Khan served—is his subordinate, Col. Dana Pittard, who commanded Khan’s brigade in Diyala Province, the violent epicenter of the unfolding insurgency and what, a year later, would become a full-blown sectarian civil war. Looking at the picture more than 12 years later, Batiste says the memory of that day came back to him. “I made it a priority to attend as many of those memorial services as I could,” he says. “But I do remember that one.”
Batiste was in the receiving line after the ceremony, he recalls, and he choked up because he had come to realize the man they were honoring that day—years before his parents got into a war of words with Donald Trump over the death of their Muslim-American son—was one of the finest soldiers under his command.
This is Khan’s story. It is also, to a very great extent, the story of America’s long experience in Iraq.
“These ceremonies are very private moments, and this one was extremely emotional,” Batiste says. “The battalion and company commanders spoke, and then some of Capt. Khan’s colleagues who served under him spoke.” They spoke, among other things, of Khan’s sacrifice, when the captain ordered his solders to hit the dirt while he moved forward to stop the bomb-laden suicide car that would kill him instantly, with a giant blast heard by “everybody at the base,” as Pittard recalls.
Says Batiste: “You could hear how well respected he was within his battalion, I remember now also, talking to one of his comrades afterwards, feeling that he was a great soldier.” Pittard agrees: “It was just raw emotion.” Khan was special, he says. “When he was still a lieutenant, we had nominated him to be aide de camp to the deputy commanding general. That means we felt that he was the outstanding lieutenant in the entire brigade. That’s who you send forward.”
M. Scott Mahaskey/Army Times
The terrible irony, says Pittard, is that Khan died—he was the first casualty in his unit—just as the 3rd Brigade was starting to make real inroads into stabilizing the province. Indeed, the suicide bomber who killed Khan may well have been retaliating for that success. By the fall of 2004, they were doing so well in quelling the Sunni insurgency and winning over Sunni moderates, says Pittard, that the general in overall command of U.S. forces in Iraq, Gen. George Casey, made a fatal mistake later in the year, and decided to move resources elsewhere: “But it was too early to move that much combat power away from Diyala. The insurgency was able to get re-embedded. By the end of 2006, Diyala had blown up. When I returned in 2006 and 2007, I got to watch that nightmare.” (Casey, now retired, did not respond immediately to a request for comment.)
The larger issue, says Batiste, was too few resources in Iraq, period. The sum total of all those wrenching memorial services—those casualties during his year commanding the 1st ID around Baqubah (where Khan was killed)—had a lot to do with the shocking decision he made a year later: Batiste rejected an offer of three stars and one of the premier commands in the U.S. military at the time: V Corps, which was being rotated back into Iraq. He would have been the top general in Iraq in charge of day-to-day combat operations. Instead, Batiste resigned from the Army. “It was gut-wrenching,” he told me. “I loved soldiering.” But he could no longer stay silent, he says, about the decisions being made in Washington—the withholding of soldiers and resources, the state of denial of what was really happening at Donald Rumsfeld’s Defense Department, that was making Iraq so dangerous for soldiers like Khan.
“The 1st Infantry Division did a hell of a job in Iraq. I couldn’t have been prouder of all those soldiers,” says Batiste, who is today president and CEO of a military and civilian armor-supply company in Buffalo, N.Y. “But we went into Iraq without a coherent strategy. And just as important as the strategy, we did not have the resourcing or any rehearsing of what to do after the fall of Saddam Hussein. The lack of foresight at the U.S. government level, I think our soldiers paid for that.”
At the moment of Khan’s death at Forward Operating Base Warhorse, malign forces were just starting to gather all at once—like the first heavy wisps of a hurricane wind—in the town of Baqubah and the province as a whole, which lies just east of Baghdad and borders Iran. “There was more and more evidence that the fault line was going through Baqubah,” says Batiste. “The early indications of Al Qaeda in Iraq were in Baqubah, where [its founding leader Abu Musab al] Zarqawi was later tracked and killed. There was a lot of Shia support coming from Iran there, and the Shias were exercising power while the Sunnis were feeling disenfranchised. It was a microcosm of everything that was going wrong in Iraq.”
Turmoil in Baqubah, Iraq Scenes from Buriz, Iraq filmed June 17, 2004 by Politico photographer M. Scott Mahaskey. Mahaskey was an embed working with Military Times (Gannett) at the time when he filmed this battle. The area, not far from Baqubah, Iraq, was within the battle space where Cpt. Khan was killed in action on June 8, 2004. (Used with permission of Military Times).
L. Paul Bremer III, head of the Coalition Provisional Authority governing Iraq at the time, also remembers that period of early summer as critical; Khan was killed at the height of uncertainty about Iraq’s future, only three weeks before the handover of sovereignty that Bremer orchestrated but which was so physically dangerous that the Americans bolted two days before the official ceremony so they could fool the enemy and avoid a terrorist attack. “There had been IEDs, but we were also starting to see car bombs,” Bremer recalled in an interview. Within a few months, from April to early June—when Capt. Khan was killed—Iraq began to come apart at the seams. “Zarqawi had started up Al Qaeda. Moqtada Sadr had radicalized the Shias. On the 24th of June three car bombs exploded in Mosul. … And when four Blackwater guys were killed on the airport road, I ordered all civilian traffic stopped. On June 7, we got hit by mortar fire in the CPA, and on June 12, the acting [Iraqi] foreign minister was assassinated. That whole period was certainly very unsettled.”
Why was the situation gettting out of control? Bremer—though he earned the ire of Batiste and other commanders for disbanding the Iraqi army as one of his first decisions—agrees with Batiste’s assessment that the troops on the ground were too few and didn’t have enough support. “Absolutely, yes. About this time, I sent Rumsfeld a top secret memo, which I sent by personal courier, to suggest we needed two divisions.” He heard nothing back. “They [the Pentagon] were trying to assert that the Iraqi army and the ICDC [Iraqi police] could take the place of the First Airborne, which I kept saying over and over was fantasy. During the earlier part of the spring uprising, in April and early May of 2004, I repeatedly said we had serious problems.” But Rumsfeld, both Bremer and Batiste say, would not listen.
Batiste said he sought to communicate his concerns that there were too few U.S. troops on the ground to play anything but whack-a-mole with the enemy. “I was extremely vocal within my chain of command,” he says, speaking to both his then-superior, Multinational Forces commander Gen. Rick Sanchez, and Centcom commander Gen. George Casey. At one point, he also brought it up with Rumsfeld himself and Deputy Defense Secretary Paul Wolfowitz. “I do remember speaking to both those guys about the frustrations of picking up a brigade element of 3,000 to 5,000 troops in contact with the enemy and moving to another location in Iraq 200 to 300 miles away to deal with an emergency. When you do that, you create an immediate vacuum. … It’s the whack-a-mole game.”
M. Scott Mahaskey/ Army Times
Pittard, for his part, is less willing to blame the lack of resources for the ultimate failure to secure Diyala—and then Iraq itself. “Sure, we could have used more. But there’s never been a commander in combat who didn’t say you could use more resources.” Despite that, he insists, the First ID—which unlike other U.S. Army units had been trained in counterinsurgency the previous year in Kosovo—was making major political and military progress in stabilizing Diyala. It was all just starting to come together that June, when Capt. Khan was killed. “We knew going in center of gravity would be people. … We had gone in having had a nine-month rehearsal in Kosovo.”
As a result, much of what the brigade—of which Khan’s 201st Forward Support Battalion was an integral part—was focused on was winning over local Iraqis, Pittard says. “Our predecessor unit might have had 10 to 15 Iraqis working at the base. By the time Capt. Khan was killed, we employed well over 1,000. That helped to stop the mortar attacks, because our Iraqis would tell their fellow Iraqis: ‘What are you doing, trying to kill us? We work there. And by the way, we’re now making money.' … The insurgency was losing force. But the learning curve for all that was the first 90 days. And he was killed in the first 90 days.”
The car bomb that killed him, adds Pittard, was hardly a total surprise—but it was the first one to hit the Warhorse base. “We were of course clearly aware of car bombs. They had been going off in Baghdad. The barriers that we set up were like those in [the Green Zone in Baghdad] , based on trying to fight car bombs. We had already killed or wounded more drivers than I wanted to do. I’m sure that’s what Capt. Khan was thinking of at that time. He was hoping he didn’t have to put a .50 caliber shell through the windshield of that car. That driver was not going to be allowed to come onto Warhorse. But Capt. Khan didn’t have the time to make that decision.”
The Iraqis working at the base were just as grief-struck as Khan’s Army comrades, says Pittard. “Among all the Iraqis there—whether Sunni, Shia, the imams, or tribal sheiks—the fact that he had been a fellow Muslim caused a real stir.”
Image Gallery: Captain Khan’s Soldiers Fight On (Click to open)
And perhaps the saddest thing of all is that the First ID, at that stage, was so close to imbuing Khan’s death with some real meaning, Pittard insists. They were fighting the good fight. Whatever the dispute over the decision to go into Iraq in the first place, the group of dedicated Americans that made up the division was on the verge of some success in making life better and more free and peaceful in Diyala Province, Pittard says.
Indeed, the radical Ansar al-Islam cell that sent in the driver who killed Khan may well have been reacting to that American success. Why does Pittard believe that? Because the Americans were listening in on the terrorists’ conversations on secure communications, he says. “They were yelling over their communications, ‘We need some help here.’ Even the price on my head went up. When we started it was $15,000. I was actually somewhat insulted by that. But by September, based on the number of moderates who had left the insurgency, they really felt we were making too much progress. The bounty on my head went up to a million dollars.”
But in the end, the American efforts were futile. A combination of bad decision-making at the top of the U.S. occupation, and the rising Sunni-Shia divide in Baghdad and Diyala, doomed those efforts, Pittard says. “The election of January 2005 was pivotal. We assured the Sunni moderates that this can work. We had the highest percentage of Sunnis voting in the entire country. The key difference was the shenanigans played by the Shia-led Iraqi provisional government in Baghdad”—to whom Bremer had handed over power the previous June. Mysteriously, the government managed to eliminate from the ballot the odds-on favorite, the Sunni governor of Diyala, enraging the Sunni population. “That was last straw for the moderate Sunnis in Diyala,” says Pittard.
The Americans in command in Baghdad and Washington then compounded things by withdrawing the combat troops, he says. “I don’t think Gen. Casey understood the magnitude of that election. Everyone thought it was fine. You remember the purple thumbs and all. But I told him, Sir, there’s a problem. He said, ‘It’s OK, they’ll work it out.' So he took out our two brigades and replaced them with [less than] one brigade—just two battalions. Hindsight’s 20-20, but we should have left the two brigades. They would have secured the province.”
But Humayun Khan, of course, would never know that.
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The hot stove season is around the corner. For those who can’t wait – and don’t have a rooting interest in this World Series – here’s a countdown of the top 20 free agents this offseason (one a day to get you into November):
12. RHP Ervin Santana
Lowdown: Cast off to Kansas City without much return, Santana enjoyed a career resurgence after posting a 5.16 ERA in 2012 with the Angels. As thin as the pitching market is, Santana’s electric stuff figures to generate all kinds of consolation-prize interest after turning in a career-low ERA and the second-best WHIP of his career.
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A correlation between digoxin plasma concentrations and systolic time intervals in hospitalized congestive heart failure patients.
Plasma digoxin level is at best an indirect measure of pharmacological response to digoxin in patients being treated for congestive heart failure. Systolic time interval (STI) measurement reflecting left ventricular function at the physiological site of action of digoxin, is both more direct and non-invasive. With a portable instrument to measure systolic time intervals, the measurement can also be convenient for hospital staff. A portable electrocardiogram (ECG) machine was modified to mimic the capabilities of a large, multichannel model. Upon satisfactory evaluation, it was employed in the collection of systolic time interval data from five hospitalized patients undergoing digoxin treatment. An attempt was made to show a relationship between STI indices and digoxin plasma concentrations. Additionally, a statistical comparison was made of change in STI (delta STI) and plasma digoxin concentration both before and after drug administrations. The change in pre-ejection period (delta PEP) values show a significant difference over the changes in total electromechanical systole (delta QS2) and the changes in left ventricular ejection time (delta LVET). In three congestive heart failure patients, the time course of the change in plasma concentration showed good correspondence with delta PEP.
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Human cytomegalovirus (HCMV) is a widespread human pathogen that infects 50-95% of the adult population worldwide. In healthy individuals, primary HCMV infection is cleared from all tissues except the submandibular salivary gland (SMG), where it can persist for several months. After this period of persistence, HCMV eventually becomes latent for the life of the host. Reactivation of latent HCMV is rare in individuals with healthy immune systems, but is far more common in individuals with compromised immune systems. These include people with congenital or acquired immune deficiencies, organ transplant recipients, or any patients taking immunosuppressive drugs. In immune compromised individuals, primary HCMV infection or reactivation of latent infection can cause serious and even fatal health complications. Even when it is not fatal, reactivation of HCMV in the salivary gland can cause several oral pathologies that greatly limit quality of life, such as increased dental cavities, periodontal infections, and chronic dry mouth. HCMV can also be passed from mother to fetus during pregnancy, and the resulting neonatal HCMV infection can result in severe birth defects. Due to the strict species tropism of the CMV family members, HCMV cannot be studied in an animal model. However, murine cytomegalovirus (MCMV) is very similar to HCMV in terms of genetics, structure, and mechanisms of infection. Thus, MCMV is an appropriate animal model to study HCMV infection. Like HCMV, MCMV persists in the SMG of the host before becoming latent. Also like in humans, murine natural killer (NK) cells are crucial for the early containment of MCMV. NK cells are innate immune cells that respond to MCMV infection by directly killing infected cells, and by releasing pro-inflammatory cytokines such as IFN-? that stimulate further immune responses. Recently, the Brossay laboratory has discovered that the NK cells of the SMG are hyporesponsive to MCMV infection: they are activated upon infection, but their IFN-? response is diminished compared to NK cells of the spleen, liver, and blood. The SMG is a delicate tissue, vulnerable to irreversible damage by either unchecked viral replication or an extensive inflammatory immune response. My hypothesis is that the diminished effector response of the SMG NK cells allows MCMV to persist in this organ at levels too low to cause direct damage, while at the same time preserving the SMG tissues from extensive inflammatory damage. In Aim 1, I will determine whether SMG NK cells control MCMV replication to low levels. In Aim 2, I will determine whether SMG NK cells can be induced to respond to MCMV infection, or whether their hyporesponsive phenotype is intrinsic and irreversible. The research described in this proposal will elucidate the factors allowing MCMV to persist in the SMG of the mammalian host. This knowledge could eventually lead to the development of more effective therapies against this ubiquitous human pathogen.
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1. Field of the Invention
The present invention relates to an organic electroluminescent element (hereinafter referred to as an organic EL element) capable of inducing luminescence by the recombination of an electron and a hole when they are injected into an organic compound layer of the EL element.
2. Discussion of Background
In recent years, in line with the trend toward diversification of information processing equipment and space-saving for location of such equipment, there is an increasing demand for a plans display element which can be operated with a low electric power and occupies a small space as compared with the cathode ray tube (CRT) display. A liquid crystal has been already proposed as such a plane display element, but much attention has been paid to an organic EL element of a self-emitting type because the EL element can indicate the information clearly and can be driven with a direct-current low voltage system.
A luminescent layer of the organic EL element is of a single-layered type, or a laminated type which comprises a carrier transporting layer and a luminescent layer. The luminance of the laminated luminescent layer is higher than that of the single-layered one. The above-mentioned carrier transporting layer for use in the laminated luminescent layer serves to transport a hole or an electron.
A variety of triphenylamine compounds, which have been developed for an organic electrophotographic photoconductor, are proved to be usable as materials for transporting the hole (hereinafter referred to as hole transporting materials) Some of them are found to have relatively high thermal stability in the amorphous condition.
On the other hand, some of oxadiazole compounds, triazole compounds and peryleneimide compounds are know as electron-transporting materials, but there are few compounds that have no absorption in the visible region. Further, the electron-transporting materials with high thermal stability are very rare among the above-mentioned compounds.
From the viewpoint of the layer structure of the EL element, the organic EL element comprising an electron-transporting layer has the advantages that various hole transporting materials can be selected for a luminescent layer, an exciton can be trapped in the small luminescent layer, and the deactivation of the exciton caused by the mutual action between the exciton and a cathode can be prevented. Thus, the organic EL element with a high luminance can be obtained using a variety of luminescent materials with different fluorescent colors. However, because the conventional electron-transporting materials do not have high stability, the durability of the conventional EL element comprising the electron-transporting layer is regarded as poor.
For instance, there is known an organic EL element using an oxadiazole compound as a luminescent material and electron-transporting material, as stated in "Nippon Kagaku Kaishi 1991, (11), pages 1540-1548". However, the problem of the film stability of the above-mentioned oxadiazole compound for use in the EL element remains unsolved, so that there is no organic EL element that shows high luminance and high reliability at the present stage.
It is considered that the poor film stability of the oxadiazole compound results from the crystallization thereof Some of the conventional oxadiazole compounds cannot be made into an amorphous film, and others can be formed into an amorphous film, but the amorphous film thus formed induces crystallization during a long period of storage. Therefore, there is an increasing demand for an organic EL element employing an electron-transporting material which has good film-forming properties and does not easily induce crystallization.
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Into the mainstream
Farm-raised salmon becoming a bigger fish story
As habitats were threatened and supplies of wild fish dwindled, investment in salmon farming began to rise in the 1980s. Then, as streams were protected and wild fish began a comeback, farming caught on worldwide in a big way. By 1999, more salmon almost 200,000 tons of it was farmed than caught in the wild.
Salmon is the fastest-growing segment of seafood, climbing 23 percent in 1999 alone to land in third place on America's favorites list, behind tuna and shrimp. That rapid rise, from fifth place throughout the early '90s, is largely attributable to farming.
Menus and seafood cases are brimming year-round with the telltale carrot-colored farmed fillets, easily distinguishable from the more flavorful wild pink, red and sometimes white wild Pacific varieties whose seasons stretch from March to November, including the highly anticipated Copper River kings due about May 20.
But here's the turn that's as strange as a salmon's odds-defying swim against the current, sometimes for 1,000 miles, to get to its birthplace to spawn: Concern has spread to farming.
Environmentalists worry that farming can threaten water in some systems when uneaten feed, wastes and antibiotics pile up. The hardy fish also can compete unfairly with and spread diseases to wild fish when they escape from farms.
And consumers worry about experiments with genetically modified fish and feed additives. All this is leading to labels that tell how the fish are raised and what exactly is, or isn't, fed to them.
Another twist: Farmed fish has become preferable for many chefs and diners because of its convenience, availability and steady price even though discerning palates prefer the taste of wild fish.
"I think when you add chemicals to any processed fish it changes the taste of it,'' says Joel Knox, founder and president of Inland Seafood in Atlanta.
One of the country's top distributors of farm-raised salmon, he claims to "hate every pound of it.''
To appreciate the great taste of salmon, wild or farmed, Knox offers these tips. The main things to remember: If there's skin on the fillets, cook with the meat side toward the direct heat first; don't use too many competing or strong flavors in sauces; and do not overcook.
Salmon's high fat content makes it a good choice for any quick-cooking method grilling, broiling, baking, pan-searing and poaching.
What's simpler than this way to get the best of wild salmon: Cook fillets skin side down for 7 to 8 minutes in a 475-degree oven, then sprinkle with a little sea salt.
We've caught some recipes, though, that can enhance wild or farmed salmon when you want to do a little more.
Removing the Skin
If the salmon fillets are not skinned, remove the skin. Place the fillet with the thickest part toward you and insert the flat blade of a thin knife between the skin and the flesh. Gently move the flat blade along the skin, pushing the flesh away from you. When you're about halfway through, flip the fillet over and continue to use the flat of the knife to pull the skin away from the flesh. Don't worry if the thin part tears a little; you can always trim this off and save it for scrambled eggs in the morning.
Removing the Pin Bones
Often, farm-raised salmon fillets are processed with pin bones removed. If not, drape the fillet over a curved surface, such as an upturned bowl. The bones will pop up and can be removed easily with a pair of tweezers or your fingertips.
Salmon With Pinot Noir and Thyme
Makes 4 servings
4 (4- or 5-ounce) salmon fillets with skin, about 3/4 inch thick
3/4 teaspoon coarse salt, divided
3/4 teaspoon freshly ground pepper, divided
1 tablespoon olive oil
1 cup pinot noir
4 fresh thyme sprigs, plus more for garnish
1 tablespoon unsalted butter
Sprinkle salmon fillets with 1/2 teaspoon each of the salt and pepper.
In a large frying pan over high heat, warm the olive oil. Add the salmon, meat side down, and cook until browned, 2 to 3 minutes. Turn the fish and cook skin side until browned and crisp, about 3 to 4 minutes. The salmon will be slightly rare at the center. Continue cooking for another minute or so until done to your taste. Transfer the fish to a warmed platter and cover to keep warm.
Add the wine and the thyme sprigs to the pan and bring to a boil over high heat. Cook until reduced by half, about 5 minutes. Remove the thyme sprigs and stir in the remaining 1/4 teaspoon each salt and pepper and the butter.
Pour the sauce over the salmon. Garnish with additional thyme sprigs and serve immediately.
Adapted from "The Pacific Northwest," Williams-Sonoma New American Cooking series (TimeLife, $22.95)
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Farrah Abraham on Learning She Was Pregnant: "I Ended Up Turning a Little Crazy"
Farrah Abraham's time on Teen Mom is wrapping up, and she's ready to impart some final words of wisdom, y'all. As we know, Farrah got pregnant at the tender age of 16, and she thinks her pregnancy resulted from boredom. "I think some of the smaller cities or towns where we're from, there's not that much to do there, and I think also that's why most teenagers have sex, is 'cause we're bored," she tells The Mom's View.Farrah was shocked when she found out about her pregnancy –– especially because she was on birth control at the time. "I was first crying, and then I ended up turning a little crazy and started yelling at the woman [her gynecologist], just because I was taking birth control," Farrah says. "I was trying to avoid getting pregnant so, so strongly."Farrah's baby daddy, Derek, died in a car accident when she was eight months pregnant, but Farrah's determined to make him part of her daughter Sophia's life. "Every year, we go to Derek's grave site and also we take pictures, we record it, and I have a whole baby box with Sophia with letters and pictures telling how and why things happened, and why I'm here today alone," Farrah says.Awww, Farrah! There's no doubt that this gal is a great mom, and she's confident that Baby Sophia will be proud of her at the end of the day. "She'll be proud of me and she'll make the right choices, and we can have the best mother and daughter relationship," Farrah says.Check out the rest of Farrah's interview with The Mom's View here!Source:Teen Mom's View
Behind the Scenes of Farrah Abraham and Sophia's Book Cover Shoot (PHOTOS)
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What is Nearby 57106:
Need Directions?
Price reduced on this one owner custom built ranch home!! With over 3,100 finished square feet, this home offers so many great features which include trayed and vaulted ceilings, main floor laundry, huge master bedroom, stainless steel appliances, solid wood six panel doors, beautiful tile flooring, formal dining, open main floor living, huge 3 car finished garage, great landscaping, all this in a wonderful southwest location, and only a block from Galway Park!! The lower level is all finished with 2 large bedrooms, an office/storage, a full bath, and a fantastic large open family room with gas fireplace. This home is in immaculate condition, and is ready for the next owner!
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Our data are all contained within the paper and Supporting Information files.
Introduction {#sec001}
============
Atherosclerosis is a chronic inflammatory disease in which monocyte recruitment plays an important role in disease initiation, plaque progression, and clinical events such as chronic ischemia, plaque rupture and thrombosis \[[@pone.0173224.ref001]\]. Chemokines are chemoattractant cytokines that direct the migration of specific leukocytes to sites of inflammation or infection and are increasingly implicated in atherosclerosis \[[@pone.0173224.ref002]\]. Chemokines are small 8- to 11-kDa proteins that are divided into four structural subfamilies (C, CC, CXC and CX~3~C) based on the placement and number of cysteine residue at the N terminal \[[@pone.0173224.ref003]--[@pone.0173224.ref005]\]. The ability of chemokines to bind to multiple receptors and also for receptors to bind to multiple chemokines indicate some redundancy in chemokine signalling. However, chemokine/chemokine receptors interactions are specific within each chemokine group \[[@pone.0173224.ref006]\].
To date, the CC-chemokine family has been strongly implicated in atherosclerosis with a host of CC-chemokines including MCP-1 (CCL2), RANTES (CCL5) and MIP-1α (CCL3) detected in human atherosclerotic lesions \[[@pone.0173224.ref007]\]. The importance of CC-chemokines in atherosclerosis has been reported in studies using transgenic or knockout murine models. For example, targeted deletions of the CCL2 gene or its receptor (CCR2) resulted in reduced atherosclerotic lesion formation \[[@pone.0173224.ref008]\]. Furthermore, studies using an amino-terminal deletion mutant of CCL2 \[[@pone.0173224.ref009]\] and a modified CCL5 peptide (Met-RANTES/CCL5) \[[@pone.0173224.ref010]\] found reductions in atherosclerotic lesion development in apoE^-/-^ mice. Broad-spectrum inhibition of the CC-chemokine class with the vaccinia viral protein '35K' was also found to inhibit macrophage recruitment, lipid deposition and atherosclerotic plaque size \[[@pone.0173224.ref011]\]. More recently the importance of CX~3~CL1 and its receptor (CX~3~CR1) in atherosclerosis has emerged. CX~3~CL1 has been detected in macrophages, foam cells and medial smooth muscle cells (SMC) of atherosclerotic human coronary arteries \[[@pone.0173224.ref012]\]. Furthermore, apoE^-/-^/CX~3~CR1^-/-^ mice have significantly smaller atherosclerotic lesions \[[@pone.0173224.ref013], [@pone.0173224.ref014]\]. These studies provide concrete evidence that chemokines from the CC and the CX~3~C subclasses play a key role in atherosclerosis. This raises the possibility that broad-spectrum chemokine inhibition across the CC and CX~3~C chemokine subfamilies would be very effective in preventing atherosclerotic disease progression. M3 is a 44-kDa protein encoded by the murine gamma herpesvirus 68 (MHV-68) that serves to evade the host immune response by binding and inactivating chemokines \[[@pone.0173224.ref015]\]. Unlike CC-chemokine inhibitor 35K, the M3 protein is capable of sequestering members from all chemokine classes, specifically key chemokines involved in atherosclerosis progression including CCL2, CCL5 and CX~3~CL1, while displaying selectivity in its binding to individual members \[[@pone.0173224.ref016]\]. M3 may have improved efficacy over single chemokine inhibition or the targeting of a single chemokine class due to redundancies in chemokine signaling.
We therefore sought to evaluate the effect of broad-spectrum chemokine inhibition using the M3 protein on atherosclerosis in the apolipoprotein (apo) E^-/-^ mouse model. We found that M3 suppressed chemokine activity *in vitro* and *in vivo*. In two different models of atherosclerosis development in which the rate of lesion progression was varied by diet (high-fat or chow), overexpression of M3 in apoE^-/-^ mice caused reductions in plaque size and macrophage content. These changes differed between the two models indicating that the effects of M3 were dependent on the rate of plaque progression. Chemokine blockade with M3 may be a promising strategy for the treatment of atherosclerosis, although some consideration needs to be given to the aggressiveness of the onset.
Materials and methods {#sec002}
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Generation of M3 adenovirus {#sec003}
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A recombinant adenovirus M3 (AdM3) was generated as described previously \[[@pone.0173224.ref017]\]. Briefly, the M3 open reading frame was cloned into a pshuttle CMV (ps) plasmid along with a myc-His epitope tag and homologously recombined with the AdEasy viral plasmid to create 'AdEasyM3'. Linearised AdEasyM3 was then transfected into Ad293 cells (293AD cell line, AD-100, Cell Biolabs Inc, USA) to create recombinant AdM3 virus. Purified adenovirus particles (vp) were isolated from the cell pellet of AdM3 infected Ad293 cells by CsCl gradient ultracentrifugation and desalted using CL-4B sepharose columns (Sigma-Aldrich, USA). Purified virus was diluted 1:1 with a mix of 80% fetal bovine serum (FBS) and 20% glycerol and aliquots of 1x10^11^ vp were frozen at -80°C for *in vivo* experimentation. A control recombinant adenovirus encoding enhanced green fluorescent protein (AdGFP) was prepared as described above. Anti-c-Myc tagged agarose-conjugated beads (Sigma-Aldrich, USA) were used to isolate soluble c-Myc-tagged M3 from AdM3 viral media. The media was run through anti-c-Myc agarose in a column and eluted with 0.1 M ammonium hydroxide into 1 N acetic acid to neutralize and snap frozen at -80°C.
Isolation of human monocytes {#sec004}
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White-cell concentrates were obtained from the peripheral blood of healthy human volunteers (Red Cross Blood Bank), and monocytes were removed within 24 h of collection by density gradient separation of the white blood cells on Lymphoprep (Axis-Shield, UK) followed by counterflow centrifugation elutriation using a Beckman Avanti J-26 XPI centrifuge equipped with a JE-5.0 elutriation rotor and a 4.0 mL elutriation chamber (Beckman Instruments Inc., USA) at 21°C, as described previously \[[@pone.0173224.ref018]\]. Collected fractions were examined by a Cytospin system (Shandon, USA) and Wrights' stain (DiffQuik; Laboratory-Aids, Australia). Monocyte purity of \>90% and viability of \>95% by Trypan Blue exclusion were confirmed by light microscopy, and the monocytes were resuspended in serum-free RPMI and used immediately for chemotaxis studies.
*In vitro* testing of chemokine activity using chemotaxis assays {#sec005}
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CCR2-, CCR5- and CX~3~CR1-directed cell migration was assessed in 8 μm pore size transwell membranes (ChemoTX, 6.0 mm diameter, 8 μm pore size, Receptor Technologies, UK). 293T cells were co-transfected (Fugene^®^6, Roche Diagnostics, Germany) with plasmids encoding CCR2, CCR5 or CX~3~CR1 plus GFP to facilitate visualization. Transfected cells (1x10^6^/membrane) were harvested and allowed to migrate overnight toward purified recombinant CCL2, CCL5 or CX~3~CL1 (Research Diagnostics Inc, USA) in the presence of increasing concentrations of M3 protein (0--500 ng/mL) placed in the lower chamber. Migrated cells were fixed and quantified by computer analysis of GFP fluorescence (green cell pixel count) in microscope images using Image-Pro^®^ software (v9.0.4, Media Cybernetics, USA). Each experimental sample was analyzed in triplicate, and 3 separate images quantified per membrane. Cell migration of human elutriated monocytes in response to recombinant chemokines was also tested *in vitro* using the Boyden chamber method as described earlier. To the bottom chambers, purified M3 protein (100ng/ mL) was added to chemotaxis media with recombinant key inflammatory chemokines CCL2, CCL5, CX~3~CL1 as well as CXCL12, a chemoattractant that is not inhibited by M3 protein. To the upper chamber, 5x10^4^ cells/100 μL of Calcein AM (5nM)-labelled monocyte suspension was placed in each well and allowed to migrate towards the lower chamber for 1 h. Migrated cells were fixed in mounting medium with DAPI to counterstain for nuclei, and quantified by computer analysis of GFP fluorescence (green cell pixel count) relative to DAPI fluorescence (blue cell pixel count) in microscope images using Image-Pro^®^ software (v9.0.4, Media Cybernetics, USA). Each experimental sample was analyzed in triplicate, and 3 separate images quantified per membrane.
Animals and gene transfer {#sec006}
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All experimental procedures and protocols were conducted with approval from the Sydney Local Health District Animal Welfare Committee (Protocol Number: 2011/018) and conformed to the Guide for the Care and Use of Laboratory Animals (United States National Institute of Health). Approval was granted for the *in vivo* use of adenoviruses from the Royal Prince Alfred Hospital Institutional Biosafety Committee (IBC Code: 14--031). All procedures were performed under methoxyflurane anesthesia, and all efforts were made to minimize suffering. ApoE^-/-^ mice were used for this study to assess the effectiveness of M3 protein in two models that were subjected to different rates in the development of atherosclerosis by varying the diet. Model 1---a high fat diet (HFD)-fed model for more aggressive, rapid promotion of atherosclerosis over a 6-week period ('rapid promotion') and; Model 2---a chow-fed model for less aggressive progression of atherosclerosis over a 12-week period ('slow progression'). For the rapid promotion model, 4-week-old mice were fed a HFD (22% milk fat, 0.15% cholesterol; SF00-219, Specialty Feeds, Australia) for 6 weeks in total (*n* = 10--12/group). Two weeks after commencement of the HFD AdM3 or AdGFP (1x10^11^ vp) were administered by tail-vein injection and mice continued on the HFD for a further 4 weeks. For the slow progression model, AdM3 or AdGFP (1x10^11^ vp) were administered intravenously to 8-week-old apoE^-/-^ mice. Mice were fed on a normal chow diet (Specialty Feeds, Australia), for 12 weeks post-adenoviral injection (*n* = 10--12/group). The stage of atherosclerosis at which M3 gene transfer was initiated and ended was estimated to be similar between the two models.
Following sacrifice, hearts were fixed overnight in 4% paraformaldehyde in phosphate buffered saline (PBS) then transferred to 70% ethanol the following day before embedding in paraffin. Descending thoracic aortas were thoroughly cleared of surrounding fat and connective tissue and stored in 70% ethanol at 4°C. Plasma was isolated from whole blood by centrifugation, aliquoted and frozen at -80°C. The livers, kidneys, spleens, lungs and aortic arches were also excised and immediately frozen at -80°C.
Plasma lipids {#sec007}
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Total cholesterol concentration was determined enzymatically on mouse plasma using the Cholesterol E kit (Wako Diagnostics, USA). HDL cholesterol concentration was determined by enzymatic assay following polyethylene glycol precipitation of apolipoprotein B containing lipoproteins. LDL levels were determined by subtracting total HDL from total cholesterol \[[@pone.0173224.ref019]\].
Viral DNA expression {#sec008}
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Genomic DNA was extracted from liver, spleen, kidney and lung using the Wizard^®^ Genomic Purification Kit (Promega Corporation, USA). All real-time PCRs were performed on 500 ng of genomic DNA using iQ SYBR green fluorophore (BioRad, Australia) with primers used to detect M3 (F `5’-GACCTAGCTGGCCTGGATTC-3’`, R `5’-TGTACTGTTCCTCCAAC-3’`), GAPDH (F `5’-GGCATCACTGCAACTCAGAA-3’`, R `5’-TTCAGCTCTGGGATGACCTT-3’`) and 36B4 (F `5’-CAACGGCAGCATTTATAACCC-3’`, R `5’-CCCATTGATGATGGAGTGTGG-3’`). Relative M3 expression was normalized to reference genes murine GAPDH or 36B4 using the ^ΔΔ^CT method.
Circulating M3 protein levels {#sec009}
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Circulating M3 protein was isolated from 250 μL mouse plasma using the anti-c-myc immunoprecipation kit (IP0020, Sigma-Aldrich, USA) and probed for M3 protein with a rabbit anti-M3 antibody (1:2000, kind gift from Professor Pedro Simas, Portugal), followed by a goat monoclonal anti-rabbit secondary antibody conjugated with horse-radish peroxidase (HRP) at 1:1000 (sc-2030, Santa Cruz Biotechnology, USA). Additional controls including media from AdM3 (positive) plasma from mice injected with AdGFP (negative plasma control) were run in parallel.
Detection of GFP in liver sections {#sec010}
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Fresh frozen livers were sectioned at 5 μm, and stained with Vector Mount for fluorescence plus DAPI (Vectorlabs, USA). Presence of GFP fluorescence in microscope images were taken using Image-Pro^®^ software.
Aortic arch phospho p65 expression {#sec011}
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Phosphorylated p65 levels were determined by Western immunoblotting in homogenized aortic arch tissue (30 μg). Membranes were probed with anti-phospho p65 antibody (1:1000, ab86299, Abcam, UK) followed by a goat monoclonal anti-rabbit secondary antibody conjugated with HRP at 1:1000 (sc-2030, Santa Cruz Biotechnology, USA). The membranes were then stripped and re-probed with anti-p65 (total) antibody (1:1000, ab32536, Abcam, UK) followed by the same secondary antibody as above.
*Ex vivo* testing of chemokine activity using chemotaxis assays {#sec012}
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Chemotaxis of human elutriated monocytes in response to mouse plasma was tested *ex vivo* using the Boyden chamber method. Monocytes were fluorescently labeled with calcein AM (5 nM, Invitrogen, USA) then re-suspended at a cell density of 5x10^5^ cells/mL in chemotaxis media (25 mM HEPES, 0.1% bovine serum albumin in RPMI media). To the bottom chambers, 600 μL of chemotaxis media and 2 μL of mouse plasma were added. To the upper chamber, 5x10^4^ cells/100 μL of monocyte suspension was placed in each well and allowed to migrate towards the lower chamber for 1 h. The number of migrated monocytes was determined as described earlier.
Histology and immunohistochemistry {#sec013}
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Serial transverse sections (5 μm) were taken through the aortic sinus. To quantify lesion area, 3 sections were chosen that were proximal, middle and distal to the top of the aortic sinus and stained with Milligan's trichrome to highlight the plaque for determination of total plaque area. Collagen content was measured (green of trichrome stain) and extracellular lipid or cholesterol clefts by acellular regions of the plaque that remain following the dissolution of cholesterol crystals from degraded macrophage foam cells during the paraffin embedding process in aortic sinus lesions \[[@pone.0173224.ref011]\]. For macrophage immunostaining, sections were incubated overnight at 4°C with purified rat anti-mouse monoclonal Mac 3 antibody at a 1:100 dilution (550292, BD Biosciences, USA) and then incubated for 30 minutes with biotinylated anti-rat IgG affinity-purified antibody at 1:200 (BA-4000, Vector Laboratories, USA). Immune-mediated inflammatory cells within the plaque were assessed by purified rat anti-mouse CD8a (550281, BD Biosciences, USA) and biotin mouse anti-mouse CD22.2 (553382, BD Biosciences, USA) antibodies at a 1:20 dilution and then incubated similarly with biotinylated anti-rat IgG affinity-purified antibody at 1:200 (BA-4000, Vector Laboratories, USA) to determine T and B cell content respectively. Sections were stained for SMC α-actin^+^ cells using a mouse monoclonal anti-α-actin antibody conjugated to alkaline phosphatase at 1:100 (A5691, Sigma-Aldrich, USA) and visualized with Vector Red alkaline phosphatase substrate (Vector Laboratories, USA). Sections (*n* = 3/mouse) were quantified from digitized microscopic images using Image-Pro^®^ software.
Circulating neutrophil and monocyte numbers {#sec014}
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To investigate the effect of systemically delivered AdM3 on circulating inflammatory cells, blood from the tail vein was collected following adenoviral delivery every week up to the fourth week in the 'rapid promotion' model and every month up to the twelfth week in the 'slow progression' model. Red blood cell lysis was performed on 200 μL of blood using cell lysis buffer (BD Biosciences, USA) before being resuspended in PBS buffer. Cells were incubated with a Zombie Aqua viability stain at a 1:100 dilution (BV510 excitation) (Biolegend, USA) to label any unviable/dead cells for 30 minutes. Cells were washed and Fc receptors blocked with an Fc block (BD Biosciences, USA) for 20 minutes. Cells were washed again and then stained with a cocktail of antibodies against CD45 (1:100 rat anti-mouse CD45-APC-Cy7, BD Biosciences), Ly6-C/G (1:100 rat anti-mouse Ly6-C/G-PerCP-Cy5.5, BD Biosciences) and CD115 (1:100 rat anti-mouse CD115-APC eBioscience) for 20 minutes. Cells were washed twice and resuspended in 300 μL of HBSS buffer. Monocytes were identified as CD45^hi^CD115^hi^ and neutrophils as CD45^hi^CD115^lo^Ly6-C/G^hi^. Monocytes were further subdivided into Ly6-C/G^hi^ to distinguish between inflammatory and patrolling monocytes. The gating strategy is presented in [S2 Fig](#pone.0173224.s002){ref-type="supplementary-material"}. All wash steps were performed with HBSS buffer (HBSS (Sigma-Aldrich) + 0.1% BSA w/v (Sigma-Aldrich) + 5mM EDTA (Sigma-Aldrich)). All flow cytometry experiments were performed on a FACSverse (BD Biosciences, USA).
RNA expression {#sec015}
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Total RNA was isolated from aortic arch samples using TRI reagent. 250 ng total RNA was reverse transcribed using the iScript cDNA synthesis kit before amplification using iQ SYBR in a Cfx384 thermocycler (BioRad, Australia). The following mouse primers were used: SMC α-actin (F `5’-TGTACCAGGCATTGCTGAC-3’`, R `5’-GAGGCGCTGATCCACAAAAC-3’`), CD68 (F `5’-GGGGCTCTTGGGAACTACAC-3’`, R `5’-GTACCGTCACAACCTCCCTG-3’`), p65 (F `5’-AGTATCCATAGCTTCCAGAACC-3’`, R `5’-ACTGCATTCAAGTCATAGTCC-3’`). Relative gene expression was normalized to GAPDH (reference gene) using the ^ΔΔ^C~t~ method.
ELISA immunoassays {#sec016}
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Mouse CCL2 and CCL5 protein levels were determined in mouse plasma (75 μL for each) and mouse CCL2, CCL5 and CX~3~CL1 protein levels were determined in homogenized liver tissue (500 μg for CCL2, 10 μg for CCL5 and 1000 μg for CX~3~CL1) in lysis buffer (80 mM Tris HCl, 10 mM NaCl, 50 mM NaF, 5 mM Na~4~P~2~O~7~, 15 mM Triton-X 100) by ELISA (R&D Systems, USA). Mouse plasma diluted 1:200 was also used to determine pro-inflammatory cytokine mouse Serum Amyloid A (SAA) protein levels (10 μL for rapid promotion model and 75 μL for slow progression model) as well as undiluted mouse plasma was used to measure TNF-alpha (Tumor necrosis factor alpha) protein concentration (200 μL in both models) by ELISA (R&D Systems, USA).
Oil Red O staining {#sec017}
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Oil Red O (Sigma-Aldrich, USA) was utilized to identify atherosclerotic lesions in the mouse thoracic aorta. Descending thoracic aortas were stained with Oil Red O solution (0.25% Oil Red O w/v, 62.5% isopropanol v/v and 37.5% ddH~2~0 v/v) and stored in 4% paraformaldehyde until they were pinned out and imaged. Using a stereological microscope, aortas were cut open longitudinally and pinned *en face* on paraffin wax with 0.1mm insect pins (Fine Science Tools, Canada) and imaged. Lesion area was determined as a percentage of total aortic area from digitized microscopic images using Image-Pro^®^ software.
Statistical analysis {#sec018}
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All data are expressed as mean±SEM. All data were compared using t-test of unpaired samples assuming equal variances or a One-way ANOVA with Bonferonni's *post hoc* test. Significance was set at *p*\<0.05.
Results {#sec019}
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M3 inhibits chemokine activity *in vitro* {#sec020}
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To investigate the effects of purified M3 protein on chemokine activity *in vitro*, CCR2-, CCR5- and CX~3~CR1-directed cell migration towards their respective chemokines CCL2, CCL5 and CX~3~CL1 in the presence of M3 protein (10--500 ng/mL) was assessed. Cell migration towards purified CCL2 ([Fig 1A](#pone.0173224.g001){ref-type="fig"}) and CCL5 ([Fig 1B](#pone.0173224.g001){ref-type="fig"}) was significantly reduced with purified M3 protein at concentrations of 50, 100 and 500 ng/ mL (*p*\<0.05 for all). Furthermore, M3 caused a marked decrease in CX~3~CR1-directed cell migration across all concentrations (*p*\<0.05 for all, [Fig 1C](#pone.0173224.g001){ref-type="fig"}). Next, we tested the effect of M3 protein on the migration of human elutriated monocytes towards recombinant chemokines CCL2, CCL5, CX~3~CL1 and CXCL12. Monocyte migration towards purified CCL2, CCL5 and CX~3~CL1 was significantly reduced in the presence of M3 protein (*p*\<0.05 for all, [Fig 1D](#pone.0173224.g001){ref-type="fig"}). M3 did not inhibit, however, the migration of monocytes towards CXCL12 ([Fig 1D](#pone.0173224.g001){ref-type="fig"}).
{#pone.0173224.g001}
Confirmation of gene transfer and inhibition of plasma chemokine activity by M3 {#sec021}
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Circulating M3 protein was detected in AdM3 mouse plasma ([Fig 2A](#pone.0173224.g002){ref-type="fig"}) at 1, 4, 6 and 8 weeks that was not present in the plasma of mice infused with AdGFP. The M3 plasma levels were high 1 week post-AdM3 infusion. This then dropped but was still detectable 4 weeks post-AdM3 and then appeared to remain at a similar level for \~4 more weeks. We also detected the presence of M3 protein in the media of Ad293 cells infected with (positive blot control). RTPCR measurements of viral M3 expression revealed that the gene transfer occurred predominately in the liver and not in other organs such as the spleen, kidney or lung ([S1 Fig](#pone.0173224.s001){ref-type="supplementary-material"}). Gene transfer was also confirmed for the AdGFP control as green fluorescent cells were detected in liver sections of AdGFP infused mice. To determine the effect of M3 expression on plasma chemokine activity *ex vivo*, a migration assay was performed in which human elutriated monocytes were allowed to migrate towards plasma from either AdM3- or AdGFP-infused mice. Monocyte migration was significantly reduced towards AdM3 mouse plasma in the 'rapid promotion' model (29.6%, *p*\<0.05, [Fig 2B](#pone.0173224.g002){ref-type="fig"}). A non-significant decline in monocyte migration towards AdM3 mouse plasma was detected in the 'slow progression' model.
{ref-type="sec"}" for details. Successful gene transfer and expression following adenoviral delivery was determined by Western immunoblotting. **A.** Culture media from Ad293 cells infected with AdM3 as well as plasma from mice infused with AdGFP and AdM3 were assessed from 4 to 12 weeks post adenoviral delivery following immunoprecipitation with anti-c-myc agarose beads. **B.** Chemokine activity was measured using the Boyden chamber migration assay. Calcein-AM labelled monocytes were allowed to migrate towards plasma from mice infused with AdGFP or AdM3 (*n* = 10--12/group). Data are mean±SEM. \**p*\<0.05, \*\*\*\**p*\<0.0001.](pone.0173224.g002){#pone.0173224.g002}
M3 reduces atherosclerotic plaque size when the rate of plaque development is less rapid {#sec022}
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There were no effects on body weight or plasma lipid levels between groups ([S1 Table](#pone.0173224.s005){ref-type="supplementary-material"}). In the 'rapid promotion' model there were no changes in atherosclerotic plaque area between mice infused with AdM3 and AdGFP ([Fig 3A](#pone.0173224.g003){ref-type="fig"}). However, in the slow progression model, AdM3 infused mice had significantly smaller atherosclerotic lesions (45.3%, *p*\<0.05), compared to AdGFP control mice ([Fig 3B](#pone.0173224.g003){ref-type="fig"}).
{ref-type="sec"}" for details. Images are representative pictures of aortic arches (upper panels) and Trichrome stained aortic sinus sections (lower panels). Quantification of total plaque area (μm^2^) was determined from 3 sections/mouse, spanning the entire aortic sinus for **A.** the 'rapid promotion' model and **B.** the 'slow progression' model. Data are mean±SEM. \**p*\<0.05.](pone.0173224.g003){#pone.0173224.g003}
M3 reduces plaque macrophage content and p65 activation when the rate of plaque development is more rapid {#sec023}
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Histological analysis showed that macrophage content in the aortic sinus of AdM3 mice was strikingly reduced (46.8%, *p*\<0.01) in the 'rapid promotion' model ([Fig 4A](#pone.0173224.g004){ref-type="fig"}). In contrast, there was no change in plaque macrophage content in the 'slow progression' model ([Fig 4B](#pone.0173224.g004){ref-type="fig"}).
{ref-type="sec"}" for details. Upper panels are representative images of Mac-3^+^ macrophages (brown staining) in aortic sinus sections. Quantification of macrophage staining within plaques (μm^2^) for **A.** the 'rapid promotion' model and **B.** the 'slow progression' model. Phosphorylated p65 levels were measured in aortic arch samples for **C.** the 'rapid promotion' model and **D.** the 'slow progression' model. p65 mRNA levels were determined in aortic arch samples for **E.** the 'rapid promotion' model and **F.** the 'slow progression' model. Data expressed as mean±SEM. \**p*\<0.05, \*\**p*\<0.01, \*\*\*\**p*\<0.0001.](pone.0173224.g004){#pone.0173224.g004}
Consistent with the reduction in macrophage content, there was a significant reduction in levels of phosphorylated (phospho) p65, the active subunit of NF-κB, in the aortic arches of AdM3 mice (37.3%, *p*\<0.0001) in the 'rapid promotion' model ([Fig 4C](#pone.0173224.g004){ref-type="fig"}). There were, however, no changes in phospho p65 in the 'slow progression' model ([Fig 4D](#pone.0173224.g004){ref-type="fig"}). These findings were further supported by a decrease in the aortic arch mRNA levels of p65 of AdM3 mice (27.2%, *p*\<0.05) in the 'rapid promotion' model ([Fig 4E](#pone.0173224.g004){ref-type="fig"}). Conversely, in the 'slow progression' model there was an increase in p65 (33%, *p*\<0.05) in the aortas of AdM3-infused mice ([Fig 4F](#pone.0173224.g004){ref-type="fig"}). There were no changes in plaque T or B cell content between groups. Neither were there changes in plaque collagen or extracellular lipid content ([S3 Fig](#pone.0173224.s003){ref-type="supplementary-material"}).
M3 prevents a decrease in inflammatory monocytes in the rapid promotion model four weeks' post-gene transfer {#sec024}
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To assess the effect of M3 on circulating leukocytes, neutrophils and monocytes were measured longitudinally in the whole blood of both models ([Fig 5A and 5B](#pone.0173224.g005){ref-type="fig"}). At all times points after adenoviral delivery there were no significant differences in the total number of circulating neutrophils or monocytes in either model. However, 4 weeks' post-injection AdM3 treated mice had a 57.8% (*p*\<0.05) increase in inflammatory monocytes in the rapid promotion model, compared to AdGFP control mice. This suggests that AdM3 is preventing a decrease in inflammatory monocytes.
{ref-type="sec"}" for details. Circulating neutrophils (CD45^hi^CD115^lo^Ly6-C/G^hi^) and monocytes (CD45^hi^CD115^hi^) were measured for Ly6-C/G by flow cytometry every week up to 4 weeks in **A.** the 'rapid promotion' model and every month up to 12 weeks in **B.** the 'slow progression' model.](pone.0173224.g005){#pone.0173224.g005}
M3 increases plaque SMC content in the 'slow progression' atherosclerosis model {#sec025}
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No significant differences were seen in plaque SMC content in the 'rapid promotion' model ([Fig 6A](#pone.0173224.g006){ref-type="fig"}). Consistent with this, there were no changes in SMC α-actin mRNA expression in the aortas of AdM3 infused mice ([Fig 6B](#pone.0173224.g006){ref-type="fig"}). However, in the 'slow progression' model there was a trend for an increase in plaque SMC content with M3 overexpression (67.4%, *p* = 0.13, [Fig 6B](#pone.0173224.g006){ref-type="fig"}), which was supported by a significant increase in aortic SMC α-actin mRNA expression (60.3%, *p*\<0.05) in this model ([Fig 6D](#pone.0173224.g006){ref-type="fig"}).
{ref-type="sec"}" for details. Images are representative sections of plaque α-actin^+^ SMCs (red staining) in aortic sinuses. SMC α-actin staining was quantified as a percentage of total aortic sinus plaque area and SMC α-actin mRNA levels were determined in aortic arch samples for the 'rapid promotion' model **(A---B)** and the 'slow progression' model **(C---D)**. Data expressed as mean±SEM. \**p*\<0.05.](pone.0173224.g006){#pone.0173224.g006}
M3 reduces lipid deposition in descending aortas {#sec026}
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To determine the effects of M3 on lipid deposition, excised aortas were stained with Oil Red O. Lipid content in thoracic aortas of AdM3-treated mice was reduced in both models, reaching significance in the rapid promotion model (66.9%, *p*\<0.05) ([Fig 7](#pone.0173224.g007){ref-type="fig"}) while a trend was seen in the slow progression model (*p* = 0.077).
{ref-type="sec"}" for details. Images are representative sections of Oil Red O stained descending thoracic aortas. Oil Red O staining was quantified as a percentage of total thoracic aorta area for the 'rapid promotion' model and the 'slow progression' model. Data are mean±SEM. \**p*\<0.05.](pone.0173224.g007){#pone.0173224.g007}
M3 modulation of chemokine levels in the plasma and tissue {#sec027}
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Systemic overexpression of M3 was found to strikingly reduce CCL2 liver protein levels in both models (rapid promotion: 72.8% and slow progression: 67.3%, *p*\<0.05 for all, [Fig 8A and 8B](#pone.0173224.g008){ref-type="fig"}). No significant differences were seen in CCL5 liver protein levels with M3 in either model ([Fig 8C and 8D](#pone.0173224.g008){ref-type="fig"}). However, M3 increased CX~3~CL1 liver protein levels (37.8%, *p*\<0.05) in the rapid promotion model but not in the slow progression model ([Fig 8C](#pone.0173224.g008){ref-type="fig"}). Assessment of circulating chemokines found that there were no significant changes in either CCL2 or CCL5 in the plasma for either model of atherosclerosis progression with M3 ([Fig 8G--8J](#pone.0173224.g008){ref-type="fig"}). There were no changes in the plasma levels of SAA or TNF-α between groups ([S4 Fig](#pone.0173224.s004){ref-type="supplementary-material"}).
{ref-type="sec"}" for details. CCL2, CCL5 and CX~3~CL1 protein levels were assessed in the liver **(A-F)** and CCL2 and CCL5 in the plasma **(G-J)** by ELISAs. Data are mean±SEM. \**p*\<0.05, \*\*\*\**p*\<0.0001.](pone.0173224.g008){#pone.0173224.g008}
Discussion {#sec028}
==========
In this study, we investigated the effectiveness of the broad-spectrum chemokine inhibitor M3 on atherosclerosis progression in two models with differing rates of plaque development including: a (1) HFD-fed 'rapid promotion' model and (2) chow-fed 'slow progression' model. M3 exhibited varying effects in the two different models. Adenoviral overexpression of M3 significantly reduced lipid deposition in the descending aorta in the 'rapid promotion model' and reduced macrophage content in aortic sinus plaques. M3 protein also reduced aortic sinus lesion size and increased plaque SMC content in the 'slow progression' model. The changes in plaque size and macrophage content by M3 in the two models are likely to have been mediated through the inhibition of chemokine activity. *In vitro* studies found that M3 protein suppressed chemokine-receptor directed migration and human primary monocyte migration towards CCL2, CCL5 and CX~3~CL1, and plasma samples from AdM3 mice induced less monocyte migration than plasma from AdGFP viral control mice. Our findings suggest that variations in the aggressiveness of disease onset influence the effectiveness of M3 to alter plaque composition and limit atherosclerosis. This indicates that competitive inhibition of a few key chemokines across all the chemokine subclasses with M3 may not be as robust as inhibiting an entire class of chemokines (e.g. the CC-chemokine class with 35K) or the complete removal of a single chemokine/chemokine receptor (e.g. murine knockout models) for the suppression of atherosclerosis.
In the present study, we have demonstrated that chemokine-receptor directed migration towards purified CCL2, CCL5 and CX~3~CL1 was significantly reduced in response to increasing concentrations of purified M3 protein. In support of this finding, we also showed that cell migration of human monocytes towards key inflammatory chemokines was suppressed in the presence purified M3 protein, but this effect was not observed in migration towards CXCL12, a chemoattractant that is not inhibited by M3. This is consistent with studies that have reported M3 inhibition of CCL2, CCL5 and CX~3~CL1 but not CXCL12 \[[@pone.0173224.ref016]\]. Furthermore, we found that M3 protein, secreted into the plasma of AdM3-injected mice, suppressed plasma chemokine activity, as fewer monocytes migrated towards plasma from AdM3-injected mice. This reduction in plasma chemokine activity was significant in mice of the 'rapid promotion' model and is comparable with other studies using a similar chemokine blockade approach with the CC-chemokine class inhibitor '35K' \[[@pone.0173224.ref011]\]. The more modest reduction in plasma chemokine activity in the 'slow progression' model is likely due to the longer gene transfer period post-AdM3 infusion. Plasma from the mice in the 'slow progression' model was collected 12-weeks post-AdM3 delivery, compared to 4-weeks for the 'rapid promotion' model. After 12-weeks the expression of M3 may be declining. This is indicated by our detection of M3 protein using Western blotting in which there is a decline after one week and the reduction in AdM3 gene expression in the livers of the 'slow progression' mice. Despite this, the levels of M3 over the 12-week time period were sufficient to significantly reduce atherosclerotic lesion size in the aortic sinus and increase plaque SMC content.
This study found that M3 was more effective at inhibiting a slower rate of atherosclerosis than a higher rate of atherosclerosis. The chow-fed 'slow progression' model is more representative of physiological plaque development than HFD-fed rapid plaque progression, suggesting that M3 may be an effective strategy to limit atherosclerosis. In support of this there was also a reduction in lipid deposition in the descending thoracic aorta of the mice of the 'rapid promotion' model, which is also a site in which the progression of atherosclerosis is slower. The lack of effect on plaque size in the 'rapid promotion' model at the site of the aortic sinus, in which plaque formation is relatively fast, indicates that M3 may not be as effective as other chemokine inhibition strategies that have managed to suppress the more aggressive atherosclerosis generated by HFD feeding \[[@pone.0173224.ref008]--[@pone.0173224.ref011]\].
It was counter-intuitive to find no changes in lesions size in the aortic sinus in the HFD-fed 'rapid promotion' model, despite a reduction in plaque macrophage content. It was postulated that this may be due to an increase in the infiltration of other cell types such as T cells and B cells \[[@pone.0173224.ref020]\]. This is possible, as M3 does not inhibit the chemokines that direct T and B cell migration (e.g. CCL12, CCL19, CCL25) \[[@pone.0173224.ref021]\]. We did not find, however, any changes in plaque T or B cell content with M3. The effectiveness of M3 to decrease plaque macrophage content in the 'rapid promotion' model but not in the 'slow progression' model may be attributed to the higher rate of macrophage recruitment in HFD-fed mice, therefore enabling a greater opportunity for inhibition with M3. Support for a role of M3 in the inhibition on macrophage recruitment has also been identified in the gut \[[@pone.0173224.ref022]\]. The potential contribution from circulating and inflammatory monocytes was also assessed in an attempt to explain these observations. We found that 4 weeks post adenoviral delivery, M3 unexpectedly prevented a decrease in the proportion of circulating inflammatory monocytes in the 'rapid promotion' model, compared to the AdGFP control. This suggests that a higher number of monocytes would infiltrate the neointima leading to an increase in macrophage content. The opposite, however, occurred and there was a reduction in plaque macrophage content. We hypothesize therefore that the inhibition of key chemokines by M3 (e.g. CCL2, CCL5) prevents the adherence of inflammatory monocytes to the endothelium, allowing them to accumulate in the circulation, rather than traversing the endothelium and entering the plaque.
Although our observations are spread over two different models with varying rates of plaque development, the reduction in plaque size in the aortic sinus and the descending aorta, and macrophage content via chemokine inhibition with M3 are consistent with previous animal knockout and transgenic studies, as well as double decoy chemokine studies. The knockout and transgenic studies provide convincing proof that individual chemokine-chemokine receptor signalling pathways are important in atherosclerosis, particularly the CCL2/CCR2 \[[@pone.0173224.ref008], [@pone.0173224.ref017]\], CCL5/CCR5 \[[@pone.0173224.ref023]\] and CX~3~CL1/CX~3~CR1 \[[@pone.0173224.ref024]\] pathways. However, in these studies only a single chemokine or chemokine receptor was targeted, a strategy which is likely to be limited by redundancy in chemokine signalling. In a different approach, adenoviral-mediated gene transfer of the broad-spectrum CC-chemokine inhibitor 35K was found to cause an 85% reduction in macrophage recruitment and a 55% decrement in aortic sinus lesion size, compared with its controls in HFD-fed apoE^-/-^ mice \[[@pone.0173224.ref011]\]. This suggests that overcoming redundancy issues, at least in the case of the CC-chemokine class, can be a very effective strategy for inhibiting atherosclerosis. Comparing the chemokine binding proteins M3 and 35K, our results suggest that targeting fewer key chemokines from all four chemokine subfamilies, as with M3, may not be as robust as blocking the entire CC-chemokine class, as with 35K, for the suppression of atherosclerosis. With its ability to block chemokines from other classes such as the CX~3~C class, M3 may, however, be advantageous in pathologies such as thrombosis and restenosis. CX~3~CL1 is reported to increase SMC cell-cell adhesion of leukocytes and promote aortic SMC proliferation--both key drivers of neointimal hyperplasia-post stent deployment \[[@pone.0173224.ref025]\]. Consistent with this, the development of intimal hyperplasia following wire injury is suppressed in the M3 transgenic mouse \[[@pone.0173224.ref015]\]. CX~3~CL1 also plays a role in the activation of platelets, which increase inflammatory cell adhesion to the endothelium and promote thrombus formation \[[@pone.0173224.ref026]\]. Therefore, whilst the effects of M3 on atherosclerosis are more modest than other strategies it may be highly efficacious in preventing other inflammatory-driven pathologies.
Analysis of chemokine protein levels in mouse liver tissue showed that M3 strikingly reduced CCL2 protein concentrations in both models with no effect on CCL5. There were also no significant changes in circulating CCL2 and CCL5 in the plasma. These results suggest that M3 is binding CCL2 in the tissues and then sequestering it into the circulation where it is then cleared \[[@pone.0173224.ref027]\]. M3 appears to have had no effect in CCL5 *in vivo*, despite our *in vitro* data showing M3 inhibits CCR5-directed cell migration and other studies demonstrating the binding of M3 to CCL5 \[[@pone.0173224.ref028]\]. It has been reported that M3 binds CCL2 with a significantly higher affinity than CCL5, which is likely to be a contributing factor to this apparent lack of effect \[[@pone.0173224.ref028]\]. There were no changes in circulating chemokine levels, despite a reduction in chemokine activity. It may have been expected that circulating chemokine levels would decrease, however, previous studies using chemokine binding proteins support our findings and have found either no change or increases in circulating chemokines levels \[[@pone.0173224.ref011], [@pone.0173224.ref029]\]. It has been suggested that this is due to compensatory increases in chemokine expression in response to the inhibition, however, the new chemokines are likely to be rapidly bound to their inhibitor and inactive, then cleared or remain bound in the circulation \[[@pone.0173224.ref011]\]. Furthermore, it has been shown that chemokines bound to chemokine binding proteins are inactive, yet still detectable by ELISA \[[@pone.0173224.ref027]\]. In contrast to the other chemokines, CX~3~CL1 protein levels increased in the liver in AdM3 infused mice. CX~3~CL1 is a unique membrane-bound chemokine and is unlikely to be sequestered into the circulation but rather remain at the site of the liver, unable to be cleared when bound to M3 \[[@pone.0173224.ref030]\]. Inhibition of CX~3~CL1 by M3 in the liver, the site at which the gene transfer occurs, may be causing a compensatory increase in CX~3~CL1 liver expression, however, the CX~3~CL1 which is present in higher levels may not be active due to being bound to M3 protein.
Changes were detected in the aortic arch expression of the active subunit of NF-κB (p65), a key transcription factor in the promotion of inflammation and atherosclerosis. There was a significant decrease in aortic arch phospho p65 levels and mRNA expression in the 'rapid promotion' model, but no changes in phospho p65 and an increase in p65 mRNA in the 'slow progression' model. Genetic evidence in mice has revealed complex roles for NF-κB in inflammation that suggest both pro- and anti-inflammatory roles for the NF-κB pathway \[[@pone.0173224.ref031]\]. While it regulates pro-inflammatory cytokine production, leukocyte recruitment, and cell survival, NF-κB can also promote leukocyte apoptosis and contribute to the resolution of inflammation \[[@pone.0173224.ref032]\]. It is also suggested that NF-κB contributes to the feedback control of inflammation to affect the magnitude and duration of the inflammatory response \[[@pone.0173224.ref033]\]. Furthermore, the levels of inflammation were likely to be higher in the HFD-fed 'rapid promotion' model, as shown by our measurements of SAA, providing a greater opportunity for M3 to inhibit p65. This result also parallels the reduction in plaque macrophage content in AdM3-infused mice seen exclusively in the 'rapid promotion' model.
Four weeks of HFD feeding induced lesions with very low levels of α-actin positive SMC as it is likely too early in the atherosclerotic process for significant smooth muscle and/or collagen remodelling to have occurred after AdM3 gene transfer, making it difficult for the differences to be detected. However, an increase of SMC content in the 'slow progression' model suggests that M3 may be improving plaque stability in a more long-term disease progression. The characterization of other components of the plaque that are representative of plaque stability including collagen content and extracellular lipid content were, however, unchanged by M3 protein.
Adenoviral overexpression of M3 protein in apoE^-/-^ mice has varying effects depending on the rate of plaque progression ([Fig 9](#pone.0173224.g009){ref-type="fig"}). We propose that in the rapid promotion model M3 inhibits chemokine activity, resulting in the suppression of inflammatory monocytes adhering to the endothelium, causing them to accumulate in the circulation rather than traverse the endothelium and enter the plaque. This leads to a reduction in plaque macrophages and a suppression in lipid deposition in the descending thoracic aorta, which develops later, but not in the aortic sinus that would contain plaque of a more advanced stage. In this more aggressive inflammatory model, aortic phosphorylated p65 was lower which may also have contributed to the reduction in macrophage content. In the more gradual slower progressive model, we saw an increase in plaque SMCs indicating improved plaque stability with M3, with an overall reduction in atherosclerotic lesion area in the aortic sinus and descending thoracic aorta. Despite the decrease in lesion area, overexpression of M3 in this model had no effect on circulating monocytes or plaque macrophage content. The mechanisms for this are unclear but may be explained by the decline in M3 protein levels at the later time points of this study (represented by the attenuated inhibition of chemokine activity) and the overall lower levels of inflammation in this chow-fed model.
{#pone.0173224.g009}
Conclusion {#sec029}
==========
In summary, adenoviral-mediated overexpression of M3 suppresses chemokine activity *in vitro* and *in vivo*. Chemokine inhibition by M3 caused different effects on atherosclerotic plaque size and composition that was dependent on the rate of plaque progression. These findings support a role for broad-spectrum chemokine inhibition with M3 to limit atherosclerosis, but consideration of the aggressiveness of the disease may need to be considered for maximum efficacy.
Supporting information {#sec030}
======================
###### Confirmation of successful *in vivo* gene transfer and *ex vivo* inhibition of chemokine activity by M3.
Successful gene transfer and expression following adenoviral delivery was determined *in vivo*. **A.** M3 viral DNA in liver (Li), spleen (S), kidney (K) and lung (Lu) tissues was detected by real-time PCR. **B.** Livers of AdGFP and AdM3 infused mice were sectioned (5 μm) and viewed for green fluorescence. Data are mean±SEM. \**p*\<0.05, \*\**p*\<0.01,\*\*\*\**p*\<0.0001, *n* = 10--12 mice/treatment group.
(TIF)
######
Click here for additional data file.
###### Gating strategy used in flow cytometry analysis to detect different subsets of lymphocytes.
In this sample gating, cells were first gated for lymphocytes (FSC-A vs. SSC-A). Lymphocytes were then analysed for their uptake of the Zombie Aqua viability stain (BV510 excitation) to determine live vs. dead cells. Viable cells were further selected for singlets by gating sequentially first on a FSC-H vs. FSC-W and then on a SSC-H vs. SSC-W plot. **A.** Single cells were gated to determine CD45^hi^CD115^hi^ monocytes and **B.** further subdivided into Ly6-C/G^hi^ or Ly6-C/G^lo^ to distinguish between inflammatory and patrolling monocytes respectively. **C.** Neutrophils were determined as CD45^hi^CD115^lo^Ly6-C/G^+^ cells.
(TIF)
######
Click here for additional data file.
###### Plaque T Cell, B Cell, collagen and cholesterol cleft content.
Histological analysis was performed on aortic sinus sections. **A.** CD8 positive T cells and **B.** CD22 positive B cells, **C.** collagen and **D.** cholesterol clefts. Data expressed as mean±SEM, *n* = 10--12 mice/treatment group.
(TIF)
######
Click here for additional data file.
###### Assessment of pro-inflammatory cytokines mouse SAA and TNF-alpha in plasma.
Levels of pro-inflammatory cytokines were measured in mouse plasma. Data expressed as mean±SEM, *n* = 10--12 mice/treatment group.
(PPTX)
######
Click here for additional data file.
###### Plasma lipids and body weight measures.
Mouse plasma lipid concentrations were determined enzymatically using a commercial kit. The mean weights of each treatment group of animals at the time of sacrifice were recorded for both the **A.** rapid promotion and **B.** slow progression model. Data expressed as mean±SEM, *n* = 10--12 mice/treatment group.
(PPTX)
######
Click here for additional data file.
The authors wish to thank the Biological Facilities staff for assisting in all aspects of animal handling procedures such as animal health monitoring, breeding, weekend checks, and all aspects of animal husbandry, as well as Mr. Pat Pisansarakit for the isolation of human monocytes and expertise in tissue culture.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist and none of the authors have industry relationships to report.
[^2]: **Conceptualization:** CB.**Data curation:** DR AR JTMT.**Formal analysis:** DR JTMT CB.**Funding acquisition:** CB DR.**Investigation:** DR AR JTMT RH SC.**Project administration:** DR CB.**Supervision:** CB JTMT.**Validation:** CB DR AR LV RH SC JTMT.**Visualization:** DR CB.**Writing -- original draft:** DR CB.**Writing -- review & editing:** CB DR AR LV RH SC JTMT.
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Background
==========
Despite the availability of new, active drugs, metastatic breast cancer (MBC) remains an incurable disease, and the treatment of it is still controversial. In fact, the best treatment for patients pretreated with antracyclines, a subset very common in clinical practice, is hotly debated, given the extensive use of doxorubicine and epirubicine in metastatic, adjuvant and neoadjuvant settings.
Nevertheless, today the treatment of MBC is a rational choice, as different studies have shown that the use of polichemotherapy in this subset of patients appears to improve long term remission, relapse-free survival and overall survival \[[@B1]-[@B4]\].
In recent years, novel drugs have emerged as important agents in the treatment of MBC patients, because of their safety and efficacy in generating symptom relief, in reducing disease progression and in prolonging survival.
Among these new agents, taxanes have become the standard therapy in patients pretreated with antracyclines. In this subset of patients paclitaxel as a single agent has generated response rates ranging from 6% to 48% \[[@B5],[@B6]\] and these results improved when the drug was used as first line treatment in metastatic disease\[[@B7]\]. Actually a number of newer cytotoxic agents have been introduced in clinical trials to evaluate novel, safe and active taxane-based combinations in the treatment of MBC, extensively pretreated with antracyclines.
Gemcitabine is a cytidine nucleoside analogue with proven activity in advanced breast cancer.
In previously treated MBC patients it has produced response rates ranging from 12% to 29%, and it was tolerated satisfactorily \[[@B8],[@B9]\], while in first line schedules the response rate reported was 14--37% \[[@B10]\].
The paclitaxel and gemcitabine combination is justified by their different mechanism of action and by the lack of overlapping toxicities. In MBC this combination has been evaluated in phase II studies using a three -weekly schedule of treatment with paclitaxel given at 175 mg/mq on day 1 and gemcitabine given at 1000--1250 mg/mq on days 1,8, showing an interesting response rate ranging from 45% to 55% \[[@B11],[@B12]\]. Recently an interim analysis of a large phase III study demonstrated that the combination of paclitaxel plus gemcitabine as first-line treatment was more efficacious than paclitaxel alone in MBC, according to the different clinical variables considered (progression-free survival, response rate, pain relief and QOL)\[[@B13]\].
In a phase II study the combination of paclitaxel and gemcitabine was explored in a biweekly schedule by Colomer et al. in 1998 in a subset of patients who had not received prior treatment for MBC \[[@B14]\]. In this trial the response rate was impressive with an overall response of 69% (24% CR), and was well tolerated.
The same authors have recently updated these data in untreated MBC patients, with an overall response rate of 71% (26% CR). Moreover, in the same study it was demonstrated that the efficacy of this schedule could be reduced by elevated levels of HER2 \[[@B15]\].
According to these results a phase II study was started to evaluate the efficacy and tolerability of a biweekly schedule of paclitaxel and gemcitabine in MBC patients pretreated with antracyclines.
Methods
=======
Eligibility
-----------
To be elegible for the study, patients were required to have histologically confirmed breast cancer, metastatic or locally advanced disease, bidimensionally measurable lesions, performance status ≥ 70%, age 18--75 ys, adequate bone marrow, hepatic and renal function (neutrophil count ≥ 1500/μL, platelets ≥ 100,000 μL, hemoglobin ≥ 10 g/L, bilirubin ≤ 2 mg/dl, creatinine 1.5 ≤ mg/dl, and alanine/aspartate amino transferase level ≤ 3 times above normal). Prior chemotherapy, excluding gemcitabine and taxanes, radiotherapy and endocrine-therapy were permitted. All the patients were required to have received prior chemotherapy with antracyclines, in neoadjuvant, adjuvant and metastatic setting. Patients were excluded from the study in cases of: brain metastases, peripheral neuropathy and vasculopathy, bone metastases as the only site of disease, history of active cardiac disease, previous malignant neoplasia, pregnancy and breast feeding, any antineoplastic treatment within 8 weeks of entering the study
.
Treatment plan
--------------
Study treatment consisted of the infusion of gemcitabine and paclitaxel according to a biweekly schedule. Patients received paclitaxel (150 mg/mq) in 3-hours infusion, followed by gemcitabine (2000 mg/mq) given as a 60 min i.v. infusion. Patients received standard premedication with i.v. dexamethasone (20 mg) and antiemetic treatment 1 hour before the start of therapy with paclitaxel, plus orphenadrine and cimetidine.
Patients were scheduled to receive a maximum of 8 cycles and chemotherapy was stopped in case of progression, patient refusal and unacceptable toxicity. Patients who had received at least one course of chemotherapy were evaluated for toxicity and at least two courses for efficacy. The toxicity and activity of the schedule were evaluated according to the WHO criteria. \[[@B16]\]. All the measurable lesions were evaluated at baseline and after every two courses in order to document any response, stable disease or progressive disease.
Complete response was defined as the disappearance of all measurable lesions, partial response as the decrease of more than 50% of the sum of the products of all the measured lesions with no occurrence of new lesions, stable disease as a reduction ranging from 25% to 50% of the sum of the products of all the measured lesions with no occurrence of new lesions and progressive disease as an increase \> 25% of the sum of the products of all the measured lesions or the occurrence of new lesions.
All the patients were required to give written informed consent before the start of treatment and the study was conducted according to the approval of the local ethical board (Azienda Policlinico Umberto I -- Ethical Committee)
Results
=======
Patients characteristics
------------------------
Twenty-five consecutive patients with metastatic breast cancer and measurable disease were recruited by the Department of Experimental Medicine La Sapienza University of Rome in order to assess the tolerability and efficacy of a biweekly schedule of paclitaxel and gemcitabine. All the patients had received prior chemotherapy with antracyclines in adjuvant and non adjuvant settings and sixteen of them have been treated for metastatic disease. All the patients were evaluated for toxicity and efficacy. The main characteristic of the patients are shown in table [1](#T1){ref-type="table"}.
The median age was 51 years and the youngest patient enrolled was 39 years old. The majority of patients presented a WHO performance status of 0 (80%). All the patients had received prior treatment with antracyclines (44 % in adjuvant setting). At diagnosis lung and liver involvement was detected in 15 patients (60% with dominant visceral disease).
Efficacy
--------
A total of 148 cycles were given in an output basis with a median of seven per patient.
Four patients (16%) achieved a complete response, 12 (48%) a partial response, with an overall objective response rate of 64%. Stable disease was documented in 5 patients (20%) while progressive disease occurred in 4 patients(16%).
After an average follow-up of 18 months, the median duration of response in the subset of responders was 11.5 months with twelve patients alive.
Table [2](#T2){ref-type="table"} shows the response rates observed in the study.
Toxicity
--------
Toxicity data were available for all the patients recruited. Treatment was well tolerated in almost all the patients with infrequent occurrence of 3--4 grade toxicity.
The most frequent toxicity was haematological : two patients experienced grade 4 neutropenia and one grade 4 thrombocytopenia; grade 3 neutropenia occurred in 24% of patients; thrombocytopenia in 16% and anemia in 12%. No febrile neutropenia was observed and none of the patients received platelet transfusion. No toxic death or hospitalisation occurred.
Grade 2 peripheral neuropathy was frequent, mainly as grade 1--2 (38% of cases).
Complete alopecia occurred in almost all the patients treated.
Dose reduction or delays were necessary in less than 10% of chemotherapy cycles.
A summary of the toxicity data is reported in Table [3](#T3){ref-type="table"}.
Discussion
==========
Although advanced and relapsing breast cancer is generally considered an incurable disease, many opportunities have been explored in recent years to achieve an efficient palliation with the combined use of chemotherapy, radiotherapy, endocrine therapy and supportive treatment Systemic chemotherapy must be considered the treatment of choice in this subset of patients, in particular when endocrine resistance occurs. Antracyclines still constitute the cornerstone of chemotherapeutic approaches in advanced breast cancer, while the problem of the best schedule of treatment when an antracycline resistance occurs still has to be resolved.
In monochemotherapy, paclitaxel has shown interesting activity in doxorubicin-refractory metastatic breast cancer (28--48% response rate) and many phase II and III trials have investigated its optimal combination with other anticancer drugs. Among the new agents investigated in the treatment of advanced breast cancer, gemcitabine has demonstrated unexpected activity with response rates ranging from 15% to 40% when used as single agent in first- and second-line therapy. Moreover, the choice of this drug in the treatment od relapsing and metastatic breast cancer is conditioned by its toxicity profile. According to these data, the combination of paclitaxel and gemcitabine in patients with advanced and metastatic breast cancer and pretreated with antracyclines has recently been investigated in some trials. Nevertheless, the best schedule of treatment with a combination of the two drugs is still under investigation (weekly, biweekly, etc\...), and other studies are necessary to address this question, in particular when gemcitabine and paclitaxel are used in triplet schedules, in combination with other traditional or innovative drugs.
For this reason we investigated the toxicity and the clinical activity of the combination of gemcitabine and paclitaxel according to the schedule reported by Colomer \[[@B14]\] with a biweekly infusion of the two drugs in untreated patients with metastatic breast cancer.
In our study the dose of gemcitabine was lower than that reported by Colomer (2000 mg/mq vs 2500 mg/mq), using gemcitabine with precaution in our group of heavily pretreated patients.
In spite of this reduction the response rate in our experience was surprisingly high, although the patients had received two and more lines of treatment in the past. Moreover, only sixteen patients experienced progressive disease during the treatment, confirming the activity of the combination of gemcitabine and paclitaxel in the palliative therapy of metastatic breast cancer.
The favourable toxicity profile of this schedule was confirmed by the satisfactory results of other studies, and led to testing of this schedule in combination with new drugs with different biological activity (trastuzumab, tyrosine kinase and VEGF inhibitors). Moreover, these data suggest that the introduction of the combination as first line treatment of MBC should be explored more extensively only after its activity as second line treatment in better understood and the best schedule of infusion has been identified.
Thus, some questions arise from this and other studies. With this novel combination which schedule of treatment in the best, weekly or biweekly? With the gemcitabine/paclitaxel combination which is the right dose and is intensification or acceleration of the dose possibility? Is this combination a valid alternative in first-line treatment of patients not candidate to receive chemotherapy with antracyclines for clinical and biological reasons?
Conclusion
==========
The study reported in this paper presents an evident methodological limit: the number of patients enrolled; in spite of the strong evidence in literature activity of the combination of gemcitabine and paclitaxel in metastatic breast cancer, the papers recently published in this field present the same limit.
This evidence data support further testing of this combination in a larger randomized phase III clinical trial.
Unfortunately because there is not a standard in the choice of the best chemotherapeutic treatment of metastatic breast cancer resistant to anthracyclines, it is difficult to design a randomized trial to compare this novel association (on weekly or biweekly schedule) with a control scheme of treatment. Moreover this clinical limitation is complicated by the extensive use of taxanes in first line chemotherapy of breast cancer and therefore the gold standard after anthracyclines/taxanes failure is fat to be identified.
Nevertheless it is evident that there is a strong indication to start also with a III phase trial to compare the weekly with the biweekly schedule ; moreover it will be very interesting to evaluate the role of emerging prognostic factors (e.g. HER2/neu gene amplification, VEGF, e-cadherin\...) in patients treated with this association with the aim to select chemosensitive patients.
Competing interests
===================
The author(s) declare that they have no competing interests.
Authors\' contributions
=======================
ST designed the study, performed the statistical analysis, followed the patients, drafted the manuscript and coordinated the submission. AR, MDS, GC, ET followed the patients. FT, GPS, LF performed the statistical analysis, revised the literature, followed the patients and involved in the final revision of the manuscript. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2407/6/137/prepub>
Acknowledgements
================
The Authors wish to thank Dr. Evelina Miele of the Department of Experimental Medicine and Pathology for her invaluable technical and scientific contribution to the preparation of the paper, and Dr. Vittoria De Luise of the Department of Experimental Medicine and Pathology for her collaboration.
Figures and Tables
==================
######
Characteristics of patients (n.25)
**Characteristic** **n(%)**
--------------------------------- ----------
Age (years)
*Median* 51
*Range* 39--69
P. S 0--1 20(80%)
P. S 2 5 (20%)
Histology
Infiltrating ductal ca 19 (76)
Infiltrating lobular ca 6 (14)
Estrogen status
ER+ 14 (56)
ER - 7 (28)
Unknown 4 (16)
Dominant sites of metastases
Nodes 10(40)
Soft tissues 2(8)
Bone 5(20)
Liver 8(32)
Lung 7(28)
Pretreatment with antracyclines
Adjuvant CT 11(44)
Not adjuvant CT 14(56)
######
Response rates
**Response** **No of patients (%)**
--------------------- ------------------------
Complete response 4 (16)
Partial response 12(48)
Overall response 16(64)
Stable disease 5 (20)
Progressive disease 4(16)
######
Hematological and non-hematological toxicity(% patients)
**Toxicity** **Grade 3** **Grade 4**
----------------- ------------- -------------
Anaemia 12 \-
Neutropenia 24 8
Trombocytopenia 16 4
Neuropathy \- \-
Mucositis \- \-
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Wynyard Waterfront Master Plan
The rural township of Wynyard sits at the mouth of the Inglis River, on the North West Coast of Tasmania. Over time, the town centre of Wynyard has become disconnected from the river, its wharf, and the beachfront beyond. Cumulus Studio was engaged by the Waratah Wynyard Council to complete a master plan to restore this connection and ‘bring the wharf to town’.
The project includes upgraded and additional boardwalks along the river, play spaces, picnic areas, meeting places and an amphitheatre. A clock helps to provide a landmark and orienting device between the main street and the riverfront, and pedestrian access is improved from Goldie Street to the foreshore, connecting with a boardwalk along the water’s edge.
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The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Introduction {#s1}
============
Viral pathogens affect their eukaryotic host partly by interacting with the proteins of the host cells [@pone.0112034-Arnold1]. Virus-host PPIs are crucial for better understanding of the mechanisms and pathogenesis of infectious diseases [@pone.0112034-Zhou1]. Several computational methods have been proposed to predict protein-protein interactions, but most are designed for intra-species PPIs and only a few for inter-species PPIs. Widely used machine-learning methods for PPIs are SVM, Naïve Bayes and Random forest [@pone.0112034-Cui1], [@pone.0112034-Jansen1], [@pone.0112034-Lin1]. Shen et al. used protein sequence information to predict human PPIs by employing SVM with a kernel function and a conjoint triad method, in which the best model predicted with an average accuracy of 83.90% [@pone.0112034-Shen1]. Guo et al. predicted yeast PPIs with an accuracy of 88.09% using auto covariance (AC) and support vector machines (SVM) [@pone.0112034-Guo1]. In contrast, Wu et al. predicted yeast PPIs by mining the knowledge of functional associations from the GO-based annotations [@pone.0112034-Wu1]. Jansen et al. has developed a Bayesian networks approach to predict PPIs in yeast [@pone.0112034-Jansen1], while Lin et al. shows that Random Forest (RF) model may be more effective than Bayesian networks for predicting PPIs [@pone.0112034-Lin1]. In addition, a number of computational methods are also available in order to predict PPIs based on domain information [@pone.0112034-BinnyPriya1]--[@pone.0112034-Memievic1]. However, relatively few methods have so far been proposed to predict interspecies (specifically host-pathogen) PPIs [@pone.0112034-Cui1], [@pone.0112034-Dyer1]--[@pone.0112034-Doolittle1]. For example, Cui et al. used relative frequency of amino acid triplets of protein sequence to predict the interactions between two types of viruses (hepatitis C virus and human papillomaviruses) and human proteins [@pone.0112034-Cui1]. Proposed SVM methods of Cui et al. had an accuracy yield over 80%. Dyer et al. also proposed a method to predict physical interactions between human and HIV proteins based on a number of features, such as domain profiles, protein sequence k-mers and properties of human proteins in a human PPI network [@pone.0112034-Dyer1]. At a precision value of 70%, their method achieved recall (sensitivity) values of 40%.
In this paper, we have made an attempt to predict viral-host (inter-species) PPIs based on three well-known supervised machine-learning methods, namely SVM, Naïve Bayes and Random Forest using significantly diverse biological information like protein sequence, domain-domain associations, disorder regions, degree and amino-acids composition of viral and human proteins. The viral-host PPIs dataset were obtained from VirusMINT, a viral protein interaction database [@pone.0112034-Chatraryamontri1]. We have shown that only four features can able to predict viral-host PPIs with high degree of accuracy, which is comparable to the existing prediction models for viral-host PPIs. Furthermore we have shown that the viral protein amino acids composition (methionine, serine and valine) plays an important role in viral-host PPIs. An attempt was made to predict unknown PPIs between hepatitis B virus (HBV)-human proteins and hepatitis E virus (HEV)-human proteins using our proposed SVM optimal model. Predicted significant protein pairs were grouped using hierarchical clustering analysis (HCA) and validated using gene ontology enrichment analysis. Overall, the proposed support vector machines (SVM)-based machine learning technique was able to predict unknown viral-host protein interaction pairs with reasonable accuracy, which may be subjected to experimental validation.
Materials and Methods {#s2}
=====================
2.1 Datasets {#s2a}
------------
### 2.1.1 Data preparation {#s2a1}
The dataset used were obtained from "VirusMINT: a viral protein interaction database" (<ftp://mint.bio.uniroma2.it/pub/virusmint/MITAB/current/2012-10-26-mint-viruses-binary.mitab26.txt>) [@pone.0112034-Chatraryamontri1]. VirusMINT database emphasises on interaction between human and some of the medically significant viruses: human immunodeficiency virus 1 (HIV-1), simian virus 40 (SV40), hepatitis B virus (HBV), hepatitis C virus (HCV), papilloma virus. Unique and positive 1,146 viral-host PPIs were derived from initial 2,707 interactions, after eliminating 1,224 repetitive interactions (Vprot A-Hprot B and Hprot B-VprotA) and 337 interacting protein pairs not having any "InterPro" domain hit. Out of these, 1,035 interactions were found between viral and human proteins and 111 interactions between viral proteins and proteins of others species including mouse, rat, dog and bovine. Furthermore, non-redundant interaction analysis based on the homologous viral proteins present in training and testing sets showed that 0.77% of the viral-human PPIs were redundant (shown in [Table S1](#pone.0112034.s010){ref-type="supplementary-material"}). We used cd-hit-2d webserver (<http://weizhong-lab.ucsd.edu/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit-2d>) at 85% sequence identity level to find the homologous proteins present in the training and testing sets [@pone.0112034-Li1]. Since, large numbers of viral-human PPIs were distinct (99.23%) and there were only few (1,035) viral-human PPIs in the initial set, we considered all 1,035 positive interactions between the viral and human proteins as training and testing datasets in our 5-fold cross-validation study ([Table S2](#pone.0112034.s011){ref-type="supplementary-material"}).
### 2.1.2 Negative training and testing dataset {#s2a2}
Ben-Hur et al. [@pone.0112034-BenHur1] proposed that in the case of predicting protein-protein interactions, a simple uniform random choice of non-interacting protein pairs yield an unbiased estimate of the true distribution. In absence of experimentally proven non-interacting protein pairs, which are considered as an ideal negative dataset, we choose random 1,035 viral-human protein pairs that were not found in the positive training and testing datasets in our study as the negative dataset. In order to avoid prediction bias, we generated a negative dataset with the same number of viral-human PPIs (positive:negative = 1:1) as the positive dataset ([Table S3](#pone.0112034.s012){ref-type="supplementary-material"}).
### 2.1.3 Blind dataset {#s2a3}
111 positive interactions between viral and non-human species proteins, which were not used in 5-fold cross-validation, were considered as a blind dataset to avoid overfitting problem in building our optimal model for predictions ([Table S4](#pone.0112034.s013){ref-type="supplementary-material"}). Non-redundant interaction analysis, based on the homologous proteins present in the training and blind sets showed that 8.11% interactions between the viral and non-human species proteins were redundant (shown in [Table S1](#pone.0112034.s010){ref-type="supplementary-material"}). Therefore we removed 9 redundant interactions between the viral and non-human species proteins from the blind dataset. Like the negative training and test dataset, 102 negative viral and non-human species protein pairs were also generated ([Table S5](#pone.0112034.s014){ref-type="supplementary-material"}).
### 2.1.4 Independent dataset {#s2a4}
In order to predict unknown viral and human PPIs, we focused on some of the medically significant viruses, such as hepatitis B and hepatitis E. Instead of taking all the proteins of hepatitis B, we concentrated on the proteins of hepatitis B virus genotype C that is prevalent in the eastern India [@pone.0112034-Datta1]. Thus, reviewed 4 hepatitis B virus proteins (genotype C) with InterPro domain hits were obtained from Swiss-Prot [@pone.0112034-UniProt1]. Begum et al. observed that hepatitis E virus genotype 4 'e' is prevalent in the Northern India [@pone.0112034-Begum1], while Caron et al. found that genotype 1 of hepatitis E virus is most prevalent in the Asian countries [@pone.0112034-Caron1]. Hence, reviewed 3 hepatitis E virus proteins (genotype 4 'e') and 3 hepatitis E virus proteins (genotype 1) with InterPro domain hits were retrieved from Swiss-Prot. Reviewed 17,615 human proteins with InterPro domain hits were also retrieved from Swiss-Prot.
2.2 Machine Learning Techniques (MLT) {#s2b}
-------------------------------------
We focused on three well-known supervised machine learning methods, such as SVM, Naïve Bayes and Random Forest that were used for predicting PPIs [@pone.0112034-Cui1], [@pone.0112034-Jansen1], [@pone.0112034-Lin1].
### 2.2.1 SVM {#s2b1}
Support Vector Machines (SVM)-based method is defined over a vector space where the problem is to find a decision surface that maximizes the margin between data points in the two classes. We have used SVM^light^ tool provided by T. Joachims [@pone.0112034-Joachims1], which allows users to select various parameters and various kernel functions like linear, polynomial, radial basis function (RBF), sigmoid to find optimal parameters for each task.
### 2.2.2 Naïve Bayes {#s2b2}
Naïve Bayes model computes subsequent probabilities for a given hypothesis (present/absence) assuming that the features that describe data instances are conditionally independent. Its performance is comparable to other supervised learning methods. We used Waikato Environment for Knowledge Analysis (WEKA) machine learning tool box to perform Naïve Bayes classification [@pone.0112034-Hall1]. Since WEKA does not allow us to select different parameters set for Naïve Bayes classification, we used the default parameters set.
### 2.2.3 Random Forest {#s2b3}
Random Forest (RF) classifier "grows" several Decision Trees (DTs) simultaneously where each node uses a random subset of the features. Each tree in the Random Forest classifies the new object, and "votes" for that class. The forest elects the classification based on majority vote (over all the trees in the forest). We obtained Random Forest (RF) classifier from the WEKA machine learning tool box. Optimal parameters were used for evaluation of the method.
2.3 Feature Vectors {#s2c}
-------------------
We focused on forty-four features of protein pairs to produce feature vectors ([Table S6](#pone.0112034.s015){ref-type="supplementary-material"}). First, occurrence frequency of viral-host domain-domain association was used since domain-domain association plays an important role in protein-protein interactions [@pone.0112034-Memievic1]. Second, common domains observed in virus and host proteins were chosen and represented as binary format \[0,1\] (absence and presence of common domain observed in virus and host proteins in a particular protein pair represented by 0 and 1, respectively). Third, maximum degree of viral or human protein for a given viral-human protein pair was selected. Degrees of human proteins were collected from APID2NET (a Cytoscape plugin) and viral protein degrees were collected from viral-host PPIs. APID2NET provided us all possible PPIs from BIND, BioGrid, DIP, HPRD, IntAct and MINT databases [@pone.0112034-HernandezToro1]. Fourth, average percentages of disorder regions of protein pairs were selected, because intrinsically disordered proteins were found to be implicated in numerous cellular processes including signal transduction, transcriptional regulation and PPIs. We used "ESpritz: accurate and fast prediction of protein disorder" to gather percentage of disorder regions of proteins [@pone.0112034-Walsh1]. Finally, amino acid compositions of viral and host proteins were selected as a fifth to twenty-fourth and twenty-fifth to forty-fourth features of our proposed feature vectors. Since, Roy et al. proposed that amino acid composition (AAC) monomers feature is crucial for predicting PPIs [@pone.0112034-Roy1].
2.4 Infer domain-domain associations {#s2d}
------------------------------------
We inferred viral and host domain-domain associations from interacting protein pairs. Our goal was to find the frequency of a certain viral and host domain-domain association present in protein pairs. We collected all protein related "InterPro" domains from Protein Knowledgebase, UniProtKB (<http://www.uniprot.org/>) [@pone.0112034-UniProt1]. After retrieving the "InterPro" domain information, we computed viral domain-human domain association matrix (rows and columns represented host and viral domain names, respectively), using similar approach proposed by Sprinzak et al. [@pone.0112034-Sprinzak1]. The range of domain-domain association varies between 30 and 1, where 0 represents no association. We tried with two domain-domain association scores, such as Maximum Domain-Domain Association Score (MDDAS) and Average Domain-Domain Association Score (ADDAS). The MDDAS and ADDAS were calculated using following equations:
2.5 Amino acid composition {#s2e}
--------------------------
Amino acid composition is the percentage of each amino acid present in a protein. Percentage of all twenty natural amino acids was calculated using the following equations:
2.6 Feature selection {#s2f}
---------------------
The feature selection was performed by regression for categorical data method with beta coefficient \>0.00 and p-value\<0.05 for selection of best features using SPSS statistical analysis software, version 20 (SPSS, Chicago, IL, USA). The beta coefficient value is a measure of how strongly each "predictor variable" influences the "criterion variable". The higher beta coefficient value implies greater impact of the "predictor variable" on the "criterion variable".
2.7 5-fold cross-validation {#s2g}
---------------------------
We used 5-fold cross-validation to estimate performance of all methods. In 5-fold cross-validation, the dataset has been partitioned into 5 equally (or nearly equally) sized segments or folds. Consequently, 5 times of training and testing were performed such that each time a different fold of the data is held-out for testing while the remaining four folds are used for training. The overall performance of a method was calculated using average performance over five folds.
2.8 Performance measures {#s2h}
------------------------
### 2.8.1 Threshold Dependent {#s2h1}
Sensitivity (also referred to as recall), specificity, accuracy, PPV (Positive Prediction Value, also referred to as precision), Matthew's correlation coefficient (MCC) and F1 score were computed on 5-fold cross validation step. All the performance measures were based on a balanced dataset of 1∶1 positive vs. negative examples. Sensitivity, specificity, accuracy, PPV, MCC and F1 score were calculated by the following equations:
### 2.8.2 Threshold independent {#s2h2}
From Receiver Operating Characteristic (ROC) plot**,** area under ROC curve was computed on 5-fold cross validation step.
2.9 Hierarchical clustering analysis (HCA) {#s2i}
------------------------------------------
Hierarchical clustering analysis was done using TIBCO Spotfire software [@pone.0112034-TIBCO1]. The input matrix was viral-host SVM prediction scores obtained from the best optimized model. Following parameters were used for HCA: complete linkage clustering method, cosine correlation distance measure, average value ordering weight, scale between 0 and 1 normalization and empty value replace by 0 for both (row and column) dendrogram.
2.10 GO Enrichment analysis {#s2j}
---------------------------
The Database for Annotation, Visualization and Integrated Discovery (DAVID) web server was used to identify significantly enriched gene ontology (GO) annotation terms in predicted interacting human protein partners of hepatitis B and E viruses [@pone.0112034-Huangda1]. We consider only GO biological process annotation terms of level greater than 2 with significant false discovery rate (FDR) value\<0.05.
Results and Discussion {#s3}
======================
3.1 Selection of optimal features {#s3a}
---------------------------------
We started with 44 features of a specific viral-host protein pair and tried with different subsets of features in order to achieve maximum accuracy with nearly equal sensitivity and specificity of our proposed method ([Table S6](#pone.0112034.s015){ref-type="supplementary-material"}, [S7](#pone.0112034.s016){ref-type="supplementary-material"}). Interestingly, we observed that four features with beta coefficient\>0.00 and p-value\<0.05 showed reasonably decent accuracy of 71%, sensitivity of 67% and specificity of 74% in proposed SVM method (shown in [Table 1](#pone-0112034-t001){ref-type="table"}). As shown in [Table 2](#pone-0112034-t002){ref-type="table"}, selected four features achieved slightly higher accuracy than all the forty-four features used together. Although disordered regions play a significant role in protein-protein interactions, it was not selected as the best feature based on our feature selection with regression (beta coefficient and p-value). Methionine residue interacts with aromatic residues and plays a specific role in stabilization of protein structure and may be associated with number of mutation and age related diseases [@pone.0112034-Valley1]. Serine residues are crucial for serine/threonine protein phosphatises and control many cell functions [@pone.0112034-DepaoliRoach1], while valine residue was shown to play a vital role in modulating syncytium formation during infection [@pone.0112034-Wilson1].
10.1371/journal.pone.0112034.t001
###### List of best 4 features selected based on categorical regression method.
{#pone-0112034-t001-1}
Features Beta Bootstrap (1000)Estimate of Std. Error df F Sig.(P~Value~)
---------------------------------------- ------- ---------------------------------------- ------- --------- ----------------
Average domain-domainassociation score 0.511 0.016 1.000 982.607 0.000
Virus Methionine 0.070 0.021 1.000 10.911 0.001
Virus Serine 0.106 0.021 1.000 25.838 0.000
Virus Valine 0.094 0.023 1.000 16.829 0.000
10.1371/journal.pone.0112034.t002
###### Comparison of performance between selected best 4 features vs all 44 features.
{#pone-0112034-t002-2}
Method All Features Selected Features
--------------- -------------- ------------------- ----------- ----------- ---------- -----------
Naïve Bayes 67.48 0.66 56.72 68.50 0.71 54.35
SVM **68.00** **0.72** **65.04** **71.00** **0.73** **69.41**
Random Forest 71.69 0.77 67.13 72.41 0.76 66.39
3.2 Performance of SVM, NB and RF using 5-fold cross validation {#s3b}
---------------------------------------------------------------
In order to achieve optimal sensitivity, specificity and accuracy, we tried different kernels and parameters using SVM. The linear and polynomial kernel function showed high specificity, but low sensitivity, whereas the sigmoid kernel function exhibited poor sensitivity ([Table S8](#pone.0112034.s017){ref-type="supplementary-material"}). In contrast, the radial basis function (RBF) showed reasonable sensitivity of 67%, specificity of 74% and accuracy of 71% as shown in [Table 3](#pone-0112034-t003){ref-type="table"}. We tried with different parameters in WEKA for Random Forest (shown in [Table S9](#pone.0112034.s018){ref-type="supplementary-material"}). SVM had nearly equal sensitivity (67%) and specificity (74%), whereas Naïve Bayes and Random Forest showed lower sensitivity (37.49% for NB, 55.66% for RF), but higher specificity (99.52% for NB, 89.08% for RF) ([Table 4](#pone-0112034-t004){ref-type="table"}). As shown in [Table 4](#pone-0112034-t004){ref-type="table"}, Random Forest perform better in terms of accuracy, MCC and area under ROC curve, whereas SVM perform better in terms of sensitivity and F1 score. We are more concerned about the recall and precision, since they are directly proportional to the true positives. As shown in [Table 4](#pone-0112034-t004){ref-type="table"}, recall score of SVM (67%) is better than RF (55.66%), while precision score of RF (82.26%) is better than SVM (72%). Therefore we computed the F1 score. F1 score of SVM and RF shows that, SVM (69.41) performs slightly better than RF (66.39). Therefore, we used the best SVM model for further study.
10.1371/journal.pone.0112034.t003
###### SVM based performance on testing dataset (5-fold cross-validation) using parameters t = 2 (RBF kernel), and g = 1, c = 0.1, j = 2.
{#pone-0112034-t003-3}
Threshold Sensitivity (%) Specificity (%) Accuracy (%) PPV (%) MCC
----------- ----------------- ----------------- -------------- --------- ----------
0.8 37 91 64 76 0.32
0.7 46 89 67 78 0.39
0.6 52 85 68 76 0.40
0.5 59 80 69 75 0.42
**0.4** **67** **74** **71** **72** **0.44**
0.3 69 70 70 69 0.42
0.2 73 65 69 68 0.41
0.1 76 59 68 65 0.38
0 80 51 66 62 0.35
-0.1 81 46 64 60 0.32
-0.2 83 40 62 58 0.28
-0.3 85 36 60 57 0.26
-0.4 87 29 58 55 0.23
-0.5 89 25 57 54 0.20
-0.6 89 20 54 53 0.15
-0.7 91 16 53 52 0.12
-0.8 91 11 51 51 0.08
10.1371/journal.pone.0112034.t004
###### Comparison of performance measures among Naïve Bayes, SVM and Random Forest methods on testing dataset using 5-fold cross-validation technique in our study.
{#pone-0112034-t004-4}
Methods Sensitivity (%) Specificity (%) Accuracy (%) PPV (%) MCC Area under ROC curve F1 Score (%)
--------------- ----------------- ----------------- -------------- ----------- ---------- ---------------------- --------------
Naïve Bayes 37.49 99.52 68.50 98.80 0.47 0.71 54.35
SVM^light^ **67.00** **74.00** **71.00** **72.00** **0.44** **0.73** **69.41**
Random Forest 55.66 89.08 72.41 82.26 0.48 0.76 66.39
3.3 Assessment on blind dataset using SVM based method {#s3c}
------------------------------------------------------
In order to avoid bias in the performance of our proposed model, we tested it on blind dataset, not used in training or testing. Consequently, 204 protein pair between viral proteins and non-human species (mouse, rat, dog, bovine etc.) was considered as a blind dataset. We used the same parameters and cut-off (threshold) for each approach. As shown in [Table 3](#pone-0112034-t003){ref-type="table"}, threshold value of 0.4 generated reasonable accuracy on the test dataset using 5-fold cross-validation technique in our study. At this threshold value, sensitivity of 64%, specificity of 83%, and accuracy of 74% was achieved on the blind dataset.
3.4 Comparison with other predictions methods for virus-host PPIs {#s3d}
-----------------------------------------------------------------
Dyer et al. developed a method to predict HIV-human PPIs using SVM classifier with linear kernel on different combinations of protein features, including domain profiles, protein sequence k-mers and properties of human proteins in a human PPI network [@pone.0112034-Dyer1]. They predicted PPIs with a precision of 70% and a recall (also referred to as sensitivity) value greater of 40% using a combination of protein sequence four-mers, protein domains and PPI network information with 1∶25 ratio of positive example (PE) to negative example. We obtained only 332 positive interactions instead of 1028 interactions reported by Dyer et al. between human and HIV proteins [@pone.0112034-Dyer1]. As shown in [Table 5](#pone-0112034-t005){ref-type="table"}, our proposed method achieved the sensitivity of 87% whereas Dyer et al. achieved 40%.
10.1371/journal.pone.0112034.t005
###### Comparison of proposed method with other viral-host PPIs prediction methods.
{#pone-0112034-t005-5}
Performance Mesaure Dyer et al. Dataset[\*](#nt101){ref-type="table-fn"} Performance Mesaure Cui et al. Dataset[\*](#nt101){ref-type="table-fn"}
--------------------- ------------------------------------------------------ --------------------- ----------------------------------------------------- ------- ------- -------
Sensitivity (%) 40.00 87.05 Accuracy (%) 78.00 80.00 82.00
\*Partial dataset.
Cui et al. worked on a similar problem of HCV-human and HPV-human PPIs using an SVM model with RBF kernel and relative frequency of amino acid triplets of a protein sequence. They have used 11 HCV (lead to 695 interactions) and 9 HPV proteins (lead to 252 interactions) [@pone.0112034-Cui1]. From the available datasets in the supplementary tables, we can extract 1 HCV protein (leads to 10 positive and 9 negative interactions) and 1 HPV protein (leads to 9 positive and 7 negative interactions) from Swiss-Prot. Our proposed method achieved accuracy of 80% on this sparsely available dataset (shown in [Table 5](#pone-0112034-t005){ref-type="table"}), whereas Shen et al. and Cui et al. achieved accuracy of 78% and 82%, respectively [@pone.0112034-Cui1], [@pone.0112034-Shen1].
3.5 Prediction of unknown HBV-human and HEV-human PPIs {#s3e}
------------------------------------------------------
Hepatitis B virus and hepatitis E virus proteins were used in order to predict unknown viral-human PPIs. All possible combinations of hepatitis B virus and human protein pairs (17615 \* 4) were predicted by our proposed model ([Table S10](#pone.0112034.s019){ref-type="supplementary-material"}). The predicted SVM score greater than 0.58 of hepatitis B virus-human protein pairs (n = 8411) was used for HCA. As shown in [Figure 1](#pone-0112034-g001){ref-type="fig"}, P0C688 (gene name P) was far apart from the other three hepatitis B viral proteins, out of which P31868 (gene name S) and Q81102 (gene name C) are closely associated. Similarly, all combinations of hepatitis E virus and human protein pairs (17615 \* 6) were predicted by the proposed model ([Table S11](#pone.0112034.s020){ref-type="supplementary-material"}). The highly predicted SVM score of hepatitis E virus and human protein pairs were used for HCA. In HEV-host interaction pairs (n = 20,375) clustering analysis, Q9IVZ7 (gene name ORF3 and genotype 4) was far apart from the other five hepatitis E viral proteins, out of which Q9IVZ8 (gene name ORF2, genotype 4), Q9IVZ9 (gene name ORF1, genotype 4), P33424 (gene name ORF1, genotype 1) and P33426 (gene name ORF2, genotype 1) were closely associated ([Figure S1](#pone.0112034.s001){ref-type="supplementary-material"}).
{#pone-0112034-g001}
Finally, the human proteins present in high confidence (red area) of hierarchical clustering analysis (Shown in [Figure 1](#pone-0112034-g001){ref-type="fig"} and [Figure S1](#pone.0112034.s001){ref-type="supplementary-material"}) were used for further gene ontology enrichment analysis. The analysis on interacting human protein partners of hepatitis B virus (Shown in [Figure 2](#pone-0112034-g002){ref-type="fig"} and [Figure S2](#pone.0112034.s002){ref-type="supplementary-material"}, [S3](#pone.0112034.s003){ref-type="supplementary-material"}) showed probable functions of viral "X protein" (UniProtKBId: P0C686), "C protein" (UniProtKBId: Q81102) and "P protein" (UniProtKBId: P0C688) (shown in [Table 6](#pone-0112034-t006){ref-type="table"} and full data on [Table S12](#pone.0112034.s021){ref-type="supplementary-material"}-[S14](#pone.0112034.s023){ref-type="supplementary-material"}). As shown in [Table 6](#pone-0112034-t006){ref-type="table"}, HBV "C proteins" probably plays a significant role in membrane docking, while "X protein" and "P protein" function in cell killing and modulating metabolic processes of host proteins, respectively.
![A network of HBX-human protein interactions predicted by our proposed method.\
The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HBX protein is represented by cyan node. The significant gene ontology enriched human proteins are representing by salmon node, whereas other human proteins are representing by slate grey node.](pone.0112034.g002){#pone-0112034-g002}
10.1371/journal.pone.0112034.t006
###### The Gene Ontology Biological Process enrichment analysis on interacting human protein partners of HBV proteins using DAVID server.
{#pone-0112034-t006-6}
HepatitisB virusprotein GOterm∼BiologicalProcess Human protein
------------------------- ---------------------------------------- --------------------------------------------------------------------------------------------------------------------------
C GO:0022406∼membrane docking SCFD1, SCFD2, VPS45, STXBP1, STXBP2, STXBP3
GO:0006835∼dicarboxylic acid transport SLC1A4, SLC1A5, SLC1A2, SLC1A3,SLC1A1
GO:0006865∼amino acid transport SLC1A4, CPT1B, SLC1A5, SLC1A2,CPT2, SLC1A3, XK, SLC1A1
X GO:0001906∼cell killing DEFA6, DEFA5, DEFA4, DEFA3, DEFA1
GO:0009620∼response to fungus DEFA6, DEFA5, DEFA4, DEFA3, DEFA1
GO:0006952∼defense response YWHAZ, DEFB4A, CD74, IL17C,IL17D, IL17A, IL17B, DEFA6, AOAH, DEFA5, DEFA4, IL17F, DEFA3, DEFA1
P GO:0051186∼cofactor metabolic process NAMPT, ACO2, HMGCR, ACO1,IREB2, GIF, PNP, SOD2, SDHA,GSS, MTHFS, PGLS, PANK2, PANK3, FXN, PANK1, NARFL, CTNS, NAPRT1, FH
GO:0006732∼coenzyme metabolic process NAMPT, ACO2, HMGCR, ACO1, PNP, SOD2, SDHA, GSS, MTHFS, PGLS, PANK2, PANK3, PANK1, CTNS, NAPRT1, FH
Significant biological process annotation terms were filtered by FDR (false discovery rate) \<0.05.
Similar study as hepatitis B virus proteins was also done with hepatitis E virus proteins (Shown in [Figure S4](#pone.0112034.s004){ref-type="supplementary-material"}-[S9](#pone.0112034.s009){ref-type="supplementary-material"}), where ORF1 (genotype 1) is probably involved in many biological processes including regulation of cytoskeleton organization, nitrogen compound biosynthetic process and translation (Shown in [Table S15](#pone.0112034.s024){ref-type="supplementary-material"} and full data on [Table S16](#pone.0112034.s025){ref-type="supplementary-material"}-[S21](#pone.0112034.s030){ref-type="supplementary-material"}).
Conclusion {#s4}
==========
Here, we proposed three supervised machine learning-based techniques for predicting viral-host (across species) PPIs by incorporating potential biological information of protein pairs including domain-domain associations score, degree, percentage of disorder regions and amino acid compositions. Initially, we started with 44 features and predicted four best features, which were domain-domain association and methionine, serine and valine amino acid composition of viral proteins using categorical regression model (beta coefficient\>0.00 and p-values\<0.05). There are biological interpretations of these residues of viral proteins for their importance in viral-host PPIs. For example, methionine, serine and valine may be involved in stabilization of the protein structure, serine/threonine protein phosphatases and modulating syncytium formation during infection, respectively. It was observed that Random Forest perform better in terms of accuracy, MCC and area under ROC curve, while the proposed SVM method performs better in terms of sensitivity and F1 score. Performance of the proposed SVM method was evaluated on the blind dataset of 204 viral-host protein pairs (102 positive and 102 negative viral-host protein pairs), which achieved a sensitivity of 64%, specificity of 83%, and accuracy of 74%. In addition, unknown HBV-human and HEV-human PPIs were predicted using optimised SVM model and were grouped by HCA and further validated by GO enrichment analysis. Hepatitis B virus interacting human proteins show distinct GO biological process terms; for example, "X-protein" probably interferes with cell defence mechanism, whereas "P-protein" binds to metabolic pathways. The predicted viral-human PPIs give us hint about the possible role of viral proteins in the pathogenesis process.
Supporting Information {#s5}
======================
######
**Hierarchical clustering of highly predicted SVM score of HEV-human protein pairs.** Hierarchical clustering analysis was done using TIBCO Spotfire software with complete linkage clustering method, cosine correlation distance measure, average value ordering weight, scale between 0 and 1 normalization and empty value replace by 0 for both (row and column) dendrogram. The high, average and low SVM predicted scores are marked in red, white and blue, respectively.
(PDF)
######
Click here for additional data file.
######
**A network of HBC-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HBC protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node, whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**A network of HBP-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HBP protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node, whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**A network of HEORF1 (Genotype 1)-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HEORF1 (Genotype 1) protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**A network of HEORF2 (Genotype 1)-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HEORF2 (Genotype 1) protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**A network of HEORF3 (Genotype 1)-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HEORF3 (Genotype 1) protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**A network of HEORF1 (Genotype 4)-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HEORF1 (Genotype 4) protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**A network of HEORF2 (Genotype 4)-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HEORF2 (Genotype 4) protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**A network of HEORF2 (Genotype 4)-human protein interactions predicted by our proposed method.** The network visualized by Cytoscape 3.0.2 [@pone.0112034-Smoot1]. The HEORF2 (Genotype 4) protein is representing by cyan node. The significant gene ontology enriched human proteins are representing by salmon node whereas other human proteins are representing by slate grey node.
(PDF)
######
Click here for additional data file.
######
**Statistic of homologous protein present in the training and testing sets as well as from the blind datasets.**
(XLSX)
######
Click here for additional data file.
######
**Positive interactions dataset used in this study to build optimal model for prediction.** The positive interactions dataset used in the study were obtained from VirusMINT.
(XLSX)
######
Click here for additional data file.
######
**Negative interactions dataset used in this study to build optimal model for prediction.** The negative interactions dataset used in the study were chosen using random protein pairs which are not found in interacting protein pairs.
(XLSX)
######
Click here for additional data file.
######
**Positive interactions dataset used in this study as a positive blind dataset.** The positive blind dataset used in the study were obtained from VirusMINT.
(XLSX)
######
Click here for additional data file.
######
**Negative interactions dataset used in this study as a negative blind dataset.** The negative blind dataset used in the study were chosen using random protein pairs which are not found in interacting protein pairs (positive blind dataset).
(XLSX)
######
Click here for additional data file.
######
**All 44 input features.**
(XLSX)
######
Click here for additional data file.
######
**SVM performance measures based on different subsets of features.** Optimal parameters were used for respective subset of features.
(XLSX)
######
Click here for additional data file.
######
**Several SVM kernel-wise performance measures (sensitivity and specificity) on different models.** Optimal parameters and threshold were used for respective kernel. In Model 1, 1^st^, 2^nd^, 3^rd^ and 4^th^ folds were used for training and 5^th^ fold was kept for testing. In Model 2, 1^st^, 2^nd^, 3^rd^ and 5^th^ folds were used for training and 4^th^ fold was left out for testing. In Model 3, 1^st^, 2^nd^, 4^th^, 5^th^ folds were used for training and 3^rd^ fold for testing. In Model 4, 1^st^, 3^rd^, 4^th^, 5^th^ folds were used for training and 2^nd^ fold was used for testing. In Model 5, 2^nd^, 3^rd^, 4^th^, 5^th^ folds were used for training and 1^st^ fold was kept aside for testing.
(XLSX)
######
Click here for additional data file.
######
**Different parameters used in Random Forest using WEKA.**
(XLSX)
######
Click here for additional data file.
######
**Predicted scores of HBV-human protein-protein association by proposed optimal model.**
(XLSX)
######
Click here for additional data file.
######
**HEV-human protein-protein association predicted scores by proposed optimal model.**
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HBV X proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HBV C proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HBV P proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HEV proteins using DAVID server.** Significant biological process annotation terms were filter by FDR (false discovery rate)\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HEV ORF1 (Genotype 1) proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HEV ORF2 (Genotype 1) proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HEV ORF3 (Genotype 1) proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HEV ORF1 (Genotype 4) proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HEV ORF2 (Genotype 4) proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
######
**GO enrichment analysis on interacting human protein partners of HEV ORF3 (Genotype 4) proteins using DAVID server.** Significant biological process terms were chosen by P~Value~\<0.05.
(XLSX)
######
Click here for additional data file.
This project was supported by Indian Council of Medical Research \[extramural project (IRIS ID: 2013-1551G)\]. SS thanks Department of Biotechnology for Ramalingaswami fellowship (BT/RLF/Re-entry/11/2011).
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: SD SS. Performed the experiments: RKB SS. Analyzed the data: RKB SS SD. Contributed to the writing of the manuscript: RKB SS SD.
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During manufacture of an electrical or opto-electronic package, electrical or optical devices or integrated circuits are frequently attached to a substrate. This is often accomplished by forming bond pads on the electrical or optical device or integrated circuit. The bond pads are placed in contact with corresponding portions of the substrate that have solder bumps or solder balls. A heating step then causes the solder to reflow and attach the device to the substrate.
The formation of the solder bumps is a critical step in the manufacturing processes used in fabricating the packages; manufacturing processes frequently require both accurate dimensions and placement of the solder bumps, and several techniques have been developed for the formation of the solder humps. An exemplary technique is a lift-off process. In this technique, a resist layer is formed on the substrate and patterned to form windows which expose selected portions of the substrate. The solder hump material is deposited on the substrate by, for example, sputtering. The resist is then lifted off by immersion of the substrate and resist in a solvent which attacks the exposed portions of the resist in the sides of the windows and ultimately removes all of the resist, as well as the metal on the resist, from the substrate. The lift-off is facilitated with windows having a negative taper; that is, the windows are bigger at the bottoms than they are at the tops. The negative taper exposes more resist to the solvent than does a window with no taper. The lift-off technique suffers from the drawbacks of poor resist adhesion to the substrate and limited solder bump height due to small resist thickness; the solder bump height can not exceed the resist thickness.
Electrodeposition is another technique that has also been used to form solder bumps. Electrodeposition may be done with patterned resists or other patterned insulating surfaces that expose a conducting surface; electrodeposition does not occur on insulating surfaces, but only on conducting surfaces. The areas where solder bumps are to be formed are connected to a current source by patterned conductors on the substrate surface. However, the patterned conductors have a finite resistance which, of course, becomes larger as the dimensions of the conductors shrink. The conductors become smaller as device dimensions decrease and more contacts are made to the devices and integrated circuits. The plating rate, and ultimately the amount of solder plated, at any point is proportional to the electric field at that point. Due to the voltage drop caused by the finite resistance, the electric field is not necessarily the same at all the areas being plated and the solder bumps may have varying heights.
Varying solder bump heights are undesirable liar several reason. For example, self-alignment of devices with respect to the substrate is difficult with bumps having different size; that is, the solder bumps should all have the same amount of solder to permit accurate self-alignment. Additionally, many devices and integrated circuits require alignment only with respect to the plane of the substrate; that is, only with respect to the patterned conductors on the substrate. There are, however, devices, such as lasers, that must be attached to a substrate with accurate alignment in the direction perpendicular to the plane of the substrate; that is, in the z direction. Accurate alignment in the z direction is difficult to obtain if the solder bumps have varying heights.
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It would be advantageous to be able to provide a device that amplifies a current in response to a control current in some CMOS devices. For example, current-mode sense amplifiers for use in reading-out memory cells have faster read times than voltage-based devices. Unfortunately, conventional CMOS fabrication techniques are not well-adapted for fabricating current-to-current gain devices such as bipolar junction transistors. Normal CMOS processing does not provide full junction isolation of the type needed to construct bipolar junction transistors. Hence, to construct such transistors in CMOS, 3 to 5 additional masking steps and additional implant/diffusion steps are required. This increases the cost of the devices and requires larger die sizes.
Broadly, it is the object of the present invention to provide an improved CMOS transistor design.
It is a further object of the present invention to provide a CMOS device that controls a current in response to another current.
It is yet another object of the present invention to provide a CMOS device that mimics the behavior of a bipolar junction transistor.
These and other objects of the present invention will become apparent to those skilled in the art from the following detailed description of the invention and the accompanying drawings.
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Karnataka election results live: Congress set to form government; JD(S) pushes BJP to number 3
Bangalore: The Congress has emerged as the single-largest party in Karnataka and at 11:15 am on Wednesday, seemed set to form government, perhaps even on its own as it led in 117 seats in the 224-seat Assembly.
If all leads do not convert to results, the party says it is open to talking to the Janata Dal (Secular) which
is the other big gainer of the day and is now placed at number 2. After
five years of turbulent rule in Karnataka, the BJP is history.
JD(S) leader HD Kumaraswamy, however, ruled out any alliance with the Congress and said they will sit in the Opposition. "We will fight against Congress like we fought against BJP. We will sit in the Opposition; Congress will not come to our house, there are other small parties," he said. (Read)
The JD(S) also said that while it did all the hard work to highlight the BJP's failings in the last five years, the Congress was getting the fruits of that effort.
Mr Yeddyurappa has effectively split the BJP's vote, but has failed to convert that into gains for his newly floated Karnataka Janata Party. But he is hanging in there, offering help in government formation if his likely clutch of seats are needed. (Read: BJP says split hurt the party)
In 2007, the BJP had won 110 - and made up the difference with the help of Independents to form government. Party leaders had hoped for about 80 seats this time, but right now the party seems unlikely to be anywhere near that, faring poorly even in urban strongholds like Bangalore. (Watch: Modi has no magic, says Congress' Siddaramaiah)
"This accidental fluke victory of the Congress because of the split in BJP votes," said the BJP's Ravi Shankar Prasad.
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Work same as Body through Rnd 4: 8{10-12} dc, 8{10-12} Clusters, and 8{10-12} sps.
MITTSBody
Foundation (RS): Work 32{40-48} Fsc; being careful not to twist piece, join with slip st to first Fsc to begin working in rnds. Loop a short piece of yarn around any stitch to mark Foundation as right side.
Rnd 1: Ch 5 (counts as first dc plus ch 2, now and throughout); dc in same st as joining, skip next 3 Fsc, work (Cluster, ch 3, Cluster) in next Fsc, skip next 3 Fsc, * (dc, ch 2, dc) in next Fsc, skip next 3 Fsc, work (Cluster, ch 3, Cluster) in next Fsc, skip next 3 Fsc; repeat from * around; join with slip st to first dc: 8{10-12} dc, 8{10-12} Clusters, and 8{10-12} sps.
Rnds 2 thru 4{5-5}: Turn; slip st in next Cluster and in next ch-3 sp, ch 5, dc in same sp, work (Cluster, ch 3, Cluster) in next ch-2 sp, * (dc, ch 2, dc) in next ch-3 sp, work (Cluster, ch 3, Cluster) in next ch-2 sp; repeat from * around; join with slip st to first dc. Do not finish off.
Thumb Opening
Row 1: Ch 3 (counts as first dc, now and throughout), turn; * (dc, ch2, dc) in next ch-3sp, work Cluster, ch 3, Cluster) in next ch-2sp; repeat from * around, dc in next dc (joining dc), do not join: 10{12-14} dc, 8{10-12} Clusters, and 8{10-12} sps.
Row 2: Ch 3, turn; * (dc, ch2, dc) in next ch-3sp, work (Cluster, ch3, Cluster) in next ch-2sp; repeat from * across to last 2 dc, skip next dc, dc in last dc.
Row 3: Ch 3, turn; * (dc, ch2, dc) in next ch-3sp, work (Cluster, ch3, Cluster) in next ch-2sp; repeat from * across to last 2 dc, skip next dc, dc in last dc; join with slip st to first dc to begin working in rnds.
Next Rnd: Turn; slip st in next 2sts and in next ch-3sp, ch5, dc in same sp, work (Cluster, ch3, Cluster) in next ch-2sp, * (dc, ch2, dc) in next ch-3 sp, work (Cluster, ch3, Cluster) in next ch-2sp; repeat from * around; join with slip st to first dc: 8{10-12} dc, 8{10-12} Clusters, and 8{10-12} sps.
Last 3{44} Rnds: Turn; slip st in next Cluster and in next ch-3sp, ch5, dc in same sp, work (Cluster, ch3, Cluster) in next ch-2sp, * (dc, ch2, dc) in next ch-3 sp, work (Cluster, ch3, Cluster) in next ch-2sp; repeat from * around; join with slip st to first dc. Finish off.
Cuff
Rnd 1: With right side facing and working in chs at base of Fsc, join yarn with hdc in ch at base of first Fsc (When instructed to join with hdc, begin with a slip knot on hook. YO, holding loop on hook, insert hook in stitch or space indicated, YO and pull up a loop, YO and draw through all 3 loops on hook) hdc in next ch and in each ch around; join with slip st to first hdc: 32 {40-48} hdc.
Rnds 2-10: Ch 2, front hdc around same st as joining, back hdc around next st, (front hdc around next st, back hdc around next st) around; skip beginning ch-2 and join with slip st to first front hdc. Finish off.
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Q:
Swift - How can I save input data into Core Data?
I am trying to store user input to my core data but i am getting an error:
My code:
import UIKit
import CoreData
class AddFriendViewController: UIViewController, UIPickerViewDelegate, UIPickerViewDataSource {
let managedObjectContext = (UIApplication.sharedApplication().delegate as! AppDelegate).managedObjectContext
@IBOutlet weak var fName: UITextField!
@IBOutlet weak var lName: UITextField!
@IBOutlet weak var mobile: UITextField!
@IBOutlet weak var gender: UIPickerView!
@IBOutlet weak var address: UITextField!
var pickerDataSource = ["Male", "Female"];
var genderPick:String = "";
//getting picker data here
@IBAction func addFriendBtn(sender: AnyObject) {
let entityDescription = NSEntityDescription.entityForName("Friends", inManagedObjectContext: managedObjectContext)
let friends = Friends(entity: entityDescription!, insertIntoManagedObjectContext: managedObjectContext)
friends.firstName = fName.text!;
friends.lastName = lName.text!;
friends.mobile = mobile.text!;
friends.gender = genderPick;
friends.address = address.text!;
var error: NSError?
managedObjectContext!.save(&error) // error occurs here
if let err = error {
showMessage("Error While Adding to Core Data")
} else {
showMessage("Save to Core Data Successfully")
}
}
func showMessage(msg: String)
{
let alert = UIAlertController(title: "Message", message: msg, preferredStyle: UIAlertControllerStyle.Alert)
alert.addAction(UIAlertAction(title: "Okay", style: UIAlertActionStyle.Default, handler: nil))
self.presentViewController(alert, animated: true, completion: nil)
}
}
Im getting an error in this line of code: managedObjectContext!.save(&error). The error is "cannot force unwrap value of non-optional type 'NSManagedObjectContext'"
A:
var theError: NSError?
do {
try managedObjectContext!.save()
} catch {
theError = error
print("Unresolved error \(theError), \(theError.userInfo)")
}
According to Apple Doc, You need to add error handling code when you invoke save function.
Handling Errors in Swift:
In Swift, this method returns Void and is marked with the throws
keyword to indicate that it throws an error in cases of failure.
You call this method in a try expression and handle any errors in the
catch clauses of a do statement, as described in Error Handling in The
Swift Programming Language (Swift 3) and Error Handling in Using Swift
with Cocoa and Objective-C (Swift 3).
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After a years wait (and well worth the wait!), the new tank is here. Absolutely Gorgeous
I provided the foam mock-up and bike and Evan Wilcox improved on it and made it a reality. He is truly one of the few masters in the world able to do this kind of work.
We agreed that Evan can make this tank for others now that he has the templates and can reproduce it. Call him and get on his wait list if desired. For any future SC3 builds we do - this will be the tank we install.
For the exception of the new rear shocks coming next week, the bike is all together with the new triples, steering damper, forks, Braking caliper, front hub, gas tank, and crash bar.
Took it for a nice ride up in the hills (hwy9, 35, page mill - for the locals), and was very happy with the handling. The bike felt more stable at all speeds.
For the gas tank, I was shooting for 6 gallons by design. Today was the test fill to see how close it came - 6.3 gallons with room to spare! That should be good for at least 200+ miles before reserve. When Evan did the tank, he added a bit more volume by adding some crowning here and there.
The front Braking caliper also is a big improvement. Two finger pull does the job.
I may have missed it earlier, but what master cylinder are you using? What diameter piston is it?
Thanks for sharing this build with us bike building dreamers.
Click to expand...
Thanks. Im using the stock Sportster master cylinder. I didn't measure the piston diameter, so don't know. I thought id try it first to see how it would work, and it works well. I did a seal rebuild on it while it was apart too.
The new Ohlins shocks & springs arrived. 15.28" long and 4.8" stroke equates to 8+ inches at the rear wheel. The Works shocks were 15" long, but the springs were stiffer and had less sag. So, the plan is hit the same swingarm angle but with slightly more sag.
On the reg/rec relocation, maybe move it straight up the front down tube above your upper crash bar tube. That would get it up to a more crash protected spot and up above the solid part of the fender so water, mud, and debris could not collect on it.
Worth a look anyway.
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In the manufacture of integrated circuits (chips) it is well known that it is desirable to encapsulate the chip in order to protected the chip from mechanical damage and contamination. Encapsulation techniques are also known to passivate the chips an enhance their long term performance. There are a variety of well known techniques available for encapsulating chips. These techniques include mounting chips within a cavity of a substrate or a die structure, wire bonding chips to a lead frame and then enclosing the package with a lid. Another technique includes mounting chips to a lead frame, wire bonding the chips to the lead frame and then passivating the chips and a portion of the lead frame in a molded plastic or plastic epoxy body. Yet another technique for packaging and passivating chips includes “flip-chip” bonding to a printed circuit board and then covering the chips with a plastic resin.
There are several applications where the above mentioned packaging and passivation techniques are inadequate because the materials used to form the packaging are opaque and/or do not provide an optical window of suitable quality for optical applications. For example, such packaging is unsuitable for EPROM devices. An EPROM device is a read-only memory device. The program or data which is stored in an EPROM can only be erased through optical radiation (ultraviolet and/or visible) impinging on the surface of the EPROM. Conventional opaque chip packaging does not allow for such a device to be erased optically and, therefore, is unsuitable for packaging these devices. To solve this problem, makers of EPROM devices mount EPROM chips within the cavity of a ceramic package and hermetically seal the assembly with an optically transparently lid.
Micro-electro-mechanical devices (MEM devices) are another class of silicon semiconductors devices. MEM devices are useful for a variety of applications including strain gauges, accelerometers, electronic levels, and also for display light valves or other optical applications. Because of their extremely small moving parts, MEM devices are particularly susceptible to ambient conditions. Accordingly, MEM devices are traditionally sealed within the cavity of a hermetic package to control the operating environment to which the MEM is subjected.
When the MEM device is an optical MEM device, as for example in the case of display applications, the MEM device is required to be accessed optically through the packaging, wherein optical energy penetrates the package, impinges on a surface of the MEM device, and where the optical energy is reflected and/or modulated and then escapes from the package forming the optical image or signal. Though conventional ceramic packages can be hermetic, they also tend to be opaque and are, therefore, unsuitable for use with a variety of optical MEM devices.
A package which includes an optically transparent window can represent a considerable portion of the manufacturing cost for making an optical MEM device. Under certain circumstances it is important to provide a package which has an optical window of suitable optical quality which has a controlled physical relationship relative to another portion of the MEM device, such as a mechanically active portion of the MEM device or the substrate of the MEM device. Specifically, in some applications it is important to position a transparent lid at an angle relative to an optical element or elements of the MEM device to reduce surface reflections from the optically transparent window, where reflections can interfere with the intended image and/or signal.
Conventional silicon semiconductor chip packaging technology does not provide for the ability to control the physical relationship of a transparent window/lid with respect to other portions of a MEM device. Therefore, there is a need for a MEM device with an optical widow that can be controllably positioned at an angle relative to other portions of the MEM device, and in particular at an angle relative the reflective surface(s) of one or more encapsulated optical elements of the MEM device, and a method for making the same.
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Hillary Clinton said now is the time to embrace the spirit of unity, referencing the most recent terror attack in London as she praised London Mayor Sadiq Khan for his 'steady, determined leadership.'
Clinton spoke for about 15 minutes on Monday in front of several students, grass-roots organizers and political figures at a fundraiser in Baltimore for the Elijah Cummings Youth Program in Israel where she encouraged teens to 'reach out to the world and understand what is happening.'
The former Democratic presidential nominee began her speech by acknowledging the victims of the two recent terror attacks in the United Kingdom where a combined 29 people were killed.
The former first lady offered her support to Khan, who was criticized by President Donald Trump over the weekend in social media postings.
While speaking during an event in Maryland, Hillary Clinton (left) said now is the time to embrace the spirit of unity, referencing the most recent terror attack in London as she praised London Mayor Sadiq Khan (right) for his 'steady, determined leadership'
Clinton spoke on Monday in front of several students at a Baltimore fundraiser on behalf of the Elijah Cummings Youth Program in Israel where she encouraged teens to 'reach out to the world and understand what is happening'
'It's a time for steady, determined leadership, like we are seeing from local authorities in London, including the mayor of London,' Clinton said.
'This is not a time to lash out, to incite fear or to use tragedy and terror for political gain.
'Normally this would go without saying, but we are not living in normal times.'
Her remarks at the event were less political and did not seemingly focus on the November election.
The fundraiser was for the Elijah Cummings Youth Program in Israel, which is a cross-cultural collaboration between the representative and the Baltimore Jewish Council that aims to foster students from the region.
Clinton also stressed the importance of developing religious tolerance through educational initiatives.
Clinton said: 'This is not a time to lash out, to incite fear or to use tragedy and terror for political gain. Normally this would go without saying, but we are not living in normal times'
Clinton also stressed the importance of developing religious tolerance through educational initiatives
'When violence motivated by hatred from, Portland, Oregon, to College Park, ends the lives of young Americans, this program's mission of spreading tolerance is more urgent than ever,' Clinton said.
'I'm asked quite often these days, what can we do? And the answer is as varied as the questioners,' Clinton added.
'There are so many ways for us to reach out, bring people together, set some common goals and work toward achieving them, to help build that brighter future for generations to come and, yes, for building leaders by building bridges, not walls.'
The former senator also encouraged event attendees to recognize the dangers of climate change following Trump's announcement that he would pull the U.S. out of the Paris climate agreement.
Cummings' youth program pays to send Baltimore high school students to Israel as part of a study abroad program.
The Maryland representative was not in attendance at the event on Monday, as he is recovering from heart surgery at Johns Hopkins Hospital.
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Q:
Plotting piecewise function using declare function
I am trying to plot a piecewise defined function, but somehow I don't manage to get it right. See below.
I use the following code
\begin{tikzpicture}[
declare function={
func(\x)=(\x >8/10) * (6/10* \x) +
and(\x > 0.6,\x <= 0.8)* (6/9* \x ) +
(\x<=0.6) * (\x*6/8);
}
]
\begin{axis}[xmin=0,xmax=1,
ymin=0,ymax=1,
x dir=reverse]
\addplot[]{func(x)};
\end{axis}
\end{tikzpicture}
My understanding was that and(cond,cond) gives me 1 when both conditions hold and zero otherwise. Then, isn't the function I declared correct?
I see that the first part is correct.
But I don't understand why it isn't followed by a jump as supposed. I also don't understand the kink at 0.4 instead of 0.6.
I expected a function that decreases (because I reversed the x axis) and has upward jumps at 0.8 and 0.6. What am I doing wrong?
I am having the same problem with
\pgfmathdeclarefunction{func}{1}{% \pgfmathparse{...
Thanks in advance!
A:
You are not doing anything wrong. It's just that you are using the default sample number and the default domain. Adjusting them gives you the result.
\begin{tikzpicture}[
declare function={func(\x)=(\x>0.8)*(0.6*\x)+and(\x>0.6,\x<=0.8)*(2/3*\x)+(\x<=0.6)*(\x*0.75);}
]
\begin{axis}[xmin=0,xmax=1,samples=351,domain=0:1,
ymin=0,ymax=1,
x dir=reverse]
\addplot[]{func(x)};
\end{axis}
\end{tikzpicture}
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mit
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Q:
Substitution in PDE (heat equation)
Members,
i have a little question concerning the following subsitution for the heat equation $u_t=u_{xx}$.
The substitution is the following:
$$t = \zeta x^2$$
$$u(x,t)=x^cF\left(\frac{t}{x^2}\right)=x^cF(\zeta)$$
The author (Baumann, 1999: Symmetry Analysis of Differential Equations with Mathematica)concludes:
$$cF-c^2F+\frac{d F}{d\zeta}-6\zeta\frac{d F}{d\zeta}+4c\zeta\frac{d F}{d\zeta}-4\zeta^2\frac{d^2 F}{d \zeta^2}=0$$
It would be nice if someone could give me a step by step explanation for this problem.
A:
This is a self-similar transform namely $\zeta = \frac{t}{x^2}$. This problem is just a case of book keeping.
$$
\partial_x = \frac{\partial\zeta}{\partial x}\frac{d}{d\zeta}\\
\partial_t = \frac{\partial\zeta}{\partial t}\frac{d}{d\zeta}
$$
we also have
$$
\partial_{xx} = \frac{\partial\zeta}{\partial x}\frac{d}{d\zeta}\frac{\partial\zeta}{\partial x}\frac{d}{d\zeta}
$$
now we have
$$
\frac{\partial\zeta}{\partial x} = -2\frac{\zeta}{x}\\
\frac{\partial\zeta}{\partial t} = \frac{\zeta}{t}
$$
so we have
$$
\partial_x = -2\frac{\zeta}{x}\frac{d}{d\zeta}\\
\partial_t = \frac{\zeta}{t}\frac{d}{d\zeta}\\
\partial_{xx} = -2\frac{\zeta}{x}\frac{d}{d\zeta}\left(-2\frac{\zeta}{x}\frac{d}{d\zeta}\right) = 4\left(\frac{\zeta}{x^2}\frac{d}{d\zeta} -2\frac{\zeta^2}{x^3}\dfrac{dx}{d\zeta}\frac{d}{d\zeta} +\frac{\zeta}{x}\frac{d^2}{d\zeta^2}\right)
$$
Now with all these in place, I then suggest taking the derivatives with respect to the original variables but transforming $u$ i.e.
$$
u_t = \partial_t x^c F(\zeta) = u_{xx} = \partial_{xx} x^c F(\zeta)
$$
thus
$$
x^c\partial_t F(\zeta) = \partial_x\left[cx^{c-1}F(\zeta) +x^c \partial_x F(\zeta)\right] = c(c-1)x^{c-2}F(\zeta) + 2cx^{c-1}\partial_x F(\zeta) + x^c \partial_{xx}F(\zeta)
$$
or
$$
\partial_t F = \frac{c(c-1)}{x^2}F + \frac{2c}{x}\partial_x F + \partial_{xx} F
$$
now apply the transforms - then you are done.
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Jacques Davidson
Jacques Davidson (14 November 1890, in Amsterdam – 13 January 1969, in Amsterdam) was a Dutch chess master.
Before World War I, he had lived in London for a number of years. Jacques had played with his father for a stake, he had won, and though he was not paid, the idea had occurred to him that it could be profitable to play chess against wealthy Englishmen. He learned how to proceed from another Dutchman, Rudolf Loman. In the 1920s, Davidson would finish second in the Dutch championship twice, behind Max Euwe.
In 1911, he won a match against Edward Sergeant (2,5 : 0,5) in London. He tied for 3rd-5th at Tunbridge Wells 1911 (Frederick Yates won); took 15th at Cologne 1911 (Moishe Lowtzky won); tied for 2nd-3rd at London 1912 (Harold Godfrey Cole won); took 6th at London 1912 (George Alan Thomas won); tied for 4-7th at London 1913 (Edward Lasker won).
He took 2nd at Nijmegen 1921 (Euwe won); took 8th at The Hague 1921 (Alexander Alekhine won); took 16th at Scheveningen 1923 (10+10, Paul Johner and Rudolf Spielmann won); took twice 2nd, behind Euwe, in Amsterdam (1923, 1924). Davidson won at Amsterdam 1925 (Quadrangular); took 16th at Semmering 1926 (Spielmann won); took 8th at Spa 1926 (Friedrich Sämisch and Thomas won); took 2nd at Utrecht 1927 (Quadrangular, Euwe won); shared 1st with Hartingsvelt at Amsterdam 1927; tied for 5-6th at Amsterdam 1929 (Euwe won).
He played several matches; drew with Richard Teichmann at Berlin 1922, and lost to Euwe (1924, 1927) and Spielmann (1932, 1933), all in Amsterdam.
References
External links
Jacques Davidson at 365Chess.com
Grave at Zorgvlied Cemetery, Amsterdam
Category:1890 births
Category:1969 deaths
Category:Dutch Jews
Category:Dutch chess players
Category:Jewish chess players
Category:Jewish Dutch sportspeople
Category:People from Amsterdam
Category:20th-century chess players
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Pediatric erythromelalgia: a retrospective review of 32 cases evaluated at Mayo Clinic over a 37-year period.
Erythromelalgia has not been well characterized in the pediatric population. We sought to review our experience of erythromelalgia in the pediatric age group. We conducted a retrospective review of patients 18 years of age and younger with a diagnosis of erythromelalgia who were examined at Mayo Clinic in Rochester, MN, from 1970 to 2007. The records of 32 patients (girls, 22 [69%]) were evaluated. Mean age was 14.1 years (range, 5-18 years) and mean time to diagnosis was 5.2 years. Seven patients (22%) had a first-degree relative with erythromelalgia; 4 were from the same family. Physical activity was limited because of discomfort in 21 patients (66%) and school attendance was affected in 11 patients (34%). Noninvasive vascular studies, which compared temperature, laser Doppler flow, and transcutaneous oximetry in the toes, identified vascular abnormalities in 13 (93%) of 14 patients. Neurophysiologic studies with autonomic reflex screening (including quantitative sudomotor axon reflex test and thermoregulatory sweat testing) showed evidence of a small-fiber neuropathy involving the skin in 10 (59%) of 17 patients studied; there was no evidence of large-fiber neuropathy in 20 patients in whom electromyographic and nerve conduction studies were performed. Topical lidocaine was the most commonly prescribed treatment (44%). Fifteen patients were monitored for an average of 9.1 years (median, 5.0 years; range, 0.4-23.7 years). At last follow-up, 5 patients had stable disease, 4 showed improvement, two had resolution, one reported worsening of symptoms, and 3 had died (one suicide). Conclusions are limited because this was a retrospective chart review. Erythromelalgia in pediatric patients is associated with substantial morbidity and even death. The majority of cases are not inherited. Most patients studied have associated small-fiber neuropathy. The disease course is variable. A reliable and safe treatment has not been determined.
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MIAMI-DADE COUNTY, Fla. – A South Florida man turned the tables on a pair of crooks early Thursday morning after they tried to storm into his home in northwest Miami-Dade.
"I felt like if I was to die right there, they would kill my grandmother and great-grandmother, Lord forbid -- God forbid," Pherrick Thomas said.
Thomas told Local 10 News that someone knocked on his back door at 2 a.m. Thursday.
He said he opened the door to find a woman he knew standing there, Georicka Mathes, 23. Soon after, he was greeted with a gun by another man, later identfied by police as Marquan Humphrey, 26.
Georicka Mathes, 23, of Miami, faces an armed robbery charge.
"We were talking for a second, because it's 2 in the morning, I'm trying to catch up, and the next thing you know a man with a rifle ran up talking about, 'Give me everything,'" Thomas said.
A struggle unfolded at the home on Northwest 85th Street, where Thomas lives with his grandmother and great-grandmother.
The two elderly women tried to defend themselves from the man and woman invading their home.
Thomas said his grandmother fought back with a brick while he took the man's gun.
"I turned the rifle around just in time," he said. "I had enough strength from seeing them hit my grandmother to actually shoot the man."
Thomas said an officer patrolling around the corner heard the shots and responded immediately.
The two suspects left in a car but were later captured by police, authorities said. Police said a second man was also seen in the victim's backyard, but he has not been found.
Humphrey was taken to Jackson Memorial Hospital to be treated for his gunshot wound and Mathes, who police said previously dated the victim, was taken into custody.
Thomas suffered a few scratches and a gash above his eye during the incident.
"They weren't strong enough, that's all I can say," he said. "The devil wasn't strong enough tonight."
Mathes and Humphrey both face armed robbery charges.
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224 Kan. 278 (1978)
580 P.2d 86
STATE OF KANSAS, Appellee,
v.
SEAN TERRENCE DeCOURCY and KENNETH GENE CARR, Appellants. AND STATE OF KANSAS, Appellant,
v.
STEVEN RAY MOULSON, Appellee.
Nos. 49,381, 49,487
Supreme Court of Kansas.
Opinion filed June 10, 1978.
David K. Martin, of Payne and Jones, Chartered, of Olathe, argued the cause and was on the brief for the appellant DeCourcy in Case No. 49,381.
Phillip A. Miller, of McCaffree and Grimshaw, Chartered, of Olathe, argued the cause, and William Grimshaw, of the same firm, was with him on the brief for the appellant Carr in Case No. 49,381.
Emest C. Ballweg, of Prairie Village, argued the cause and was on the brief for the appellee in Case No. 49,487.
Michael B. Buser, assistant district attorney, argued the cause, and Curt T. Schneider, attorney general, and Dennis W. Moore, district attorney, were with him on the brief for the State in both cases.
*279 The opinion of the court was delivered by
PRAGER, J.:
This case involved appeals in two criminal actions which were consolidated in this court and argued together. They concern the same basic legal issues the constitutionality and interpretation of the mandatory sentencing statute (K.S.A. 1976 Supp. 21-4618). We shall consider each appeal separately.
DeCourcy and Carr
In case No. 49,381 the defendants, Sean Terrence DeCourcy and Kenneth Eugene Carr, have appealed from sentences imposed on convictions of robbery (K.S.A. 21-3426). In the district court, both defendants entered pleas of not guilty, stipulated as to the facts of the robbery, and were found guilty by the trial court on the basis of the stipulation. The stipulated facts showed that on April 7, 1977, the defendants, DeCourcy and Carr, entered a tavern in Johnson County, Kansas, played pool, and drank beer for approximately one hour. While in the tavern, the defendants discussed robbing the place. DeCourcy informed Carr that he had a rifle. DeCourcy then left the tavern and returned with a.22-caliber rifle to be used during the robbery. DeCourcy concealed the rifle under his full-length coat. After further discussion between the defendants, DeCourcy approached the manager of the tavern, exhibited the firearm, and announced that a robbery was in progress. DeCourcy and Carr then obtained money from the cash register. The manager was ordered to lie facedown on the floor. DeCourcy and Carr then fled the tavern in an automobile and went to Carr's house, where Carr placed the rifle in a crawl space underneath his house. It was stipulated that, although the holdup was a joint enterprise between DeCourcy and Carr, only DeCourcy had possession of a firearm during the commission of the offense. Following their convictions, each of the defendants made application for probation. On July 14, 1977, the defendants appeared before the trial court for sentencing. The state requested that the court impose the mandatory minimum sentence pursuant to K.S.A. 1976 Supp. 21-4618. The district court denied the defendants' application for probation without a hearing, holding that a mandatory minimum sentence was required to be served under K.S.A. 1976 Supp. 21-4618. The defendants have appealed. They do not challenge the validity of their convictions but attack *280 the imposition of a mandatory minimum sentence under K.S.A. 1976 Supp. 21-4618 and the summary denial of their applications for probation.
Both DeCourcy and Carr challenge the constitutionality of K.S.A. 1976 Supp. 21-4618 on the grounds that the statute denies them equal protection of the law under the Kansas and the United States Constitutions. This same constitutional issue was raised and determined adversely to the position of these defendants in State v. Freeman, 223 Kan. 362, 368, 574 P.2d 950 (1978) and State v. Stuart and Jones, 223 Kan. 600, 575 P.2d 559 (1978). We adhere to our decisions in those cases and, therefore, find this point to be without merit.
Defendant DeCourcy also challenges the constitutionality of K.S.A. 1976 Supp. 21-4618 as a violation of his constitutional right to due process of law under the United States Constitution. In Freeman we addressed this same basic issue and upheld the statute. In the opinion we stated that the fixing and prescribing of penalties for violating the criminal statutes of this state is a legislative function. We further held that the deterrence of the use of guns in committing crimes against persons (Article 34 crimes) is a legitimate governmental interest and the imposition of a mandatory minimum sentence bears a rational relationship to that goal. On this appeal the defendant DeCourcy argues that due process of law was violated since he was not afforded a probation hearing prior to the imposition of sentence. Probation is a matter of legislative grace and is granted as a privilege, not as a fundamental right. (Thomas v. United States, 327 F.2d 795 [10th Cir.], cert. den. 377 U.S. 1000, 12 L.Ed.2d 1051, 84 S.Ct. 1936 [1964].) Of course, once probation has been granted, it may not be revoked without notice to the probationer and an opportunity to be heard. (K.S.A. 22-3716.) Under K.S.A. 1976 Supp. 21-4618, the serving of the minimum statutory sentence is mandatory upon a finding by the sentencing court that a firearm was used in the commission of an Article 34 offense. Here the district court was without authority to grant probation. Since the defendant DeCourcy had no right to probation in this case, it was not a violation of due process of law for the district court to deny him a hearing on his application for probation.
Defendant DeCourcy next maintains that the trial court erred in finding that he had used a firearm in the commission of the *281 robbery within the meaning of K.S.A. 1976 Supp. 21-4618. DeCourcy argues that although he had possession of a firearm and exhibited it to the manager of the tavern, he never pointed the gun at the manager, never fired it, and had no intention of hurting anyone. Hence, he concludes that the gun was not "used" within the meaning of 21-4618. The statute, by its express language, prohibits the granting of probation to any defendant convicted of an Article 34 crime in which the "defendant used any firearm in the commission thereof." In our judgment, for the statute to be applicable, the state must establish, and the sentencing court must find, that the firearm was an instrumentality of the crime. In a robbery case, the statute requires proof that the firearm was used as an instrument of force to overcome the will of the victim, resulting in the transfer of possession of property from the victim to the robber. Under the facts of this case, the exhibition of the firearm to the manager of the tavern, coupled with a demand for money, constituted a use of the firearm in the commission of the robbery. Under the circumstances, K.S.A. 1976 Supp. 21-4618 was clearly applicable. The trial court properly denied probation to DeCourcy and imposed the mandatory minimum sentence for robbery as required by K.S.A. 1976 Supp. 21-4618.
The defendant Carr contends that he cannot be sentenced under K.S.A. 1976 Supp. 21-4618 because he was an unarmed accomplice and did not personally use the firearm in the commission of the crime. This undisputed fact is contained in the stipulation of facts provided by the parties. This exact point was raised and determined by this court in State v. Stuart and Jones, supra, which holds that K.S.A. 1976 Supp. 21-4618 applies only to a defendant personally armed with a firearm at the time the crime was committed and does not apply to an unarmed accomplice. In view of our decision in that case, the defendant Carr was entitled to have a hearing before the sentencing court denied his application for probation.
For the reasons set forth above, the sentence imposed by the district court on the conviction of defendant Sean Terrence DeCourcy is affirmed. The sentence of defendant Kenneth Eugene Carr is vacated and the case is remanded to the district court for a hearing on that defendant's application for probation.
Steven Ray Moulson
Case No. 49,487, the second of the consolidated cases, is a *282 direct appeal by the State of Kansas following the conviction of the defendant, Steven Ray Moulson, on two counts of robbery under K.S.A. 21-3426. After a presentence investigation, the defendant was sentenced on each count to a minimum of one year and a maximum of twenty years with the sentences to run concurrently. Defendant Moulson then made application for probation. The state opposed the granting of probation on the basis that the granting of probation to the defendant Moulson was prohibited under the mandatory sentencing provisions of K.S.A. 1976 Supp. 21-4618, since there was a stipulation by the parties that a firearm was used by the defendant in the commission of both of the robberies. Counsel for defendant Moulson challenged the constitutionality of 21-4618 on the grounds that the statute denied him equal protection of the law and provided for cruel and unusual punishment. Stated simply, it was the position of Moulson that the statute is unconstitutional because it denies to a sentencing court the power to take into consideration any mitigating circumstances in the case, including the past good conduct and tender age of the defendant. He argued that for many years the Kansas law has supported a philosophy which emphasizes the rehabilitation of persons convicted of crime as opposed to strict punishment. He maintained that K.S.A. 1976 Supp. 21-4618 ignores rehabilitation in certain cases where it can be shown that more efficient and effective means of rehabilitation are available to a convicted offender outside the penal system. Following a hearing on the defendant Moulson's application for probation, the district court declared K.S.A. 1976 Supp. 21-4618 unconstitutional and granted the defendant a five-year probation. The state appealed from the judgment of the trial court holding the statute unconstitutional and granting probation to the defendant.
The contention that K.S.A. 1976 Supp. 21-4618 violates the constitutional right to equal protection of the law was determined adversely to the position of the defendant Moulson in State v. Freeman, supra. We find that case to be controlling on the equal protection issue.
In this case, in support of the ruling of the trial court, the defendant cites Furman v. Georgia, 408 U.S. 238, 33 L.Ed.2d 346, 92 S.Ct. 2726 (1971), reh. den. 409 U.S. 902, 34 L.Ed.2d 164, 93 S.Ct. 89 (1972) and Roberts v. Louisiana, 431 U.S. 633, 52 *283 L.Ed.2d 637, 97 S.Ct. 1993 (1977). Both Furman and Roberts involved constitutional challenges to state capital punishment statutes. In our judgment, those cases are not applicable to cases involving minimum sentencing statutes such as K.S.A. 1976 Supp. 21-4618. In State v. Freeman, supra, we discussed, in depth, 21-4618 and its relation to the constitutional provisions proscribing cruel and unusual punishment. We upheld the statute. We pointed out, after discussing Furman, that the concept of cruel and unusual punishment is not rigid but acquires meaning from the evolving standards of decency which mark the progress of a maturing society. In Furman and again in Roberts, it was the method of punishment, the taking of a defendant's life, that was being considered. In our judgment, the basic issue raised by Moulson on this appeal, attacking 21-4618 on the basis that it constitutes cruel and unusual punishment, was determined adversely to his position by our decision in State v. Freeman, supra. We therefore reject Moulson's constitutional attack on the statute as being without merit. Defendant Moulson raises other issues which were not raised or determined in the trial court nor have they been briefed by the state. Under the circumstances, those issues are not properly before us in this case.
For the reasons set forth above, the judgment of the district court granting probation to the defendant Moulson is vacated and the case is remanded to the district court with directions to the district court to order execution of the minimum statutory sentence heretofore imposed by the district court on September 2, 1977.
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MRI of elastofibroma dorsi.
To determine if the MR features of elastofibroma are sufficient for diagnosis of this neoplasm. The MR studies of two patients with pathologically proven bilateral elastofibromas were reviewed retrospectively. In each patient, bilateral semilunar soft-tissue masses demonstrating signal intensities isointense to that of muscle were identified in the periscapular regions deep to the posterolateral musculature of the chest. Focal linear areas of fat were present. The biopsy specimens of both patients demonstrated a positive reaction to the elastin stains. A periscapular soft-tissue neoplasm demonstrating a pattern of linear alternating regions of high and intermediate signal intensity on T1- and T2-weighted spin-echo MRI should be sufficient to allow the diagnosis of elastofibroma.
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Q:
Test iFrame "top === self" in Python Tornado
In Tornado, you can do if statements in the HTML such as {% if true %} do stuff {% end %}. I'd like to check if the page is within an iframe.
In Javascript, it would be something like: if (top === self) { not in a frame } else { in a frame }
How can I do this in with Tornado?
A:
Javascript has access to the browser context but a templating system will only have access to the request object.
If you control the creation of the iframe in question, for instance if that is happening on another part of your site, you might be able to pass get parameters in to the templating system or something... But in general this is something you have to do with javascript. Add javascript directly to your template or (better) include a javascript file. You can expose both the iframed and the non-iframed versions of your page in the template and have javascript select which one to show once it hits the browser.
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How Alabama is the same as last year
The defense remains stout against the run
Entering last season’s national title game, Alabama’s opponents gained 2.3 yards a carry. This season, Alabama’s opponents are gaining just 2.0 yards a carry. Defensive tackle Da’Ron Payne and defensive end Dalvin Tomlinson have been nearly impossible to move, and the most success opponents have had has been outside the tackles.
Not including the 14 yards Clemson quarterback Deshaun Watson lost on sacks, he ran for 87 yards against the Crimson Tide last year. Tailback Wayne Gallman struggled (for him, but not for someone playing against Alabama), gaining just 45 yards on 14 carries. If Gallman and Watson combine for 132 yards on the ground—sacks not included—this time around, the Tigers would happily take it.
Derrick Henry was far more proven at this point last season. Heck, he’d won the Heisman Trophy. But sophomore Bo Scarbrough is about the same size, has a similar running style and might be a little faster. Henry was 6’3” and 242 pounds, and his 36 carries for 158 yards with three touchdowns helped the Tide keep pace with Clemson and gave Alabama’s defense a little rest on the sideline. Scarbrough is 6’2” and listed at 228, but teammates contend he’s closer to Henry in weight. He was the only part of the offense that worked consistently in the Peach Bowl win against Washington, and the Tide will need a similar performance to keep the Tigers from pinning their ears back and attacking Alabama quarterback Jalen Hurts.
Streeter Lecka/Getty Images
How Alabama is different from last year
The quarterback, silly
Lost in Watson’s historic performance in last year’s title game was perhaps the best game of the season from Alabama fifth-year senior Jake Coker. Even with his right tackle getting whipped on nearly every play, Coker completed 16 of 25 passes for 335 yards with two touchdowns and no interceptions. (It helped that Clemson simply neglected to cover tight end O.J. Howard on two touchdown plays that combined for 102 yards.)
Hurts must play better than he did in the Peach Bowl (7 of 14, 57 yards), but the true freshman doesn’t have to be perfect for Alabama to win. If new offensive coordinator Steve Sarkisian can find a way to let Hurts make safe, high-percentage throws to receivers Calvin Ridley and ArDarius Stewart near the line of scrimmage, then Ridley and Stewart can turn those plays into bigger gains.
Hurts also might be able to use Clemson’s aggressiveness against the Tigers. He scored the lone touchdown in a 10–0 win against LSU on a scramble, and if he gets free from a blitz, he could tuck the ball and run for a while.
The best offense is a good defense, especially when the defense is a pretty great offense
Alabama’s defense scored four touchdowns last season, all on interception returns. The Tide have 11 defensive touchdowns (six interception returns, five fumble returns) this season. What’s crazy is that Alabama defenders intercepted more passes last year (19) than they did this year (16). This group simply has a knack for making opposing offenses pay the ultimate price for mistakes. The Peach Bowl win against Washington turned on linebacker Ryan Anderson’s pick-six just before the half. Now the Tide will face Watson, who is tied for second in the nation in interceptions with 17.
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Octomom: 'I Screwed My Life Up' As the octuplets head toward 6 months, Nadya Suleman says she has deep regrets.
June 5, 2009 -- Nadya Suleman, the infamous "Octomom," expresses deep regret over her historic birth of octuplets, saying, "I screwed my life up, I screwed up my kids' life," in an interview with Radar Online.
"What the heck am I going to do? I have to put on this strong face, and I have to pretend like I don't regret it," she said.
As Radar Online cameras rolled, Suleman opened up to a friend about her "guilt" over the multiple birth and her romantic feelings for the mystery man who she says fathered the octuplets along with the six children she already had.
"I blame myself for bringing this on my own," Suleman said. "These are my choices. I coerced him into helping me every year. ... The more kids I had, the stronger connection I had with him, and I couldn't even imagine anyone else, and he knew that."
Click here to watch the Radar Online video.
Suleman, who claims to have had all her children through in vitro fertilization, said she did not tell the donor before she had more children with his sperm.
"I went behind his back, and I feel so much guilt and want to apologize to him," she said.
As for the identity of the Octodad, Suleman plans to keep quiet.
"He has his own world. He will lose everything. He will lose what he created, and it is important to him in his life. I think he is so fearful [for] what he created on his own," she said. "I live in denial, and it is my fault and that is how I live with that."
The sullen statements come just days after Suleman agreed to let a camera crew film her and her children for a reality television show.
'Regret' a Sharp Turn for Once-Happy Octomom
The Radar Online video shows Suleman with a very different demeanor than the near-beaming smiles she showed during an exclusive interview with "Good Morning America" in April as she gave a tour of the babies' nursery in her La Habra, Calif., home.
"I know I'm going to sound kind of crazy to say this, but [raising the kids] is actually a lot less stressful than I envisioned it to be," Suleman told "Good Morning America". "They're really good babies."
"I never anticipated [having] more than one [baby]. I was praying for one more," Suleman said. "If one hadn't come, I'd be happy with six."
Then, Suleman was raising the eight newborns with the constant help of at least seven nurses working in shifts -- four during the day and three each night.
The octuplets reportedly go through around 2,000 diapers every month, placing a hefty financial burden on the family.
Multiple Birth Makes History, Stirs Controversy
Likely the nation's most recognizable mother, Suleman made headlines in January when she gave birth to history-making octuplets.
Days after news of the miracle multiple birth spread from coast to coast, the public turned on Suleman when it came to light that the 33-year-old already had six children who were born, like the octuplets, through in vitro fertilization.
Now all eight of the babies, the world's longest-surviving set of octoplets, are home with Suleman and their six brothers and sisters. The last to arrive home was Jonah on April 14. Jonah weighed 1 pound 8 ounces when he was born Jan. 26.; he now weights 4 pounds 10 ounces.
"They're all here and really, really healthy," Suleman said in a Radar Online video in April. "They have very strong personalities."
Click here to watch the video or here for the first photos of the babies at home.
Nadya's Mother Criticizes Multiple Birth
Many people across the country expressed outrage at Suleman and the fertility doctor who impregnated her, saying it was irresponsible for a single woman to bring 14 children into the world without the means to care for them.
Even Suleman's mother, Angela Suleman, has been vocal about her disapproval. In Februrary, said her daughter's decision was "really unconscionable."
Click here to read Radar Online's report.
On Feb. 24, Radar Online posted video of a heated debate over the babies between Nadya and her mother.
"I will never understand," Angela said to her daughter about her decision to have in vitro. "You should have considered your other six children."
"You can't go back and alter the past. Done, done, done," Nadya replied.
Click here to watch the video.
In early March Suleman defended herself after a frantic 911 call she made when she thought she lost one of her six previous children.
Police in Whittier, Calif., say they responded eight times to emergency calls from the Suleman family.
Suleman moved out of her parents house in early March and into a larger one in La Habra, Calif. She told Radar Online she put the down payment on the home herself.
Click here to see Suleman give Radar Online a tour of the house.
Nurse Claims Suleman 'Does Not Care' About Kids
The non-profit nursing service Angels in Waiting offered to provide around-the-clock nurses to help Suleman care for the children, but there were problems from the beginning, according to reports.
A nurse who had been helping provide care for some of the octopulets said on the "Dr. Phil Show" that Suleman seemed more interested in publicity than her children .
"This woman does not care for these kids, that's my honest opinion," said Linda West-Conforti, the founder of Angels in Waiting.
Suleman fired Angels in Waiting accusing the group of being unprofessional, spying on her and reporting her to child welfare officials.
Suleman told Radar Online she nearly called police to file a restraining order against the group. In the video, she described a bizarre scene where an Angels in Waiting worker opened her purse and said how easy it would be to stuff one of her babies in there and abduct it.
Click here to watch the video.
Just days after West-Conforti claimed Suleman did not care for her children, Victor Munoz, Suleman's publicist, told Usmagazine.com she was "nuts."
"It just got to be too much," Munoz said. "Nadya got real greedy. This woman is nuts."
Suleman's first publicist, Joann Killeen, also stepped aside.
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Sccm2012 need’s some exclusions to work in the right direction and includes his own security PKI and Hashes when de deployment is running if someone like “the corporative antivirus” is scanning the inapropiate folders… it can drive to us in the common erros like 0x80004005 on task secuence or content mismatch into a normal deployment because is modifying in some way this hashes …
For the configuration manager clients the following exclusion can be added:
%windir%ccmcache
When using System Center Endpoint Protection you can use the out of the box template (SCEP12_Default_CfgMgr2012.xml) located %Program Files%\Microsoft Configuration Manager\AdminConsole\XmlStorage\EPTemplates.
In the template the following folders and filetypes are excluded:
%allusersprofile%\NTUser.pol
%systemroot%\system32\GroupPolicy\Machine\registry.pol (update 30/1/2014; in the Template \Machine\ is left out, thanks to Kim Oppalfens)
%windir%\Security\database\*.chk
%windir%\Security\database\*.edb
%windir%\Security\database\*.jrs
%windir%\Security\database\*.log
%windir%\Security\database\*.sdb
%windir%\SoftwareDistribution\Datastore\Datastore.edb
%windir%\Software\Distribution\Datastore\Logs\edb.chk
%windir%\Software\Distribution\Datastore\Logs\edb*.log
%windir%\Software\Distribution\Datastore\Logs\Edbres00001.jrs
%windir%\Software\Distribution\Datastore\Logs\Edbres00002.jrs
%windir%\Software\Distribution\Datastore\Logs\Res1.log
%windir%\Software\Distribution\Datastore\Logs\Res2.log
%windir%\Software\Distribution\Datastore\Logs\tmp.edb
for the next folders both “Program Files” and “Program Files x86″ paths are listed:
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Topic: rooster soup (Read 3459 times)
I had african geese, they were soooo funny! I could hold the girl & pet her, she loved it...the whole time the gander would stomp around, visibly shaking & hissing doing that snake deal with his neck, never getting his beak closer than an inch to any part of my body. He would tear up a glove or baggie when I dropped it. I would turn my back on him then turn around really fast to see how quickly he could stop! :evil: He would however wing slap & bite others. When he first started thinking he was a tough guy I would hold him, talking in a high syrupy voice & kiss him on his beak bump! Of course this was done in front of the goose he was trying to show off for. He was mortified & soon learned not to mess with me. It is also fun to catch the ones at the park when they think they are tough..the look on their little faces when they realize I'm not scared is priceless!! My Daughter does the same thing & the geese remember you! :-D J
Butchered one of my home grown turkeys for Thankgiving today. Due to family obligations we couldn't get all the kids and grandkids together until Tomorrow (Sunday). Size wise the turkey is the same volume as the one we had on thursdays for those who live here, but weight wize it was 5 lbs heavier. The breast is bulging and hard from flying around the pen and up and down from the roof.
My 2 daugthers are doing most of the cooking and are taking the time to fix the pumpkin pie using Cocnut milk so I can have some. They are using CM in other things as well due to my food allergies.
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Life is a school. What have you learned? :brian: The greatest danger to our society is apathy, vote in every election!
Butchered one of my home grown turkeys for Thankgiving today. Due to family obligations we couldn't get all the kids and grandkids together until Tomorrow (Sunday). Size wise the turkey is the same volume as the one we had on thursdays for those who live here, but weight wize it was 5 lbs heavier. The breast is bulging and hard from flying around the pen and up and down from the roof.
My 2 daugthers are doing most of the cooking and are taking the time to fix the pumpkin pie using Cocnut milk so I can have some. They are using CM in other things as well due to my food allergies.
Oh Brian, that goes to show you how much your Daughters love you, taking that extra effort and time to get that coconut milk to put as an ingredient so you can have some of that yummy stuff too. That is going an extra mile, and I take my hat off to these gals. What more could you ask for, not much I would say, the love of family.....say no more, it makes my heart swell....
Now, you must tell a little bit about these turkeys. I know that when we were down for the bee barbeque at the end of August, I saw these two birds. At that time they looked the same size. How on earth could one of them have gotten so ding dang much bigger than the other one. Like five pounds is a lot of weight.....They were really big birds. Do you have any idea what their finished weight was, I am curious, you know me, hee, hee.... I think back and compare these two hens to my bronze heritage tom, Richard, I think even when I was at your place they may have surpassed him in size, but that is kind of hard to tell exactly, from how my memory serves me.
What I am wondering here is: were those two bronzes that you had there the ones that they call "double breasted"? That makes for much more breast meat than a regular heritage bird. Do you know this answer? Mine is not that "double breasted" one, maybe that is why mine looked to be a little bit smaller, in my mind's eye. My Richard doesn't fly up onto the roofs or anything really high. He did fly to the chickenhouse roof a couple of times when we first got him last December, but he was only 9 months old then. Now he is one and one half years old, he was born June of 2007. I think he is too big to fly up high. He can fly up on his roost inside his house, and onto the fences, but he doesn't even bother much with flying up on things, think he knows his limits. Now the girls, they are always flying up onto high things. Have a great and most wonderful day, Cindi
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There are strange things done in the midnight sun by the men who moil for gold. The Arctic trails have their secret tales that would make your blood run cold. The Northern Lights have seen queer sights, but the queerest they ever did see, what the night on the marge of Lake Lebarge, I cremated Sam McGee. Robert Service
Some 271 million turkeys will be raised in the United States this year, according to the National Turkey Federation, and a good number of them will be consumed on Thanksgiving, after which many Americans will loll about, overstuffed, sleepy and in many cases intoxicated.
This is not what the Pilgrims had in mind.
The first Thanksgiving was a moment for the Pilgrims to thank God for allowing them to kill enough game and grow sufficient crops to get through the winter, says Anne Blue Wills, assistant professor of religion at Davidson College. Those Pilgrims would have spent much of their day in church contemplating the mercies of God's covenantal love, Wills argues.
Not until Sarah Hale, editor of Godey's Lady's Book and Magazine promoted for 50 years the idea of a regular Thanksgiving holiday did President Abraham Lincoln make it one in 1863. In the years since, the turkeys we eat have changed dramatically, and so has scientific knowledge of them. Among the things you might not know:
1. Turkeys Can FlyWild turkeys feed on the ground, which might explain the myth of their flightlessness. They can in fact soar for short bursts at up to 55 mph. But their tendency to stay on or near the ground contributed to successful hunting that brought the wild population of turkeys down to about 30,000 in the 1930s. There are now 7 million of them.
2. Dark Meat is Rare Because ...Meat is muscle. And muscle is fed by blood. In the blood is myoglobin, which binds with oxygen and stores it in muscles for when it's needed. Myoglobin also makes meat dark. Muscles that are used most, like those in drumsticks (legs), have more myoglobin. Domestic turkeys are too fat to fly, so they don't use their breast muscles much, which is why breast meat is white. The breast of a wild turkey is entirely different, darker (and far tastier for those who are game).
3. Turkey Eggs Wouldn't SellChickens are champion egg-producers. Turkeys, not so good. Turkey eggs are bigger, so their nests tie up coop space. And farmers have learned that they make more raising turkeys for meat rather than eggs. Oh, and some turkeys are protective of their eggs, making the gathering more challenging.
4. It's Not the Turkey That Makes You SleepyTurkey contains a natural chemical called tryptophan, which we need to build proteins for our bodies. Indeed, tryptophan is also related to the production of serotonin, which helps us sleep. But all meat has about the same amount of tryptophan. Cheddar cheese has a lot more. What really makes you sleepier after a Thanksgiving meal compared to other meals is eating too many carbohydrates, from potatoes to pies. Alcohol can contribute, too.
5. Dinosaurs Had Wishbones, TooThe wishbone, called a furcula, is the fusion of two collarbones at the sternum. It's where a bird's flying muscles hook up. It's elastic and great for flapping. Turns out T. Rex and the Velociraptor had wishbones, too. While they didn't fly, this fairly recent discovery is one of the many bits of evidence that shows birds evolved from dinosaurs.
Sarah Hale probably never thought about any of this back in the mid-1800s. She just wanted the nation to celebrate a pious, patriotic holiday, said Wills, the Davidson College researcher. Hale used columns and stories in her magazine to portray Thanksgiving as a triumph of domesticity and rural simplicity over urban sophistication. She urged President Lincoln to create a single day on which all states would mark the holiday as a national event.
"The message is that the simple, pure, honest rural life, away from the temptations of the city, puts you in touch with true values," Wills said. "If we can just travel back to the old home place once a year we'll be protected from temptations and evil."
While some of that spirit might remain in the holiday, much indeed has changed about American culture in general and in how people view and partake in the holiday.
"For instance," Wills said, "I don't think football games and making the day after Thanksgiving the biggest shopping day of the year ever crossed her mind."
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:rainbowflower: Light travels faster than sound. This is why some people appear bright until you hear them speak. :rainbowflower:
Jerry, well done, thank you for bringing that in here, that was a VERY interesting read, beautiful day in this great life, Cindi
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There are strange things done in the midnight sun by the men who moil for gold. The Arctic trails have their secret tales that would make your blood run cold. The Northern Lights have seen queer sights, but the queerest they ever did see, what the night on the marge of Lake Lebarge, I cremated Sam McGee. Robert Service
Dark Meat is Rare Because ...Meat is muscle. And muscle is fed by blood. In the blood is myoglobin, which binds with oxygen and stores it in muscles for when it's needed. Myoglobin also makes meat dark. Muscles that are used most, like those in drumsticks (legs), have more myoglobin. Domestic turkeys are too fat to fly, so they don't use their breast muscles much, which is why breast meat is white. The breast of a wild turkey is entirely different, darker (and far tastier for those who are game).
Since the bird I butched liked to roost atop the chicken house and had been known to fly the coop now and then the white meat was actually light brown. The breast meat was also much denser due to the flight habits of the bird. Pigeons, whose wishbone is fused with the keel bone, are nothing but red meat. The breast muscles are used in flight. My pigeons have larger hearts than either the chickens or the rabbits I butcher.
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It's Not the Turkey That Makes You SleepyTurkey contains a natural chemical called tryptophan, which we need to build proteins for our bodies. Indeed, tryptophan is also related to the production of serotonin, which helps us sleep. But all meat has about the same amount of tryptophan. Cheddar cheese has a lot more. What really makes you sleepier after a Thanksgiving meal compared to other meals is eating too many carbohydrates, from potatoes to pies. Alcohol can contribute, too.
I'm usually alseep by the time the desert is served. I don't drink alcoholic beverages and I'm allergic to most cheeses so it has to be the Turkey and the Potatoes.
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Life is a school. What have you learned? :brian: The greatest danger to our society is apathy, vote in every election!
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Q:
Can we access Session variable in Asp.Net from SIlverlight Application
Can we access Session variable in Asp.Net from SIlverlight Application
A:
There are two approaches to getting the value of Session variable.
The first is to include the value in the generated HTML of the ASPX page hosting the Silverlight application. Add the value in the InitParams <Param> tag of the silverlight object.
<param name="initParams" value="myValue=<%=Server.HTMLEncode(Session["myValue"].ToString())%>" />
Now in Silverlight code you can access this value:-
string myValue = Application.Current.Host.InitParams["myValue"];
The above is the most likely scenario. If though you need to also mutate the session value during execution of the Silverlight application and/or read a potentially changes value for the variable then things are tricker.
At this point many would probably advise the creation of some WCF to assist with this. Alternatively I might be inclined to create a .ashx file that simply accepted and/or returned some Xml that can assist with such very simple server side work.
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In electronic devices, such as mobile phones, printers, digital and video cameras, GPS devices, panel computers, personal computers, and the like, for convenience in operation, two printed circuit boards (PCBs) are generally electrically connected by a board to board electrical connector. Such a connection is used to transmit electrical power and data signals between the two PCBs.
However, these conventional electrical connectors are limited to a low levels of current, such as 2 amps. Therefore, the conventional electrical connectors are unable to meet the high transmission current demands required in modern electrical devices, such as mobile communication devices and the like.
There is a need for an electrical connector for connecting two PCBs that permits high levels of transmission current.
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News
June6, 2016
Celebrating National Gardening Exercise Day
Being around nature has always been therapeutic, but gardening especially has its advantages. It benefits your mind, body and soul. June 6th is National Gardening Exercise Day! This is an entire day dedicated to recognizing the important health benefits of gardening. Check out a few of the many advantages to be gained from gardening listed below.
Physical Exercise
There’s no question that gardening is a physical hobby, but it is also more exercise than some may expect. Any repetitive activity like digging, planting or weeding are useful forms of low-impact exercise, especially for those who find high-impact activity a challenge.
Gardening also has the advantage of being goal-oriented, meaning people are more likely to stick with it. It’s not just exercise for exercise’s sake—you are capable of literally seeing your efforts planted and growing into something beautiful.
Stress Relief
A recent Dutch study by Wageningen University and Research Center asked two groups to complete a stressful task. Afterwards, they had one group of participants garden for 30 minutes, while the other read indoors. The gardening group not only reported being in better moods, but also had remarkably lower levels of the stress hormone cortisol, which has been linked to everything from obesity, to memory or learning problems and to heart disease.
Today’s society is consistently fast-paced and surrounded by to-do lists. Taking a few minutes each week to relax and garden not only breaks the pattern, but can also relieve stress and pressure.
Brain Health
Physical activity has also been associated with increased brain health. Two recent studies tracked individuals in their 60s and 70s for 16 years, and found that those who gardened regularly had between a 36 and 47 percent lower risk of dementia and Alzheimer's than non-gardeners, even when outside health factors were taken into account.
Even being in a garden environment can be therapeutic, as many residential homes for people with dementia have implemented gardens on their grounds, to help improve patients’ dexterity and problem-solving.
Mental Health
Likewise, gardening is an excellent source of boosted mental health. Horticultural therapy is a treatment some therapists have begun using to help patients cope with issues such as anxiety and depression through gardening. Benefits stem from a combination of physical activity, awareness of natural surroundings, cognitive stimulation and work satisfaction. Patients who have utilized this type of therapy have reported a renewed desire to live, decreased anxiety and improved self-worth. Gardening therapy has been used in individuals suffering from various ailments, including depression, addiction, eating disorders and more. With success like that, there’s no reason not to partake in this special holiday.
To celebrate National Gardening Exercise Day, go out and see how you can utilize a garden to enjoy its plentiful benefits. Soak in the rainbow of colors, feel the fresh dirt between your fingers and smell the array of crisp fruits and veggies. Embrace nature, and nature will never cease in giving back to you.
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Sexy to Go Gay Romance “Top 10 Amazon LGBT Anthology”
Sexy to Go Gay Romance features nine scorching hot MM romances to make your blood sing. Whether finding love at a Jewish singles dance or while crossing the River Styx, these men remind us that love is always worth fighting for, no matter what the cost. Meet a handyman who’s an ace with his tools, a king who kindles the passion of a Greek god, and an architect who erects more than buildings. From the everyday to the fantastical, these stories explore male-male love in all its simmering intensity.
Excerpt from Matzo Ball by Avery Duran
I walked toward the hot bartender, wondering exactly what he wanted from me. He gestured for me to follow him. His long legs moved quickly, but I didn’t mind walking behind him since it meant I could watch that tight ass move in his clinging pants. The rest of him was just as sexy as I had expected, broad shoulders tapering down into a small waist that emphasized his perfect rear. He was a little taller than I, which would make him just over six feet. We walked through a door marked “Employees only” and down a long, winding hallway. I suppressed the voice in my head reminding me this was a set-up for an episode of Unsolved Mysteries.
Finally, some sense of self-preservation emerged and I asked him, “Where are we going?”
He turned around, smiled at me, and leaned against the wall. “Here is good.”
I looked around the barren hallway, “But…what am I helping you with?”
“It’s actually more of what I helped you with.”
I was stumped. I mean, the guy was hot as hell, but I was starting to feel as if I’d fallen down the rabbit hole. I really hoped he wasn’t going to try to sell me something, or rope me into a get-rich-quick scheme. The time for me to make my escape had definitely come. Pity such hotness was going to waste. “Look, buddy, I appreciate whatever you mean to do, but I’m meeting a friend and should get going.”
He started to laugh and I froze—seriously, I could not move. When he smiled, his eyes crinkled up at the corners and his whole face lit up; I had never seen anything more mesmerizing.
He put his hand out. “Wait a minute, was I reading you wrong? Because seriously, I did NOT think you were interested in giving that woman her dream of two-point-five kids and a white picket fence.” He put his hand on my shoulder. “C’mon, we can go back.”
“Well, I have to go back if I want to get paid, but I can get you out the back door—for a price.”
“What’s the price?”
One side of his mouth kicked up. “Your number and a kiss.”
Styx & Stone by Leigh Ellwood
What’s the afterlife really like? Charon the ferryman knows the River Styx like the back of his hand, and he enjoys giving newly departed Stone the full tour.
First Swallow of Spring by Asta Idonea
The first swallow of spring draws Seanán back to the fae circle each year, where he dances with the handsome fae lord, Iorweth. He knows the four rules he must follow if he wishes to be free to leave at the end of the night; however, Iorweth is growing ever more inventive in his attempts to trick Seanán into breaking them.
How Hercules Got His Bruise by Eva Lefoy
Hercules might have completed his 12 labors and become immortal, but he’s still not out to himself. Until he meets Sisyphus, a man who turns him on in ways not even a goddess can compete with. When Zeus threatens to destroy their new bond, Hercules must again rise to the challenge, this time to protect his right to love a man.
Loggerhead by Dale Lowry
Soon after they fall in love, Jake makes Eric a promise inspired by an old track uniform. But demanding work schedules at Jake’s four-star restaurant and Eric’s newspaper keep them from following through. Six years later, they take the honeymoon they never had, heading to the Florida coast in search of sea turtles – and rekindling their passion for each other in the process.
Handyman by Jodi Payne
Danny is haunted by memories of his ex, Peter, who moved out six months ago. He recognizes just how bad off he is when he wakes up to a flood in his condo, a problem Peter would have adeptly handled. Danny can’t find the insurance paperwork, he doesn’t know who he should call first, and he’s about ready to strangle his stoner neighbors. His day starts looking up, though, when the workmen arrive to deal with the water, replace his breaker box and demolish the soaked ceiling. Ken, a handyman, shows up to handle the drywall, but can Danny handle Ken?
Clipped Wings by Shiloh Saddler
When a male swan shifter is captured by a cruel master he develops feelings for another male slave. The master has plans for the shifter that go against every fiber of his being. Will the two men bow down to their station in life or take flight to keep their love alive?
Rebuilding the Future by Sam Thorne
Allen’s peace has been seriously disturbed. As the shy architect takes on the task of extending the home he inherited from his late aunt, his life becomes a battle. The building work is going fine, but he can barely contain his one-way lust for sexy foreman Declan, who’s a one-man generator of testosterone and mixed messages. As the build completion approaches, Allen’s ready to do just about anything to keep Declan around a little longer…
Man in the Mirror by A.E. Wasp
A companion story to the Paper Hearts novel, Man in the Mirror is the first short in a new erotic series set in the world of the Veterans Affairs books.
Benny hasn’t let Mikey see him naked in a week, will barely let Mikey touch him. Mikey’s going to get to the bottom of the issue if it takes all night.
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CDT New Mexico: Northern Highlands
My last night in Colorado found me on the side of a stream next to the highway, filtering water and trying to figure out where I was going to spend the night. I hadn’t done many miles that day, but having been harassed by a pack of dogs twice made it feel like a much longer day.
“13 mile highway walk to Cumbres Pass? That’ll take a long time,” a local told me. I’m pretty sure he thought I was a hobo. Granted, I look the part.
“I was hoping to get there by 11am with a late start tomorrow,” I absentmindedly mention, trying to calculate how much water I’ll need. Non-thru hikers have no concept of how far we go in a day. “Is this national forest land?”
“Uh, yeah, I think you can camp anywhere here.” And I did just that. It was a pretty stellar campsite, out of sight of the dirt road (Great Divide Alternate) I’d been following for 140 miles to bypass the snowy, frigid San Juans.
This is the actual New Mexico/Colorado border. Three miles north of this point, at Cumbres Pass on the highway, we hitch into the town of Chama after a 13 mile highway walk. Cumbres Pass is technically still in Colorado, but is just an hour’s walk on the trail from this photo (the New Mexico border). It’s a little confusing because you hitch from the Pass to Chama, in New Mexico, and then hitch back to the Pass in Colorado and then cross the New Mexico border 3 miles later to the south in the woods. By the time most of our family and friends find out we’re in Chama we’re already back on trail and have actually crossed the border in the forest. So we just said we were in New Mexico when we got to Chama and claimed victory, even though we still had 3 miles once we got back on trail at Cumbres Pass until we crossed into New Mexico on trail. If I had my maps out I could explain it better.
Story of Colorado: the night started off surprisingly warm, then around 4am became insanely cold. It was probably my coldest night on trail, and I later heard it was around 11F/-12C. When it’s that cold it’s hard to sleep, so I just tossed and turned for a few hours until it became semi-light enough for me to semi-safely walk on the highway. There was very little traffic, with a decent enough shoulder, so it could’ve been a lot worse.
It gets warmer as the sun comes up, but then when I’m on the high point that trend starts to reverse itself. It just gets colder and colder, and I put away my trekking poles so as to be able to warm up my hands with my breath.
Picked up a cool new hat in Chama after I left my old one in a hostel in southern Colorado. The old one had thousands of miles on it and I was going to replace it anyways. This was the hat that fit me the best at the dollar store, really the only store in town, and I figured why not? The locals all think I’m insane already so it doesn’t bother me.
Then the wind picks up and I just can’t anymore. It’s getting colder and colder. My fingers won’t move on their own, and my water bottles are all frozen. Fuck this I hate everything and especially the warm dry cars zooming by and why am I out here this is so stupid I just want to go home and why am I out here I should’ve just gone to medical school – the last thought jolts me out of my pity party. I need to get warm, fast.
I stop in someone’s driveway on this high, dry, and isolated highway. I don’t care how weird it looks being in the dirt, I just need to be able to feel my fingers again. I throw all my belongings on the side of the road and shove myself into my 10F/-12C down sleeping bag and put my hands in my armpits until I can move them again. Once they’re operable, I start eating my pumpkin spice belvita cookies which are basically like crack cocaine at this point sooooooo good.
This was from northern New Mexico, the first 90 miles of which are basically cold and high like Colorado. On the PCT, the transition from the southern California desert to the High Sierra took days and was quite gradual. On the CDT, I was in the pine forest in below freezing temps and an hour later was in the desert surrounded by cacti. The sudden, steep drop to the desert floor was hell on my knees. Sometimes out here I feel like I’m 26 going on 80.
I check my phone, only 4.5 miles to Cumbres Pass. You can do this you can do this you can do this, I tell myself as I think of all the obnoxious people who told me I was basically going to die in the snow in Colorado. My hatred for them is what pushes me through the last hour, gonna prove them wrong and finish this goddamn state.
“Hey kid, you want a ride to Chama?” some saint in a camper van calls out a mile from the pass, where the CDT leaves the road into the wonderful forest and leaves this hellscape behind and I’ll hitch into Chama and everything will be fine because I’ll be in town and it’ll be in New Mexico. It takes about three seconds for me to realize I’ll just hitch back to this point rather than the pass and have an extra mile on my way to the next town.
“Yes!” I almost scream, and jump in the stranger’s car.
“I live near the Arizona Trail, and come up here to take photos a lot. I pick up Arizona Trail hikers and you CDT folks all the time,” he tells me as I hug the heater. “You know how cold it is out there?”
“No, and I don’t want to know,” I respond before he can tell me. Ignorance is bliss. I don’t even catch my savior’s name. He drops me off in front of a restaurant he recommends and I rush inside into the warmth and central heating.
Immediately upon walking into the restaurant in Chama somebody asks, “Hey, were you the drum major that used two batons at Upper Arlington High School? You’re Cormac’s brother, right?” I was shocked for a few seconds before I remembered my youngest brother mentioned a friend of his from marching band is hiking the CDT southbound this year. It makes sense now that on the Idaho border a motel owner remarked that I was the fifth hiker in three days from Columbus, Ohio. Small world. He was a freshman the year after I graduated, so we never met before this. His trail name is Oilcan, and I still have no idea what his real name is.
There are 15 southbounders in Chama, the most I’ve seen thus far, with almost everyone left on trail in Colorado having gone home or hitched down here. Technically the trail doesn’t enter New Mexico three miles after Cumbres Pass, but I don’t care and am calling it a victory here. The fifth and final state!
I was concerned I’d gotten a motel room at a brothel again (I did once in rural Ethiopia by accident and it was actually quite a nice establishment). But it ended up being nice and warm, with a great heating system. Not bad for $37.50 including tax.
Sonic did a combination of hitching and snow traverses to get down here, and we hang out one last time before her friend picks her up and drives her to the Utah/AZ border to start the Arizona Trail. That or the Colorado Trail will likely be my next long-ish hike. This has been great, but taking almost five months to walk cross country is deeply exhausting.
“Receiving a care package?” a local woman asks me in the Chama post office.
“Kind of. I’m sending one to myself. Because there’s no store in Pie Town and I don’t want to starve to death in the desert,” I tell her. In retrospect, I probably looked and sounded insane.
“You better watch out for bears out there,” she tells me in a very condescending tone.
“That’s really not one of my top concerns,” I reply. Damnit, not this again. Lightning, hypothermia, and getting lost are WAY bigger concerns out here.
“Well, it should be! We’ve been having lots of issues with bears in town.” I don’t reply. If you think bear problems in a town with dumpsters and lots of easy food are reflective of bears on trail in the remote wilderness then you’re an idiot, I say in my head.
“You’re going to have trouble beating winter,” some dude tells me, which is the thousandth time I’ve heard it on this trail.
“Too late, we already did,” Sonic responds. The next 90 miles are basically Colorado, then we descend to Ghost Ranch and the desert valley and warmth (I hope?).
A Chama trail angel gave me and four others a ride back to the trail. We camped early most nights, not really in a rush now that we’ve more or less beat winter. From L to R: Stomper, Surprise, me, Murphy.
Stomper took this photo of me walking across the last stretch of real higher elevation trail in northern New Mexico. It was comparatively cold at night, but still much warmer than Colorado already.
As per CDT fashion, there are multiple routes and although I started with a group of five we quickly get separated. I take a break at what my maps note is a “remote campground,” which must be really remote if it says that out here. Usually you cross a paved road every four or five days, and then it’s a 30 mile hitch to the closest tiny town (if there are any towns nearby at all). Quite a difference from the PCT and AT, where you can usually get to a major metropolitan area within an hour from a road.
There’s a vault toilet in the campground and I head up to it. There’s something weird about the door, like it’s been recently painted. The lock isn’t working either, and there’s a cord to hold onto while you’re on the toilet so that it doesn’t fly open while you’re using it. And there’s a weird pinkish stain in the corner…
Looking closer at the door, I can still see etched into it:
DEAD CDT HIKER INSIDE
CALL COPS
OTTER
In November of 2015, an experienced hiker was caught in a snowstorm here and took refuge inside the privy. I can’t vouch for its veracity, but I’ve heard he was pretty stoned and wasn’t paying attention to weather reports. Either way, he became trapped in there by the snow with no way out. It’s not certain exactly when he died, but his journal entries continued through the middle of January. That’s months spent inside that vault toilet.
I talked to a guy who’d hiked the CDT northbound in 2016 who said they were all aware that he’d gone missing the previous autumn, and that they were to be on the lookout for remains in that area. One of the first northbounders, passing the privy, found the aforementioned etching plus a note tied to the door: “Warning there is a dead human body inside bathroom locked in….. Please Notify Authorities Immediately – NOT A JOKE.” The hiker reported what he’d found to authorities. There’s a newspaper article about the event here.
I made a box of food back in June for my father to send to me at Ghost Ranch, a Presbyterian spot in the desert where there’s no store for resupply.
I got to Ghost Ranch, a cool little spot, hours before my friends. So I just sprawled out on the front porch, charged my stuff, and relaxed.
This was my view from my spot in the photo above this one. Not much like this back in Ohio. Not pictured: getting an inch long cactus needle embedded in my thigh and pulling it out with tweezers, drenched in blood.
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2 thoughts on “CDT New Mexico: Northern Highlands”
Omg I’m laughing so much my husband wants to know why! My hands are so cold reading this that I’m hugging my hot tea cup! An inch long cactus spine covered in blood! Jeez don’t hold back Connor! I love it that you guys take time to write this stuff down (double baton drum major?! Who knew?). And I believe you guys will make it. I know you will. I send you my good juju on sweetgrass smoke that you make it.❤️
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LOS ANGELES (AP) — Betty Garrett, the vivacious Broadway star who played Frank Sinatra’s sweetheart in two MGM musicals before her career was hampered by the Hollywood blacklist, has died in Los Angeles, her son said Sunday.
Garrett was best known as the flirtatious girl in love with the shy Sinatra in "Take Me Out to the Ballgame" and "On the Town," both in 1949, and later in life she became well-known to TV audiences with recurring roles in the 1970s sitcoms "All in the Family" and "Laverne and Shirley." Garrett died Saturday at Ronald Reagan UCLA Medical Center, most likely from an aortic aneurysm, said her son, Garrett Parks.
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Q:
Regex - Remove all matches leaving the last
I need a regex to remove dots in an number but just leaving the last. Example:
12312.123132.12312.131.3131.3123.13123.1231
to
12312123132123121313131312313123.1231
I tried some expressions but none worked.
A:
In addition to the other answers, here is one more
[.](?!\d*$)
or
[.](?![^.]*$)
A:
This regex detects all but the last dot: [.](?![\w]{2,4}$)
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Up regulation of AT4 receptor levels in carotid arteries following balloon injury.
Angiotensin IV, (V-Y-I-H-P-F), binds to AT4 receptors in blood vessels to induce vasodilatation and proliferation of cultured bovine endothelial cells. This latter effect may be important not only in developing tissues but also in injured vessels undergoing remodelling. In the present study, using normal rabbit carotid arteries, we detected AT4 receptors in vascular smooth muscle cells and in the vasa vasorum of the adventitia. Very low receptor levels were observed in the endothelial cells. In keeping with the described binding specificity of AT4 receptors, unlabelled angiotensin IV competed for [125I]angiotensin IV binding in the arteries, with an IC50 of 1.4 nM, whereas angiotensin II and angiotensin III were weaker competitors. Within the first week following endothelial denudation of the carotid artery by balloon catheter, AT4 receptor binding in the media increased to approximately 150% of control tissue. AT4 receptor binding further increased in the media, large neointima and re-endothelialized cell layer to 223% at 20 weeks after injury. In view of the known trophic effects of angiotensin IV, the elevated expression of AT4 receptors, in both the neointima and media of arteries, following balloon injury to the endothelium, suggests a role for the peptide in the adaptive response and remodelling of the vascular wall following damage.
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I don’t think there actually is a collective noun for complexes, I feel sure that if there were it wouldn’t be a complexity… maybe a difficulty of complexes or a confusion of complexes…
Complexity would likely be the collective noun for puzzles or some such thing…
Anyway…
Back to complexes…
A complex, in this context, is a series of lifts or exercises that is done back to back without putting the bar / dumbbell down. For example, 8 reps each of: deadlifts, cleans, OH presses and back squats then rest and repeat
This has the advantage of being super efficient, a combination of lifting and cardio, not super boring and does all that good stuff like boost muscular endurance and metabolism and suchlike!
It has the disadvantage of being super hard and making you want to fall down!
In case you hadn’t guessed where this was going…I have been adding these into my workouts recently…
I was looking for options as I had got into a very samey pattern with lifting, which I still love, I just thought it is good to mix in some more heart-rate-raise-y whole body fun stuff!
I have been doing a mixture of barbell and dumbbell complexes, and a couple of times I have worked them as a superset as I was training with another person so we did one complex each then swapped… this, when it gets right down to it, is pure madness!
I had to play with the number of reps because I can lift a lot more on a deadlift than I can on a military press… so I upped the reps of the deadlift so I could use the same weight for all of them…switching weights sort of defeats the purpose!
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Isaiah 6:1 In the year that King Uzziah died, I saw the Lord seated on a throne, high and exalted, and the train of his robe filled the temple. 2 Above him were seraphs, each with six wings: With two wings they covered their faces, with two they covered their feet, and with two they were flying. 3 And they were calling to one another: "Holy, holy, holy is the LORD Almighty; the whole earth is full of his glory." 4 At the sound of their voices the doorposts and thresholds shook and the temple was filled with smoke. 5 "Woe to me!" I cried. "I am ruined! For I am a man of unclean lips, and I live among a people of unclean lips, and my eyes have seen the King, the LORD Almighty." 6 Then one of the seraphs flew to me with a live coal in his hand, which he had taken with tongs from the altar. 7 With it he touched my mouth and said, "See, this has touched your lips; your guilt is taken away and your sin atoned for." 8 Then I heard the voice of the Lord saying, "Whom shall I send? And who will go for us?" And I said, "Here am I. Send me!"
Today is Trinity Sunday. It’s a day to take a big, deep breath and confess the incomprehensible – God as one divine Being in three Divine Persons, a Unity in Trinity and a Trinity in Unity. Or as we say, Triune. It sure is confusing, isn’t it? We were baptized in the name of the Father, Son and Holy Spirit. We begin our worship in the name of the Triune God and we end our worship with the Benediction of a threefold blessing of our Trinity. We confess our faith in the Trinity in our Creeds and sing of His majesty in the songs “Holy, Holy, Holy” and “Glory Be to the Father,” yet we never come any closer to wrapping our minds around this concept. Something you can’t rationalize or harmonize, you can only believe and confess.
You may try to picture the Trinity, but be careful; analogies break down quickly. Some compare the Triune God to the three phases of water – solid, liquid and gas. It sounds good, but it doesn’t hold water for long. Liquid, ice, steam - three forms but its all H2O. Years ago, one of my confirmands thought she had it figured out saying it was like her dad being a soldier, a husband, and a father. Not even close. Father, Son, and Holy Spirit aren’t modes or roles. They are persons with whom one has a personal relationship.
Triangles, circles and three leaves of the shamrock have been used throughout Christian history to describe (not explain) the mystery of the Trinity. Our God isn’t a Deity we can turn into a concept, an illustration, a theory or a picture in our minds. Holy Trinity Sunday is a reminder that we can’t put God in a box or fit Him neatly inside our heads.
A Bible story that reflects the awesomeness and majesty of our Triune God took place 2755 years ago. The prophet Isaiah was given a grand vision of angels around the throne of God. “In the year that King Uzziah died (740 B.C.), I saw the Lord seated on a throne, high and exalted, and the train of his robe filled the temple.”
“Above him were seraphs, each with six wings: With two wings they covered their faces, with two they covered their feet, and with two they were flying.” Seraphs are angels. Rather unusual looking angels since they each had six wings. What did they do with these wings? “With two they covered their faces.” Covering the face was a sign of humility. “I’m unworthy to be in your presence, God.” Even holy angels recognize that they are not on God’s level. He’s too awesome, too amazing, too majestic, too mighty for them to dare to assume they can be His equals.
Isaiah was given this vision of angels around the throne of God, and his reaction of “Woe! I’m unworthy” is even more justified than that of the angels because Isaiah was a mere mortal. Isaiah cried, “Woe to me! I am ruined.” Isaiah was granted a glimpse of what’s its like to be in the presence of God – not some distant, dangerous, gurgling volcano; not a massive, pot-bellied, cross-legged statue sitting silently at the end of an incense-filled hall; not some holographic Wizard of Oz-like character; but the unchanging and unchangeable Lord who rules all angelic armies and who made and controls the universe and beyond. No wonder, in the presence of such grandeur, such glory, such greatness that Isaiah cried out, “Woe is me! I’m insignificant. I’m just a worm compared to God. I’m unworthy.”
I hope you that way. Whether we are standing on the edge of the Grand Canyon or watching an incredible sunset or holding a newborn baby, we realize that measured against the vastness of the universe and the even greater greatness of God, we’re like one tiny grain of sand on an expanse of ocean beach.
But amazingly, God wants to connect with mere mortals. So, He turns the “Woe” of “I’m unworthy” into the “Wow!” of awe. Hear the awe of the angels calling to one another: “‘Holy, holy, holy is the LORD Almighty; the whole earth is full of his glory.’ At the sound of their voices the doorposts and thresholds shook and the temple was filled with smoke.” There is awe in Isaiah’s reaction: “My eyes have seen the King, the LORD Almighty.”
God the Father doesn’t swat us away like pesky mosquitoes. He doesn’t squish us like worms. He doesn’t see us as insignificant beings. Instead He turns our “Woe! I’m unworthy” into “Wow!” of awe through His Fatherly care. Jesus described the Father’s care: “Do not worry about your life, what you will eat or drink; or about your body, what you will wear … Look at the birds of the air; they do not sow or reap or store away in barns, and yet your heavenly Father feeds them. Are you not much more valuable than they?” (Matthew 6:25-26).
Though we are unworthy of being shown any grace or kindness, yet God pours grace and kindness into our daily lives. He grants us the ability to see the beauty of a bright blue sky, to hear the laughter of our child, to taste a Culver’s butter burger, to hold our grandchild on our lap, to feel the love for our spouse grow and mature. Though we deserve to be destitute and derelicts, we are awed that God has given us loving parents, an education, a career, job satisfaction, a home, plus so much more. We bow in awe like angels covering faces, like Isaiah in God’s temple, like Moses at the burning bush, like the three disciples on the Mount of Transfiguration because God turns our “Woe” of “I’m unworthy” into the “Wow!” of awe.
God also turns the “Woe” of “I’m dirty” into the “Wow!” of pardon. “With two [wings] they covered their feet.” Why would they do that? Were they embarrassed because their shoes were worn out and scuffed? Did they have gnarly, ugly toes sticking out from their sandals? No. Back in the day when everyone walked everywhere in open toed-sandals, their feet would become filthy. That’s the meaning of this angelic wing action. Angels don’t have sin. They don’t even have dirty feet. But covering feet was symbolic of the dirt of sin that needs covering because God won’t stand for sin in His presence.
That was Isaiah’s biggest problem. “Woe to me! I am ruined! For I am a man of unclean lips, and I live among people of unclean lips.” Isaiah had a keen sense of the filth of his own sin. Do you?
God didn’t force Isaiah to try to remove the dirt on his own or to try to cover his own sin. Amazingly, God did it for him. “One of the seraphs flew to me with a live coal in his hand, which he had taken with tongs from the altar. With it he touched my mouth and said, ‘See, this has touched your lips; your guilt is taken away and your sin atoned for.’”
Jesus the Son of God touches our lips and hearts with the love that comes from the altar of His cross. He atoned for our sin and took away our guilt. That means nothing for you and is as boring as watching paint dry unless you have taken a good look at your own feet … that leads you astray down wrong paths, and your hands … that writes nasty notes and unloving anonymous entries with your computer, and your eyes … that lusts and covets what doesn’t belong to you, and your ears … that accepts gossip, and your head … that harbors hatred, entertains envy and invites insecurity. All this makes you dirty.
If we don’t have a keen sense of how dirty our sin makes us in God’s sight, we’ll never appreciate what God did about our sin. We’ll get bored with church stuff. We’ll stop reading our Bible. We’ll stop setting aside Sunday mornings and Wednesday evenings for worship. But Jesus has turned our guilt away and covered our sins with the wings of His love.
It is truly amazing that God the Son would sacrifice Himself so that we are declared clean. We lift our hearts in praise because our sins are covered like angels covering their feet, like Isaiah whose tongue was purified by a live coal, like King David whose guilt was turned away, like Peter who denied the Lord but saw His forgiving glance. Though we are undeserving, God the Son grants us free forgiveness, great grace, a redemption reprieve, our soul’s salvation, a permanent paradise, a home in heaven. God the Son turns our “Woe” of “I’m dirty” into the “Wow!” of pardon.
God also turns the “Woe” of “I’m scared” into the “Wow!” of “I’ll serve.” After having his mouth cleansed and his sins burned away, Isaiah was now eager to be a messenger. Previously, he had been a flawed messenger. We, too, are flawed messengers with plenty of excuses and some legitimate fears about serving God – like Isaiah, “I’m not good enough to be your messenger, Lord;” like Moses, “I won’t know what to say;” like Jeremiah, “I’m too young;” like Jonah, “I don’t like my intended audience.”
God didn’t reject those scaredy-cats. Instead, He pardoned their sin, and that pardon inspired them to serve. Isaiah heard the voice of the LORD saying, “Whom shall I send? And who will go for us?” And he said, “Here am I. Send me.”
We cower from speaking. We shirk our responsibility of serving. We are too busy for God’s missions. We are too occupied to contribute to God’s ministries. God the Holy Spirit ignites within us the flames of desire to serve. The Holy Spirit is sent by the Father and the Son to empower and equip us for service through God’s holy words. He motivates us to move. He empowers us to proclaim. He loves us into loving others. He cleanses our lips so we open our mouths to preach. The Holy Spirit uses the message of Holy Scripture to turn our “Woe! I’m scared” into the “Wow!” of “I’ll serve!”
The Holy Trinity is about a living relationship, communion within God and with God. Together as One the undivided holy Trinity creates, redeems, and sanctifies. Each divine Person doing His personal thing, yet always as One. When you were baptized in the name of the Father, Son and Holy Spirit, you entered into the triune love and life of God. The Father is your Father. Jesus is your Brother and Friend. The Spirit is your Counselor, Comforter and Guide. You are a member of God’s family; you live in triune communion with God – with the Father, through the Son, in the Holy Spirit.
You don’t have to understand someone to be in a relationship. Most of us are in relationships with people we can’t begin to understand. How much more so with God? You don’t have to understand the mystery of the undivided Holy Trinity; only confess and praise Him for turning out “Woe” into “Wow!” Amen.
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MANCHESTER, England - Manchester United's home game against Bournemouth in the Premier League was abandoned after a suspect package was found inside Old Trafford stadium.
After discovering the suspicious package, police announced they had carried out a "controlled explosion" on what was described as a realistic-looking "explosive device."
Bomb disposal experts carried out controlled explosion at Old Trafford on what is described as incredibly lifelike explosive device... — G M Police (@gmpolice) May 15, 2016
Greater Manchester Police added: "Full assessment now concluded and found device wasn't viable. A full search of the stadium is ongoing."
Get Breaking News Delivered to Your Inbox
A police investigation later revealed that the package was a training device accidentally left behind by a private company.
Two stands inside Old Trafford - The Sir Alex Ferguson Stand and the Stretford End - were evacuated about 30 minutes before the scheduled kickoff time of 3 p.m. local time (1400 GMT), with United saying "a suspect package was found in the North West Quadrant."
Fans in other stands initially had been allowed to stay inside the stadium.
Old Trafford evacuation pic.twitter.com/Hx9Qae1vkn — Patrick Rycroft (@PRycroft) May 15, 2016
Police are asking people to "avoid the area if possible."
The rest of the Premier League games being played on Sunday, the final day of the season, went ahead as scheduled.
Manchester United said on Twitter the match has been rescheduled for Tuesday.
European officials have been on high alert at sporting events since the November 13 Paris attacks when extremists attempted to blow themselves up inside the stadium at an international friendly between France and Germany. Security guards were able to stop the suicide bombers before they entered the stadium itself.
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Canadian Power and Sail will replace any Boaters Card, as long as they are in our directory. If your card is from another organization, and not on our system, give us a quick call. We will need the Organization, issue date and number on that card. Canadian Power and Sail is the only organization that is NASBLA approved, meaning you can go boating in both Canada and United States with our card.
Please ensure that your Date of Birth is entered correctly on the first page of the Check Out tool. A card cannot be printed without a date of birth and we cannot identify you properly without it.
Members, please log in for member pricing and to have your personal data pre-entered.
There is a $20.00 premium for this rush replacement (3 to 5 business day turn around depending on location)
Canadian Power and Sail will replace any Boaters Card, as long as they are in our directory. If your card is from another organization, and not on our system, give us a quick call. We will need the Organization, issue date and number on that card. Canadian Power and Sail is the only organization that is NASBLA approved, meaning you can go boating in both Canada and United States with our card.
Please ensure that your Date of Birth is entered correctly on the first page of the Check Out tool. A card cannot be printed without a date of birth and we cannot identify you properly without it.
Members, please log in for member pricing and to have your personal data pre-entered.
Before ordering, please ensure that your certificate was issued by Canadian Power and Sail Squadrons or Industry Canada. We replace ROCM certificates only.
Please ensure that your Date of Birth is entered correctly on the first page of the Check Out tool. A certificate cannot be printed without a date of birth and we cannot identify you properly without it.
Members, please log in for member quick identification and to have your personal data pre-entered.
Before ordering, please ensure that your certificate was issued by Canadian Power and Sail Squadrons or Industry Canada. We replace ROCM only.
Please ensure that your Date of Birth is entered correctly on the first page of the Check Out tool. A certificate cannot be printed without a date of birth and we cannot identify you properly without it.
Members, please log in for member quick identification and to have your personal data pre-entered.
There is a $20.00 premium for this rush replacement (2 to 4 business day turn around depending on location).
Before ordering, please ensure that your certificate was issued by Canadian Power and Sail Squadrons or Industry Canada. We replace ROCM only.
Please ensure that your Date of Birth is entered correctly on the first page of the Check Out tool. A certificate cannot be printed without a date of birth and we cannot identify you properly without it.
Members, please log in for member quick identification and to have your personal data pre-entered.
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Q:
Cannot find the SetCount method of QueryRequest in DynamoDb with Java
In my Android app, I use DynamoDb. I want to use a query request, which returns only amount of matching items. In documentation of Amazon I found this line:
In a request, set the Count parameter to true if you want DynamoDB to provide the total number of items that match the filter expression, instead of a list of the matching items.
But I cannot find the SetCount method in Java. Can any body help me?
A:
I've just found the answer. It's not SetCount as .NET .
QueryRequest request = new QueryRequest();
request.setSelect(Select.COUNT);
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1. Field of the Invention
The present invention generally relates to methods, systems, and carrier media for evaluating reticle layout data. Certain embodiments relate to a computer-implemented method that includes determining manufacturability, inspectability, and/or printability of reticle layout data.
2. Description of the Related Art
The following description and examples are not admitted to be prior art by virtue of their inclusion in this section.
Designing an integrated circuit involves creating a schematic design that includes individual devices arranged and coupled to perform a particular function. As integrated circuits become increasingly complex, the design of the integrated circuits also increases in complexity. For example, integrated circuits are generally designed to have smaller dimensions and greater circuit density to improve the speed and other characteristics of the integrated circuits.
The integrated circuit design may be developed using any method or system known in the art such as electronic design automation (EDA), computer aided design (CAD), and other integrated circuit design software. Such methods and systems may be used to generate the circuit pattern database from the integrated circuit design. The circuit pattern database includes data representing a plurality of layouts for various layers of the integrated circuit. Data in the circuit pattern database may be used to determine layouts for a plurality of reticles. Reticles or “masks” are used in a lithography process to transfer a pattern to a resist on a wafer. The terms “reticle” and “mask” are used interchangeably herein.
A layout of a reticle generally includes a plurality of polygons that define features in a pattern on the reticle. Typically, these polygons can be generally defined by their size and placement of the reticle. Each reticle is used to fabricate one of the various layers of the integrated circuit. The layers of the integrated circuit may include, for example, a junction pattern in a semiconductor substrate, a gate dielectric pattern, a gate electrode pattern, a contact pattern in an interlevel dielectric, and an interconnect pattern on a metallization layer.
In particular, the reticle is used to pattern a resist in a lithography process step, then the patterned resist is used to form features of the integrated circuit on the wafer. Therefore, the patterned features that are formed on a reticle and are to be transferred to the wafer reflect the characteristics of the features that are included in the integrated circuit design. For example, the features that are formed on the reticle may be based on and are used to form individual components of the integrated circuits such as those described above. The complexity of the integrated circuit design, therefore, has a direct impact on the manufacture and inspection of reticles.
Accordingly, as the complexity of the integrated circuit design increases, successful reticle manufacture becomes more difficult. For instance, as the dimensions of the integrated circuit features and the spacings between the features decrease, the dimensions and spacings of features on the reticle also decrease. In this manner, it becomes more difficult to form these features on a reticle due to, for example, limitations of the reticle manufacturing process. In a similar manner, it becomes more difficult to inspect these features due to limitations of the reticle inspection processes. Furthermore, as is known in the art, the difficulty of successfully reproducing these features on wafers increases as the dimensions and spacings decrease.
In addition, as the dimensions of integrated circuit features approach the wavelength of the energy source used to print the reticle pattern on a wafer, reticle enhancement techniques (RET) such as optical proximity correction (OPC) features are increasingly relied upon to increase the accuracy of the transfer of the reticle features to the wafer. In particular, RET features cause the pattern printed on a wafer to differ significantly from the pattern physically formed on a reticle. Examples of reticle enhancing techniques include, but are not limited to, OPC features, phase shifting regions, polarization reticles, multiple exposures, off-axis illumination, illumination shapes, and dipole illumination.
OPC features generally take the form of sub-resolution features that are formed on the reticle but which do not print on the wafer. Instead, the OPC features are designed to increase or decrease the amount of light incident on the wafer proximate certain portions of the features such as corners. The OPC features further complicate the design, manufacture, and inspection of the reticle. However, due to the assistance that these features provide in printing features with acceptable characteristics, almost all reticles today include OPC features or another type of RET features. Furthermore, optical effects such as mask error enhancement factor (MEEF) may cause additional distortion of the final image at the wafer level. MEEF may be generally defined as the ratio of the critical dimension of a feature printed in a resist to the critical dimension of a structure formed on a reticle.
Generally, prior to manufacturing a reticle, the reticle layout data that is generated from an integrated circuit design will be checked. The reticle layout data is generally checked using a design rule checking (DRC) technique and/or an optical rule checking (ORC) technique. A DRC tool checks a mask layout file for design rule violations and identifies any violations in an output file. Design rules can include, for example, minimum line spacings, minimum line widths, minimum gate widths, or other geometric layout parameters. The design rules are based on, for example, the manufacturing process to be used to manufacture the resulting design layout. An ORC tool generally analyzes the edge collection by simulating the performance expected on the wafer and determining whether the wafer structures will violate a set of fabrication tolerances. Therefore, the optical rules are based on the lithography process to be used to manufacture wafers with the reticle.
Many DRC and ORC techniques are known in the art, and the results of DRC and ORC are used to correct design rule or optical rule violations. For example, systems and methods for correcting design rule violations in a mask layout file are illustrated in U.S. Published Patent Application No. 2002/0144230 by Rittman, which is incorporated by reference as if fully set forth herein. One method described by Rittman includes correcting design rule violations in a mask layout file by comparing a feature dimension in a mask layout file with a design rule in a technology file. If the feature dimension is less than the design rule, a design rule violation is identified and automatically corrected in the mask layout file. The design rule violation in the mask layout file may be automatically corrected by adjusting the feature dimension until the feature dimension is approximately equal to or greater than a design rule in the technology file. The method is intended to automate correction of the mask layout file to eliminate new design rule violations that may be created when a layout designer manually corrects the mask layout file.
While this and other methods known in the art for correcting design rule violations in a mask layout file have proven to be somewhat successful, DRC and ORC systems currently in use suffer from several limitations. For example, typical systems generally operate entirely on the data as drawn by the layout and RET decoration programs without accounting for the changes that will occur to the data in the mask making and wafer printing processes. In many cases, the mask maker or “shop” will apply sizing corrections to the data of which the designer is unaware. These process biases may alter some of the features included in the design, especially sub-resolution OPC structures such as scattering bars, serifs, or small edge extensions such that they become so small that they disappear from the data entirely or become large enough to cause problems such as bridging in either the reticle or wafer printing process.
In a further example, the list of design rules becomes impractically long as processes become more complex. While the design rules several years ago could be written on a single piece of paper, current processes can have well over 1,000 design rules that need to be checked. Encoding the design rules into software and ensuring that they completely encompass all possible patterning errors that will reduce device yield is increasingly difficult. If the design rule developer fails to anticipate every possible violation that would impact device performance, these violations will pass through the DRC system undetected. Even ORC systems, while making use of more detailed simulation, still require manual input of long rule lists for the checking component.
DRC and ORC simulations also do not capture the details of the mask making process and offer no means of calibrating the patterns to reflect what will actually be written on the reticle. In another example, the existing checkers do not take into account the key question of whether the pattern as laid out by the design program can actually be written by the reticle processing tools. For example, the current state of the art integrated circuit designs can produce complex designs and layouts that pass the current rule checks, but cannot be manufactured either because the RET pattern is too complex to be written correctly on the reticle, inspected on the reticle, and/or printed at the wafer level. Even so called “correct by construction” approaches only optimize the original, pre-OPC layout to eliminate patterns with design rule violations. However, once the OPC structures are added, numerous design rule violations are still possible. Therefore, the existing rule checkers act on idealized data without addressing the key question of whether or not the design can be written and inspected to yield a manufacturable process from design to reticle to wafer levels.
Many design databases take the initial physical design and add incredibly complex RETs which expand the design data by more than an order of magnitude. These huge layout files include tiny jogs, edge extrusions, and minute critical dimension (CD) variations that cannot possibly be reproduced by the mask writing tools. The resulting large data files lead to very long and expensive reticle writing times which are basically wasted since the pattern cannot be reproduced with all of the tiny CD variations and sub-resolution jogs.
In yet another example, the currently available DRC and ORC systems do not take into account whether or not the pattern can be adequately inspected. For example, even if the patterns could be written, no inspection tool is available that is capable of verifying the correct writing of such patterns and no exposure tool is believed to be available that could print such patterns. Therefore, the effort is wasted. Some of the small RET implementations may actually be counterproductive since in trying to write them on the reticle only a partial pattern can be defined, which can end up creating defects on the reticle and later on the wafer. Writing such large data files on a reticle is relatively expensive. As such, it is highly desirable to detect such problems in the data before the design is committed to the mask writing process thereby saving time and money.
Accordingly, it may be advantageous to develop methods, systems, and carrier media that can be used to evaluate reticle layout data to determine the manufacturability, inspectability, and/or printability of the reticle layout data while eliminating at least some of the disadvantages described above.
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Q:
Got a wierd ROC curve and AUC of a classifier
My training dataset has 9 rows(samples) and 705 columns(features+target) (Train_5, y_train_5)
My testing dataset has 17 rows and 705 columns (I know the split is not right)(Test_5, y_test_5)
First I do this:
clf = GradientBoostingClassifier ()
fit = clf.fit(Train_5, y_train_5)
y_predicted2 = clf.predict(Test_5)
c_report = classification_report(y_test_5, y_predicted2)
print('\nClassification report:\n', c_report)
Classification report:
precision recall f1-score support
0 0.13 1.00 0.24 2
1 1.00 0.13 0.24 15
This result is normal. But when I want to draw the ROC curve, it gives me the full thing and the AUC is 1!
y_predicted = clf.predict_proba(Test_5)[:, 1]
false_positive, true_positive, _ = roc_curve(y_test_5, y_predicted)
auc = roc_auc_score(y_test_5, y_predicted)
auc
1
And this is ROC curve.
This is clearly wrong! I mean how could a classifier with 9 samples for training gives you this?? What I am doing wrong?
A:
It's not necessarily wrong. We have to ask ourselves what the axes mean. They are the true positive and true negative rates. i.e. the fraction of items that are correctly and incorrectly labeled as the "positive class".
If 8 of your 9 samples are truly positive and the last one is truly negative. This is possible. Imagine taking a slider and classifying everything to the left as positive and everything to the right as negative. Think about what your true positive and true negative rates would be (for simplicity I'll use 5 total)
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^
^Here, there's nothing to the left, so 0 things are classified correctly or incorrectly as positive. So both the axes are 0, let's move it over 1:
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^
^Here, everything to the left is positive and classified correctly, we have nothing falsly positive. this will be the case for every point along the line
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^
^ The same explanation holds true here. Let's move the slider one more time:
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^
^At this point. Everything that is actually positive has been correctly labeled as positive and everything (i.e. the one thing) that's negative is falsly labeled as positive (thus False positive). This is why these curves always start and end at the diagonals.
I mean you also could have just messed something up...
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Introduction {#s1}
============
Microorganisms dynamically interact with their environment, they are influenced by its composition and, in turn, they influence its composition. This reciprocity has an effect on bacterial gene expression, protein synthesis, and metabolite uptake and production. In the ocean the dissolved organic matter (DOM), which consists of a collection of reduced carbon compounds often containing heteroatoms (e.g. N, P, S), is the result of these interconnected processes. Photosynthetic and non-photosynthetic bacteria can release metabolites into the environment according to their physiological state [@pone.0096038-Carlson1]. Examples are compounds secreted for nutrient acquisition (e.g. siderophores), for communication (e.g. homoserine lactones), and for interspecies competition (e.g. antibiotics). Several studies have investigated the effect of the activity of photosynthetic bacteria on DOM composition (reviewed in [@pone.0096038-Carlson1] and [@pone.0096038-Kujawinski1]), whereas the composition of the DOM produced by heterotrophic bacteria is almost unknown. Special attention has been paid to metabolites of biotechnological interest, but little is known about the full suite of compounds produced by bacteria under different nutrient regimes and growth phases, resulting in a general lack of information on the influence that the metabolism of marine heterotrophic bacteria has on oceanic DOM composition [@pone.0096038-Kujawinski1].
Metabolomics is the field of science that aims to characterize and quantify metabolites, or low molecular weight molecules, originating from cellular activity under a given set of physiological conditions. This collection of metabolites is termed the metabolome [@pone.0096038-Oliver1], which can be partitioned into the so called endo-metabolome (all intracellular metabolites) and the exo-metabolome (all extracellular metabolites) [@pone.0096038-Allen1]--[@pone.0096038-Mapelli1]. Metabolomics is a "downstream" approach and reflects the final response of cells to specific environmental conditions and it completes and integrates the associated techniques of proteomics and transcriptomics [@pone.0096038-Oliver1]. Microbial metabolomic studies have already been performed for different purposes, e.g. to elucidate metabolic pathways, to investigate the response of bacterial metabolism to environmental stresses, to support bacterial identification, and to diagnose bacterial infections [@pone.0096038-Cundy1]--[@pone.0096038-Rinas1]. Such studies have the potential to provide new insights into the composition of the metabolites secreted by marine heterotrophic bacteria and into their influence on the oceanic DOM composition.
Among the different analytical techniques, high resolution accurate mass (HRAM) mass spectrometry has acquired a predominant position in metabolomic studies [@pone.0096038-Want1]. Among others, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is emerging as the most promising technology since it provides accurate mass measurement with ppm or sub-ppm error. It allows to obtain ultra-high resolved profiles with thousands of accurate masses, which in principle can be transformed into real elementary composition [@pone.0096038-Junot1]--[@pone.0096038-Marshall1]. Therefore, it permits high-throughput screening of intracellular and extracellular metabolites providing overall information on bacterial metabolism.
This technique was successfully employed to analyze the variation in the endo-metabolome during bacterial growth, in studies of metabolic diversity among different ecotypes and in analyzing bacterial response to stress conditions [@pone.0096038-RossellMra1]--[@pone.0096038-Antn1]. However, studies that analyze the bacterial exo-metabolome during growth and in response to nutrient limitation are missing. In the present manuscript we report a detailed analysis of the exo-metabolome of strain FO-BEG1, which belongs to the genus *Pseudovibrio*. These are heterotrophic *Alphaproteobacteria* distributed worldwide and they have been detected especially in association with marine invertebrates [@pone.0096038-Bondarev1], [@pone.0096038-Enticknap1]. Bacteria belonging to this genus have often been shown to produce bioactive secondary metabolites, and they are considered a potential source of new molecules of medical interest [@pone.0096038-Bondarev1], [@pone.0096038-OHalloran1]--[@pone.0096038-Kennedy1].
We investigated the composition of the secreted metabolites during bacterial growth, and we analyzed the effect of phosphate limitation. Phosphate limitation was chosen because it is a common environmental condition encountered in many marine systems [@pone.0096038-Thingstad1]--[@pone.0096038-Wu1], and it has been described to have a significant effect on primary and secondary metabolism [@pone.0096038-Martn1], [@pone.0096038-Martn2]. We report here the astonishing diversity of the exo-metabolome of strain FO-BEG1 and the drastic effect that phosphate limitation has on its composition. These data shed new light onto the complexity of the metabolites secreted by heterotrophic marine bacteria and onto the effect that their metabolic state can have on the composition of DOM in the ocean.
Materials and Methods {#s2}
=====================
Growth conditions {#s2a}
-----------------
Strain FO-BEG1 was cultivated in the carbohydrate/mineral medium (CM) as described by Shieh et al. [@pone.0096038-Shieh1] and modified by Bondarev et al., [@pone.0096038-Bondarev1]. For the phosphate surplus condition (+P~i~) phosphate was added to a final concentration of 1.4 mmol L^−1^, whereas no phosphate was added to the phosphate limited (−P~i~) medium. Under −P~i~ conditions the final phosphate concentration was 0.1 mmol L^−1^, and derived from the buffer used for preparing the vitamin solutions. Erlenmeyer flasks of 250 mL were filled with 100 mL of medium and inoculated with 100 µL of a pre-culture grown under +P~i~ conditions. Cultures were incubated at 28°C in the dark and shaken at 120 rpm. We monitored bacterial growth by means of Optical Density (OD) measured at 600 nm using an Eppendorf BioPhotometer (Eppendorf AG, Hamburg, Germany). The OD~600~ was then correlated with the cell number, determined using a Thoma chamber (Brand GmbH, Wertheim, Germany; data not shown). All experiments were performed and sampled in independent experimental triplicates.
Solid phase extraction of dissolved organic matter (SPE-DOM), dissolved organic carbon (DOC) measurements, and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) of DOM
For both −P~i~ and +P~i~ cultures, samples were collected immediately after the inoculation (T0) and in the exponential growth phase (T1). Additionally, samples at the end of the logarithmic phase (T2) and during the stationary phase (T3) were collected for the −P~i~ cultures. One more set of samples was collected also in the stationary phase (T2) of +P~i~ cultures. Cells were removed via centrifugation at 10,000 × *g* for 10 min at 5°C, the supernatant was then filtered into 150 mL combusted glass serum-bottles using Acrodisc 25 mm syringe filters with a 0.2 µm pore size GHP membrane (Pall LifeSciences, Ann Arbor, MI, USA), acidified to pH 2 with 2 mol L^−1^ HCl, and stored at 4 °C until further analyses. We collected the samples from all biological triplicates in both +P~i~ and −P~i~ conditions, with the exception of T0.
DOM of the cell-free supernatants was extracted according to the solid phase extraction of dissolved organic matter (SPE-DOM) method described by Dittmar et al. [@pone.0096038-Dittmar1]. The extraction was performed using Bond Elute PPL (Agilent Technologies, Wildbronn, Germany) cartridges with a styrene-divinylbenzene (SDVB) polymer modified with a property surface able to retain also the most polar classes of analytes. DOC content of each extract was analyzed using a Shimadzu TOC-VCPH total organic carbon analyzer (Shimadzu, Kyoto, Japan). The extracted DOM samples were then diluted with a mixture of methanol (MS grade) and ultra-pure water (50:50 v/v) to yield a DOC concentration of 20 mg L^−1^ carbon, filtered using a 0.2 µm pore size PTFE filter (Rotilabo, Carl Roth GmbH, Karlsruhe, Germany), and analyzed with a solariX FT-ICR-MS (Bruker Daltonik GmbH, Bremen, Germany) with a 15.0 Tesla magnet and equipped with an electrospray ionization (ESI) source. To maximize our analytical window, all samples were analyzed on the ESI-FT-ICR-MS in positive and negative ionization mode. We minimize the formation of adducts (and dimers of analyte compounds) by applying a gentle in-source collision-induced dissociation (CID) energy. This breaks apart larger adducts (including dimers), but no covalent bonds. All data were acquired with a time domain size of 4 megawords and with a detection range of *m*/*z* (mass to charge ratio) 150 to 2,000. For each run, 500 broadband scans were accumulated. All the mass spectra acquired under both positive and negative mode were analyzed with the Data Analysis version 4.0 SP4 software package (Bruker Daltonik GmbH).
Calibration of the mass spectra was performed as follows: one replicate of −P~i~ T3 was spiked with 0.05 ppm L-arginine (Sigma-Aldrich, Steinheim, Germany), used for the ESI-negative analyses, or with 0.05 ppm Tuning-mix (Agilent Technologies, Palo Alto, CA, USA), used for the ESI-positive analyses. The resulting mass spectra were calibrated internally with reference mass lists, and molecular formulae were assigned for the remaining peaks in the spectra using the Data Analysis software. For the ESI-negative mode, the molecular formulae were assigned to an elemental composition in the following ranges: C 1--∞, H 1--∞, O 1--∞, N 0--4, S 0--2, P 0--1, and allowing errors lower than 0.2 ppm. A mass list with more than 300 masses in the range 150--800 *m*/*z* was obtained and used to calibrate all other acquired mass spectra. Due to the diversity of the samples, the calibration list was adjusted manually to cover always the full detected mass range with at least 40 calibration points. For the ESI-positive mode, the molecular formulae were assigned to an elemental composition in the following ranges: C 1--∞, H 1--∞, O 1--∞, N 0--4, S 0--2, P 0--2, Na 0--1, allowing errors lower than 0.2 ppm. A mass list containing 25 masses covering the range 295--850 *m*/*z* was used to calibrate the other acquired mass spectra, using at least 5 calibration points. All linear calibrations resulted in an average mass error of below 0.05 ppm. Additionally, the instrument was externally calibrated with an in-house marine deep sea DOM reference sample (mass accuracy of less than 0.1 ppm). Before each sample set, blank checks with methanol/ultrapure water 1:1 were measured.
Sample comparison, molecular formulae assignment, filtration of the datasets, and statistical analysis {#s2b}
------------------------------------------------------------------------------------------------------
Comparison of the mass spectra and isotope (^13^C) identification were performed for the data obtained from both ionization modes, whereas the formulae assignment was done only for the ESI-negative mode. Sodium adducts frequently occur in ESI-positive mode and are considered in our molecular formulae assignment routine. However, other adducts, such as NH~4~ ^+^, are not easily identifiable, because those cannot be distinguished from other compounds where the same combination of elements is covalently bound. Such a distinction would require extensive additional analyses, such as fragmentation experiments (MS/MS) in the FT-ICR-MS. This was beyond the scope of this study. Because of the inherent uncertainty, we restricted our main analysis to ESI-negative data. With our ESI settings, the ionization in negative mode is highly reproducible and due to loss of H^+^. Other possible adducts (e.g. Cl^−^) can be identified by their unique isotope patterns but were not present in our mass spectra. The computational procedures were performed using an in-house Matlab routine developed by the Max Planck Research Group for Marine Geochemistry. The molecular formulae were assigned in the elemental composition in the following ranges: C 1--40, H 1--∞, O 1--∞, N 0--4, S 0--2, P 0--1, no Na, Fe, Cl and allowing a mass error of maximum 500 ppb. Only peaks with signal to noise ratio \> 4 were considered and only formulae with a minimum H/C ration of 0.3 and a maximum O/C ratio of 1 were accepted. All detected ions were singly charged, as indicated by the mass difference between isotopologues (of ^12^C versus ^13^C). Therefore, all detected *m*/*z* values were equivalent to molecular masses.
The ion intensities of the *m*/*z* detected in both ionization modes were normalized by dividing the intensity of each mass by the sum of the 500 highest intensities measured in the respective mass spectra. This normalization procedure was performed independently for each measurement. The normalization was performed after removing all singlets, i.e. masses detected only in one sample out of the seventeen analyzed. In order to have an overview of the similarity among the samples, we performed a non-metric multidimensional scaling (NMDS) for the datasets obtained in both ionization modes, using the Bray-Curtis similarity index for the calculation of the distance matrices. Minimum-spanning trees between all samples were constructed to visualize pairwise sample similarities. Nearest neighbors, i.e. the most similar samples, were identified and graphically connected.
In order to reduce contingent noise and to consider only the molecules produced by the bacteria, we further filtered both datasets using the following criteria: we removed all masses detected in the samples −P~i~ and +P~i~ T0 that did not at least double their normalized ion intensity during the experiment; we removed all masses that were not present in at least all triplicates of one condition at a specific time point; we removed all masses that could contain the isotope ^13^C. The filtered datasets were newly analyzed by means of NMDS, but the samples collected at T0 for both growth conditions were not considered, due to the significant alteration in their *m*/*z* composition derived by the filtration of the datasets. A minimum-spanning tree between all samples was newly constructed. In order to verify the statistical reliability of the clustering observed in all NMDS plots, bootstrap analyses with 1000 reiterations were performed on dendrograms constructed for the similarity matrices obtained using the Bray-Curtis index. The paired group algorithm was used for the construction of the dendrograms. NMDSs, the relative stress values, which are a measure that reflects the degree of deviation of NMDS distances from original matrix distances, the minimum-spanning trees, the construction of the dendrograms, the calculation of the cophenetic correlation coefficients, which are a measure of how faithfully the dendrograms preserve the pairwise distances between the data points, and the bootstrap analyses were carried out by means of the PAST program [@pone.0096038-Hammer1]. Subsequently, in order to identify the unique masses present per time point under both conditions and the masses shared among the growth stages, we created Venn diagrams considering only masses present in all triplicates at the respective time points.
The elemental composition and the modified aromaticity index (AI~mod~; [@pone.0096038-Koch1]) of each molecular formula assigned to the *m*/*z* detected in ESI-negative mode were used to divide them into molecular categories according to criteria modified after Šantl-Temkiv et al. [@pone.0096038-antlTemkiv1]. For this analysis, we excluded all masses for which multiple molecular formulae were obtained. We divided the molecular formulae into the following categories: peptides (if the molecular formula has an H/C ratio between 1.5 and 2, an O/C ratio lower than 0.9 and includes N), sugars (if the molecular formula has an O/C ratio equal or higher than 0.9 and an AI~mod~ lower than 0.5), saturated fatty acids (if the molecular formula has an H/C ratio equal or higher than 2 and an O/C ratio lower or equal to 0.9), unsaturated aliphatic compounds (if the molecular formula has an H/C ratio between 1.5 and 2, an O/C ratio lower than 0.9 and does not contain N), highly unsaturated compounds (if the molecular formula has an AI~mod~ lower than 0.5, an H/C ratio lower than 1.5, and an O/C ratio lower than 0.9), phenols (if the molecular formula has an AI~mod~ equal or higher than 0.5 and less than 12 C atoms), and polyphenols (if the molecular formula has an AI~mod~ equal or higher than 0.5 and 12 or more C atoms). We emphasize that this categorization is not unambiguous, and alternative structures may exist for a given molecular formula. However, this subdivision provides a helpful overview of likely structures behind the identified molecular formulae.
Metabolite and pathway annotation {#s2c}
---------------------------------
The masses detected in all three biological replicates at each time point in ESI-negative mode were putatively annotated (i.e. level 2 of metabolite identification as defined by the Metabolomics Standards Initiative [@pone.0096038-Sumner1]) using the "transformation mapping" approach [@pone.0096038-Weber1], after correcting the mass values for the H^+^ loss. This method is based on mapping an experimentally-derived empirical formula difference for a pair of peaks to a known empirical formula difference between substrate-product pairs derived from the KEGG database (Kyoto Encyclopedia of Genes and Genomes [@pone.0096038-Kanehisa1]). To reduce the number of false positive assignments only metabolites that occurred in one of the *Pseudovibrio* sp. FO-BEG1 pathways (KEGG identifier: psf) were selected for annotation (as listed in KEGG on July 2013). Furthermore, we annotated the obtained masses considering the molecules reported in the Human Metabolome Database [@pone.0096038-Wishart1] and Drug Bank [@pone.0096038-Wishart2]. An additional annotation was performed using a sub-set of compounds reported in the Dictionary of Natural Products Online [@pone.0096038-DictionaryofNatural1] obtained after performing a search based on the word "bacteria" typed in the property field "Type of organism word". Since in these three databases the pathways for the compounds are not indicated we could not apply the "transformation mapping" approach; therefore, the annotation was based on one to one matches between the detected masses and the masses of the known compounds, allowing always an error of ≤ 1 ppm.
Results {#s3}
=======
Measurement of the DOC released during bacterial growth and FT-ICR-MS analysis {#s3a}
------------------------------------------------------------------------------
Phosphate limitation repressed the growth of *Pseudovibrio* sp. FO-BEG1, leading to a final cell density 2.5--3.5 times lower than the one observed under phosphate surplus conditions ([Fig. 1A](#pone-0096038-g001){ref-type="fig"}). Under −P~i~ conditions, a slightly higher amount of solid phase extractable dissolved organic carbon (SPE-DOC) was produced during the first half of the exponential phase (T1; [Fig. 1B](#pone-0096038-g001){ref-type="fig"}). As observed in T0, the SPE extraction did not retain the provided glucose, which alone would correspond to 60 mmol L^--1^ DOC. Therefore, the measured DOC represented the organic compounds produced and secreted by *Pseudovibrio* sp. FO-BEG1. At T1 under both conditions only around 2 mmol L^−1^ of glucose was taken up by the cells (Romano et al., unpublished data), resulting in a conversion of the initial carbon source in SPE-DOC of 0.4% for −P~i~ cultures and 0.3% for +P~i~ cultures. Despite the lower growth, under −P~i~ conditions the SPE-DOC concentration increased to 267 ± 58 µmol L^−1^ and 511 ± 192 µmol L^−1^ at the end of the logarithmic (T2) and in the middle stationary (T3) phase, respectively. At both growth stages the glucose consumed was around 5 mmol L^−1^ (Romano et al., unpublished data). Consequently, in both cases the SPE-DOC represented 0.9% of the used glucose. Compared to this, the SPE-DOC concentration under +P~i~ conditions during the stationary phase was more than three times lower (144 ± 18 µmol L^−1^; [Fig. 1B](#pone-0096038-g001){ref-type="fig"}), representing 0.2% of the consumed glucose.
{#pone-0096038-g001}
The raw data obtained from the ESI-negative FT-ICR-MS analysis consisted of 23,892 masses ranging from 154 *m*/*z* to 1,930 *m*/*z*. After normalization of the ion intensities, we performed a non-metrical multidimensional scaling (NMDS) in order to evaluate the similarities among the samples ([Fig. 2A](#pone-0096038-g002){ref-type="fig"}). As the stress value of the NMDS plot was 0.06, it could be considered a good representation of the calculated distance matrix and thus of the similarity among the samples. The samples collected at T1 for each biological triplicate under both −P~i~ and +P~i~ conditions clustered together and were clearly separated from the samples collected during the rest of the growth period ([Fig. 2A](#pone-0096038-g002){ref-type="fig"}). All biological triplicates of the −P~i~ conditions collected at the end of the logarithmic phase and in the stationary phase (T2 and T3) were completely divergent from the samples collected under +P~i~ stationary phase (T2). Moreover, the samples T2 and T3 for the −P~i~ conditions also clustered separately in the plot ([Fig. 2A](#pone-0096038-g002){ref-type="fig"}). The bootstrap analysis of the dendrogram constructed for the Bray-Curtis similarity matrix revealed that the divergence among the samples described above was statistically highly significant, since during the 1000 reiterations always the same clustering occurred ([Fig. S1A](#pone.0096038.s001){ref-type="supplementary-material"}). In ESI-positive mode 17,859 masses were detected, ranging from 153 *m*/*z* to 1,999 *m*/*z*. The NMDS plot obtained for this dataset was characterized by a stress value of 0.07, therefore, it could be considered a good representation of the distance matrix as well ([Fig. S2A](#pone.0096038.s002){ref-type="supplementary-material"}). All samples had a similar clustering as the one observed in the ESI-negative NMDS plot. One of the main difference was the higher divergence between one −P~i~ replicate (−P~i~ III T1) and the other replicates collected at the same time point. However, the minimum spanning tree showed that this sample shared the highest degree of similarity with the other samples collected under the same growth stage. Additionally, the samples collected at T2 and T3 under −P~i~ conditions showed a higher degree of similarity ([Fig. S2A](#pone.0096038.s002){ref-type="supplementary-material"}). The bootstrap analysis performed on the respective dendrogram revealed that in \> 75% of the cases the samples clustered consistently with the NMDS groups, indicating that the divergences described above were statistically significant ([Fig. S3A](#pone.0096038.s003){ref-type="supplementary-material"})
{#pone-0096038-g002}
In order to consider only those metabolites that were produced by the strain under the respective conditions, we removed from the datasets all compounds that were already present at T0 and did not at least double their ion intensities during the investigated growth period. Moreover, only compounds present in all biological triplicates at a certain time point and growth condition were further considered. This filtration reduced the ESI-negative dataset to 8,381 masses ranging from an *m*/*z* value of 154 to 998. The NMDS plot ([Fig. 2B](#pone-0096038-g002){ref-type="fig"}) performed for this new dataset showed the same clustering pattern as the one constructed for the unreduced dataset ([Fig. 2A](#pone-0096038-g002){ref-type="fig"}). 7,499 masses ranging from 163 to 1,234 *m*/*z* were obtained after the filtration of the ESI-positive dataset and, as for the negative mode, the new NMDS plot constructed using these masses showed a clustering consistent with the one of the unreduced dataset ([Fig. S2B](#pone.0096038.s002){ref-type="supplementary-material"}). The only exception was the higher similarity among the samples +P~i~ T2 and the ones collected at T1 under both phosphate regimes. For both ionization modes, the bootstrap analyses performed on the dendrograms suggested that the clustering of the samples observed in the NMDS plots was statistically significant ([Fig. S1B](#pone.0096038.s001){ref-type="supplementary-material"}, [S3B](#pone.0096038.s003){ref-type="supplementary-material"}). Only for the filtered data obtained in ESI-positive mode the divergence among the samples collected at T2 and T3 under phosphate limited condition was not statistically significant, since their divergence occurred in \< 50% of the reiterations ([Fig. S3B](#pone.0096038.s003){ref-type="supplementary-material"}).
In the Venn diagram constructed considering the masses obtained in ESI-negative mode ([Fig. 3](#pone-0096038-g003){ref-type="fig"}), it was evident that the samples collected during the logarithmic growth phase under both +P~i~ and −P~i~ conditions presented 23 and 100 unique masses, respectively. These samples shared 202 masses never detected in the stationary phase. Independent of the condition and the growth phase, we detected 573 masses shared among all samples. The samples collected at the end of the logarithmic and in the stationary phase under −P~i~ conditions (T2 and T3) overall showed 1,088 unique masses never detected in the other time points, whereas in the samples collected in the stationary phase under +P~i~ conditions we detected 832 unique masses ([Fig. 3](#pone-0096038-g003){ref-type="fig"}). A highly similar distribution was observed in the Venn diagram obtained for the ESI-positive mode ([Fig. S4](#pone.0096038.s004){ref-type="supplementary-material"}). The samples collected at the end of the logarithmic phase and in stationary phase under −P~i~ conditions showed a higher number of masses (total of 2,220) than the samples collected in the stationary phase under +P~i~ conditions. In contrast to the results obtained in ESI-negative mode, a higher number of masses (108) was shared among all phosphate limited samples, and a lower number of masses (122) was shared among all samples independent of the condition or growth stage.
{#pone-0096038-g003}
The higher variability among replicates observed in ESI-positive mode was likely due to multiple ionization mechanisms, which can result, for example, in ammonium or sodium adduction, both ions present in our culturing medium.
Conversion of masses obtained in ESI-negative mode into molecular formulae and annotation of metabolites {#s3b}
--------------------------------------------------------------------------------------------------------
Of the 8,381 masses detected in ESI-negative mode after the filtration of the dataset described above, we were able to assign molecular formulae to 4,914. Isotopologues were not included in the number of assigned molecular formulae. Of these, 4,122 were unique molecular formulae, i.e. only one molecular formula could be assigned to the respective *m*/*z* value, corresponding to 49% of the *m*/*z* values present in the filtered dataset. A greater percentage of molecular formulae could be assigned to the masses obtained from samples collected at T1 under both +P~i~ and −P~i~ conditions ([Table 1](#pone-0096038-t001){ref-type="table"}). Under +P~i~ conditions an increase in the relative number of formulae containing nitrogen was observed from logarithmic to stationary phase, whereas the percentage of these compounds decreased under −P~i~ conditions ([Table 1](#pone-0096038-t001){ref-type="table"}). Interestingly, during bacterial growth under −P~i~ conditions the relative amount of molecular formulae containing sulfur increased strongly from 45% to 65% of the total assigned formulae ([Table 1](#pone-0096038-t001){ref-type="table"}).
10.1371/journal.pone.0096038.t001
###### Overview of the data obtained from the ESI-negative FT-ICR-MS analysis.
{#pone-0096038-t001-1}
+P~i~ T1 +P~i~ T2 −P~i~ T1 −P~i~ T2 −P~i~ T3
-------------------------------- -------------- -------------- -------------- -------------- --------------
detected masses 2596 4206 3112 3566 2479
unique molecular formulae 1578 (60.8%) 2426 (57.7%) 1931 (62.1%) 1876 (52.6%) 1241 (50.1%)
formulae containing nitrogen 1193 (75.6%) 2085 (85.9%) 1362 (70.5%) 1387 (73.9%) 813 (65.5%)
formulae containing sulfur 648 (41.0%) 1087 (44.8%) 859 (44.5%) 1138 (60.7%) 802 (64.6%)
formulae containing phosphorus 221 (14.0%) 374 (15.4%) 247 (12.8%) 301 (16.0%) 176 (14.2%)
The number of masses detected in all biological triplicates of each time point is shown. The data refer to the dataset obtained after applying the filtration criteria described in the Materials and Methods section. Values in brackets represent the percentages of masses to which a unique molecular formula could be assigned and the percentages of unique molecular formula containing heteroatoms. Isotopologues of assigned molecular formulae are not counted as assigned. Overall, a unique molecular formula could be assigned to 4,122 masses, corresponding to 49% of the obtained *m*/*z*.
After calculating the modified aromaticity index (AI~mod~) we assigned the obtained molecular formulae to specific molecular categories and calculated their relative abundances at different time points ([Fig. 4](#pone-0096038-g004){ref-type="fig"}). In agreement with the similarity observed in the NMDS plots, at T1 the composition of the secreted metabolites was similar in both treatments. The major components of the exo-metabolome were compounds with molecular formulae assigned to peptides and highly unsaturated molecules. Only under −P~i~ conditions, a pronounced increase of highly unsaturated, phenolic and polyphenolic compounds and a decrease in peptides and unsaturated aliphatic compounds could be observed during stationary phase.
{#pone-0096038-g004}
The ultra-high resolution of the FT-ICR-MS results in precise masses that can be compared and assigned to known compounds present in pathways described for the considered organism and collected in target databases such as KEGG (Kyoto Encyclopedia of Genes and Genomes). The metabolite names reported in the pathways of strain *Pseudovibrio* sp. FO-BEG1 in this database were used to annotate the masses obtained from the FT-ICR-MS analysis. The annotation strategy was based on mapping an experimentally-derived empirical formula difference for a pair of *m*/*z* to an empirical formula difference calculated for substrate-product pairs retrieved from KEGG [@pone.0096038-Weber1]. It was previously shown that this approach can reduce the false positive rate of putative metabolite annotation by more than fourfold in comparison to searching a compound database using a one to one match approach (peak by peak search), while maintaining a minimal false negative rate [@pone.0096038-Weber1]. A molecular name could be assigned only to a minor proportion of compounds detected in ESI-negative mode (less than 3%; [Dataset S1](#pone.0096038.s006){ref-type="supplementary-material"}). For the masses detected in ESI-positive mode, the percentage was even lower. For the reasons outlined above, we did not further consider the ESI-positive results for the annotation of metabolites. We could annotate 85 masses for the sample −P~i~ T1 and of them 55 were assigned to unique metabolites (1.8% of the detected masses in all triplicates). The number of masses assigned to unique metabolites decreased to 46 (65 total annotated masses) for the samples −P~i~ T2 and to 30 for −P~i~ T3 (37 total annotated masses), representing 1.3% and 1.2% of the detected masses in all triplicates, respectively. 49 and 64 masses could be assigned to unique metabolites (73 and 97 total annotated masses) in the samples +P~i~ T1 and +P~i~ T2 (1.8% and 1.5% of the detected masses in all triplicates), respectively. Most of the annotated compounds were intermediates in the metabolism of the amino acids lysine, tyrosine, tryptophan and phenylalanine ([Dataset S2](#pone.0096038.s007){ref-type="supplementary-material"} and [Fig. S5](#pone.0096038.s005){ref-type="supplementary-material"}). In all samples, except −P~i~ T3, several metabolites were also annotated in the pathways of the purine metabolism ([Dataset S2](#pone.0096038.s007){ref-type="supplementary-material"} and [Fig. S5](#pone.0096038.s005){ref-type="supplementary-material"}).
In order to identify possible molecules of biotechnological relevance, we performed three additional annotations using the masses obtained in ESI-negative mode and targeting the Drug Bank ([Dataset S3](#pone.0096038.s008){ref-type="supplementary-material"}), the Human Metabolome Database (HMDB; [Dataset S4](#pone.0096038.s009){ref-type="supplementary-material"}), and the Dictionary of Natural Products (DNP; [Dataset S5](#pone.0096038.s010){ref-type="supplementary-material"}). When the Drug Bank was chosen as target, the maximum number of annotated masses was 327 and was obtained for the sample +P~i~ T2. The same sample presented the highest number of annotate masses (211) also in the annotation performed targeting the DNP. Whereas, using the HMDB, 186 was the maximum number of masses annotated, and it was obtained for the data of the sample −P~i~ T1. In these databases the bio-synthetic pathways are not reported; therefore, the "transformation mapping" approach could not be applied. Consequently, the data obtained have to be considered with caution since a high number of false positive annotations can occur. The annotation performed using the HMDB was in line with the results obtained using the KEGG database, showing mostly intermediate metabolites of amino acid and nucleotide metabolisms ([Dataset S4](#pone.0096038.s009){ref-type="supplementary-material"}). Although the annotation performed using the Drug Bank database resulted in a higher number of assignments, in every sample several *m*/*z* were annotated as plant metabolites (e.g. epigallocatechin, commonly found in tea leaves; ginkgolide-A, produced by *Ginkgo biloba*) or compounds of synthetic origin (e.g. ibuprofen; [Dataset S3](#pone.0096038.s008){ref-type="supplementary-material"}). This suggests a high number of false positive annotations; therefore, these data will not be further discussed except when consistent with the other annotation approaches we applied. Finally, the annotation performed using the DNP resulted in the assignment of several masses to compounds having antibacterial, signaling, and enzymatic inhibiting activities ([Dataset S5](#pone.0096038.s010){ref-type="supplementary-material"}). Interestingly, only in the samples −P~i~ T2 and −P~i~ T3 the *m*/*z* 210.952904 was annotated as tropodithietic acid, which is a potent antibiotic produced by bacteria belonging to the *Roseobacter* clade and the *Pseudovibrio* genus [@pone.0096038-Bondarev1], [@pone.0096038-Bruhn1].
Discussion {#s4}
==========
In order to quantify and characterize the metabolites released by strain *Pseudovibrio* sp. FO-BEG1 into the medium during growth and to evaluate the effect of phosphate limitation on them, we performed an ultra-high resolution mass spectrometry analysis of the bacterial exo-metabolome. Mass spectrometry is the most widely used approach in metabolomic studies [@pone.0096038-Want1]. In particular high resolution accurate mass (HRAM) mass spectrometry instruments are receiving progressively more attention, owing to their ability to resolve highly complex samples and to yield accurate mass measurements, which allow precise calculations of the elemental composition [@pone.0096038-Junot1], [@pone.0096038-Aharoni1], [@pone.0096038-Marshall1].
When cells growing under −P~i~ conditions entered stationary phase, they released three times more solid phase extractable dissolved organic carbon (SPE-DOC) than cells growing under +P~i~ conditions ([Fig. 1](#pone-0096038-g001){ref-type="fig"}). We are confident that the SPE-DOC concentrations and the number of metabolites obtained are not biased by the presence of compounds derived from the cultivation medium because, as shown by the amount of SPE-DOC at T0, the SPE method did not retain significant quantities of organic compounds present in the medium ([Fig. 1B](#pone-0096038-g001){ref-type="fig"}). Moreover, during the filtration of the datasets we removed all *m*/*z* (mass to charge ratio) that were detected at T0 and did not at least double their ion intensities during the experiment. Therefore, all compounds originally present in the medium and not used by the cells during bacterial growth were excluded from the analyses.
It has been known for several years that low phosphate concentrations can induce the production of secondary metabolites ([@pone.0096038-Martn1] and references therein), which would suggest that under −P~i~ conditions a higher fraction of the carbon source provided was used by *Pseudovibrio* sp. FO-BEG1 for the production of such compounds. In addition, it is known that phosphate limitation can trigger membrane lipid rearrangement, with the substitution of phosphorous-containing with phosphorous-free lipids [@pone.0096038-Benning1], [@pone.0096038-Minnikin1], a phenomenon that we also observed for *Pseudovibrio* sp. FO-BEG1 (Romano et al., unpublished data). Therefore, it is reasonable to hypothesize that due to the membrane rearrangement more cytosolic metabolites could leak out from the cells, explaining the higher production of SPE-DOC under −P~i~ conditions. Consistently, nutrient leakage was also described in a marine yeast strain growing under phosphate limited conditions [@pone.0096038-Robertson1]. Other studies showed that bacteria can convert from 5 to 15% of the provided carbon into DOC [@pone.0096038-Ogawa1]--[@pone.0096038-Gruber1], which is one order of magnitude higher than observed in our experiments. However, a precise comparison is difficult because in all mentioned examples different medium composition, growth parameters, and analytic procedures were used.
Rosselló-Móra et al. [@pone.0096038-RossellMra1] and Antón et al. [@pone.0096038-Antn1] analyzed the endo- and the exo-metabolome of different *Salinibacter ruber* isolates during the classification of different ecotypes, and reported that the isolates can be distinguished by their metabolic profiles. Moreover, Brito-Echeverria et al. [@pone.0096038-BritoEcheverria1] analyzed the endo- and exo-metabolome of different *Salinibacter* strains in response to different stress conditions, and reported that the exo-metabolome was affected to a greater extent than the endo-metabolome. In all studies, the analyses were performed via FT-ICR-MS, and they are the first reports that provide information about the complexity of the bacterial exo-metabolome. In line with these observations, our analysis revealed that *Pseudovibrio* sp. FO-BEG1 produced and released at least many hundreds of compounds into the medium, and that the composition of this DOC was greatly affected by phosphate limitation. In this respect, FT-ICR-MS represents an ideal and powerful technique to unravel this complexity. We could clearly show that the exo-metabolome composition differs during different growth phases and between the two tested conditions ([Fig. 2](#pone-0096038-g002){ref-type="fig"}, [3](#pone-0096038-g003){ref-type="fig"}, [S1](#pone.0096038.s001){ref-type="supplementary-material"}, [S2](#pone.0096038.s002){ref-type="supplementary-material"}, [S3](#pone.0096038.s003){ref-type="supplementary-material"}, [S4](#pone.0096038.s004){ref-type="supplementary-material"}). These data are consistent with previous studies, which applying low resolution techniques reported that the metabolites secreted by bacteria can change during different growth phases and in response to environmental stresses [@pone.0096038-Shnayderman1], [@pone.0096038-Coucheney1], [@pone.0096038-Takahashi1], [@pone.0096038-Barreto1]. One interesting difference between the two phosphate regimes was the higher amount of compounds containing sulfur detected under −P~i~ conditions ([Table 1](#pone-0096038-t001){ref-type="table"}). This suggests that phosphate limitation also influences the sulfur metabolism of *Pseudovibrio* sp. FO-BEG1, increasing the amount of sulfur released into the environment in the form of DOM.
The presence of unique masses detected only at specific time points under both conditions shows a dynamic cycling of organic compounds. Molecules produced during the beginning of the logarithmic growth phase were then taken up again when cells entered stationary phase. A similar phenomenon was observed in a study that investigated the effect of grazing on the DOC production in a pure culture of *Pseudomonas chlororaphis* [@pone.0096038-Gruber1]. Interestingly, even though for each sampling point and each condition we identified hundreds of unique masses, we also detected 573 and 122 masses in ESI-negative and positive mode, respectively, which were always present in our samples independent of the growth stage or the growth condition ([Fig 3](#pone-0096038-g003){ref-type="fig"}, [S4](#pone.0096038.s004){ref-type="supplementary-material"}). It would be interesting to verify whether this "core" exo-metabolome is affected by other environmental changes or it represents a distinctive "metabolic signature" of the strain.
It has been suggested that the trophic status of the environment affects DOM composition via shaping the ecological processes that are responsible for its production [@pone.0096038-Kujawinski1]. Productive, nutrient rich regions have significant DOM production directly from photosynthesis, whereas oligotrophic, nutrient poor regions have significant DOM production from grazing processes [@pone.0096038-Marann1], [@pone.0096038-Nagata1]. This difference was attributed to the complexity of the microbial food web in different environments, with the oligotrophic regions having a more effective microbial loop compared to the classical food web described in the productive regions [@pone.0096038-Teira1]. Our data suggest that in order to understand DOM composition the effect of the environmental nutrient regimes on bacterial physiology should not be underestimated. As we show, it can greatly affect both the amount and the composition of the produced organic compounds.
Comparing the variation of the metabolome of *Escherichia coli* and *Saccharomyces cerevisiae* in response to carbon and nitrogen limitation, Brauer et al. [@pone.0096038-Brauer1] unexpectedly showed global metabolic trends remarkably conserved among these two distantly related microorganisms. Therefore, in order to verify the presence of shared metabolic responses, which could indicate the presence of highly conserved regulatory schemes, it would be of great interest to compare the variations of the exo-metabolome in response to nutrient limitation among different bacteria. Here we show that the nutrient regime greatly influenced the DOM secreted by *Pseudovibrio* sp. FO-BEG1 into the environment. Therefore, it is reasonable to expect that by extending these kinds of studies to different marine bacteria the influences of microbes on DOM composition in natural environments characterized by particular trophic conditions could be better understood.
The molecular formula assignment allowed us to classify the detected masses in molecular categories, giving a broad overview of the types of compounds released during growth. Under phosphate limitation, we observed a higher production of phenolic and polyphenolic compounds when cells entered stationary phase ([Fig. 4](#pone-0096038-g004){ref-type="fig"}). Production of phenol was described for the strains *Pseudovibrio* sp. D323 and L4-8 [@pone.0096038-Penesyan1], [@pone.0096038-Rou1]. The crude extract of the spent medium of the latter strain showed a strong antioxidant activity, which is consistent with our finding that strain FO-BEG1 produces different types of phenols and polyphenols, known for their antioxidant properties [@pone.0096038-Scalbert1]. Higher production of these compounds under −P~i~ conditions could be related to the increased oxidative stress that cells growing under phosphate limitation might experience [@pone.0096038-Yuan1]--[@pone.0096038-Moreau1], and which we also inferred for strain FO-BEG1 from the comparison of the protein expression between +P~i~ and −P~i~ conditions (Romano et al., unpublished data).
Some of the detected phenolic and polyphenolic compounds could be, for example, tropone derivates. These molecules are commonly produced by bacteria of the *Roseobacter* clade and can have algaecide and antibacterial activity, as, for example, the potent antibiotic tropodithietic acid (TDA; [@pone.0096038-Seyedsayamdost1], [@pone.0096038-Thiel1]). Previous experiments using high performance liquid chromatography suggested that a compound with the same retention time and UV-visible spectra as the TDA standard was produced by *Pseudovibrio* sp. FO-BEG1 under −P~i~ conditions when cells entered stationary phase (Romano et al., unpublished data). During the FT-ICR-MS analyses, we identified the *m*/*z* 210.952904 with the molecular formula assigned C~8~H~4~O~3~S~2~, which was, considering also its peculiar isotopic patterns due to the presence of two sulfur atoms per molecule, consistent with being TDA. This compound was detected only under phosphate limitation and its ion intensity increased from T2 to T3. Consistently, when the Dictionary of Natural Products (DNP) was used as target for annotating the detected masses, the previously mentioned *m*/*z* was assigned to TDA, and to thiotropocin and troposulfenin ([Dataset S5](#pone.0096038.s010){ref-type="supplementary-material"}) which are tautomers of TDA [@pone.0096038-Greer1] and are known to be produced by *Pseudomonas* spp. [@pone.0096038-Kintaka1]. In addition, in the same samples, the *m*/*z* 226,947816 was annotated as hydroxytropodithietic acid, which was suggested to derive from the hydroxylation of TDA [@pone.0096038-Liang1]. Members of the *Roseobacter* clade produce TDA together with an uncharacterized yellow pigment [@pone.0096038-Bruhn1] and consistently also the *Pseudovibrio* cultures growing under −P~i~ conditions developed an intense yellow coloration when entered stationary phase (Romano et al., unpublished data). Altogether, this data support our interpretation that TDA was produced during the stationary phase under −P~i~ conditions.
The annotation using the DNP resulted in the attribution of several masses to molecules previously described in marine bacteria, including members of the *Roseobacter* clade ([Datasets S5](#pone.0096038.s010){ref-type="supplementary-material"}; e.g. 3-(4-Hydroxy-3-nitrophenyl)propanoic acid, cyclo(glutamylglycylprolyl), cyclo(glutamylglycylserylprolyl), homo-ξ-rhodomycinone). Among those, several masses were annotated as cyclic dipeptides produced by *Roseobacter* strains isolated from marine sponges. Cyclic dipeptides are molecules with antibacterial properties and biological and pharmacological effects on cells of higher organisms. It was suggested that they could play a role in bacterial and prokaryote-eukaryote communication [@pone.0096038-Degrassi1]--[@pone.0096038-Prasad1]. In the last years several cyclic dipeptides have been isolated from marine organisms such as sponges and algae and from many marine prokaryotes, suggesting that the ability to produce these compounds is widespread among marine bacteria [@pone.0096038-Huang1]. Considering the phylogenetic and physiological similarity between *Roseobacter*- and *Pseudovibrio*-related bacteria, and the recurrent association between both cyclic dipeptides and *Pseudovibrio* with marine sponges [@pone.0096038-Bondarev1], [@pone.0096038-Huang1], it is reasonable to speculate that *Pseudovibrio* sp. FO-BEG1 released such compounds into the medium. This information offers a solid base for further chemical characterization of these compounds, which could represent new molecules of biotechnological interest.
When the KEGG database was used as target, most of the metabolites assigned to the detected masses were compounds involved in the synthesis of mainly aromatic amino acids (e.g. tyrosine, tryptophan, phenylalanine; [Dataset S1](#pone.0096038.s006){ref-type="supplementary-material"}, [S2](#pone.0096038.s007){ref-type="supplementary-material"}, [Fig. S5](#pone.0096038.s005){ref-type="supplementary-material"}) and nucleotides. Consistently, the annotation performed using the HMDB and the Drug Bank also suggested the presence of intermediates of these metabolisms as, for example, the shikimate pathway (e.g. erythrose-4-phosphate, shikimate-3-phosphate; [Datasets S3](#pone.0096038.s008){ref-type="supplementary-material"}, [S4](#pone.0096038.s009){ref-type="supplementary-material"}), which is responsible for the biosynthesis of aromatic amino acids. Release of these compounds was also observed in the analysis of the exo-metabolome of other bacterial and yeast strains [@pone.0096038-Paczia1], [@pone.0096038-Behrends1]. In conditions of "overflow metabolism", i.e. conditions with an excess of carbon or energy source or in the presence of nutrient limitation, intermediates of different metabolic pathways can be released [@pone.0096038-Kramer1]. Recent evidence suggests that this is a common phenomenon in different microorganisms when they are cultivated under conditions of non-inhibited carbon uptake [@pone.0096038-Paczia1]. Aromatic amino acids are key intermediates in the production of aromatic secondary metabolites [@pone.0096038-Herrmann1] suggesting that strain FO-BEG1 is potentially producing such compounds, which, however, are of unknown structure. Unlike observed for other microorganisms [@pone.0096038-Paczia1], no masses were annotated as metabolic intermediates of central metabolic pathways such as the tricarboxylic acid cycle. The main reason for this is that most of these metabolites have a low molecular mass, e.g. fumarate 116.07 Da, which fells outside the *m*/*z* range chosen for our analysis (150-2,000 *m*/*z*).
Among the identified compounds, a smaller number of metabolites could be annotated for the samples collected at T3 under −P~i~ conditions. However, the majority of the annotated compounds belonged to the same pathways identified in KEGG for the other samples. Under phosphate limitation, the number of formulae annotated in the pathway "tyrosine metabolism" and "tryptophan metabolism" using KEGG, and the intermediates of the shikimate pathway detected in HMDB and Drug Bank, decreased strongly from T1 to T3, indicating that these metabolites were taken up again by the cells when they entered stationary phase. This uptake of previously released metabolites was likely done to satisfy specific anabolic needs under this growth conditions.
Production and release of amino acids by bacterial communities was also reported by Kawasaki and Benner [@pone.0096038-Kawasaki1], and these compounds were shown to be important constituents of DOC in some coastal areas [@pone.0096038-Coble1], [@pone.0096038-Yamashita1], environments where also *Pseudovibrio* strains were often isolated [@pone.0096038-Shieh1], [@pone.0096038-Hosoya1]. It is worth pointing out that comparing the list of molecular formulae retrieved from our exo-metabolome study with a list of formulae detected in DOM of the deep North Pacific Ocean [@pone.0096038-Rossel1], we found only 83 shared compounds. However, comparing our data with a list of molecular formulae detected in DOM during and after a phytoplankton bloom in the North Sea (Dittmar et al., unpublished data), we detected 729 matches (18% of the masses with unique molecular formulae assigned) and 91% of them were always present in the natural samples, irrespective of the occurrence of the phytoplankton bloom ([Dataset S6](#pone.0096038.s011){ref-type="supplementary-material"}). This indicates that, at least on a molecular formula level, a large fraction of the detected compounds are indeed part of natural DOM, and their presence does not seem to be directly related to the immediate activity of primary producers. Consistently, also Kujawinski et al. [@pone.0096038-Kujawinski2] showed that some molecules detected in a pure culture of "*Candidatus* Pelagibacter ubique" were present in open-ocean DOM.
Our approach represents a high-throughput way of performing metabolomic studies, and it was adequate to capture the diversity of the metabolites released by the bacterium into the environment. However, the translation of the analytical information into existent biological knowledge by using the available tools showed two major drawbacks. The first one regards the reliability of the annotation performed using the HMDB, the Drug Bank and the DNP. By using a one to one match approach (peak by peak search), we were able to assign metabolite names to up to 8% of the detected masses. Especially using the first two databases a high number of double assignments and of non-bacterial metabolites were obtained, suggesting a high rate of false positive identifications. These results confirm previous reports that underlined the limit of single match annotation even when a mass error below 1 ppm is adopted [@pone.0096038-Weber1], [@pone.0096038-Kind1]. As pointed out previously, these approaches require the application of orthogonal filtration processes, such as isotopic abundance patterns or "transformation mapping", which can significantly decrease the number of false positive assignments, generating more reliable information which can be integrated into a biological contest [@pone.0096038-Weber1], [@pone.0096038-Kind1].
To increase the confidence in the annotation, we applied the "transformation mapping" approach, using the metabolic pathways of *Pseudovibrio* sp. FO-BEG1 reported in KEGG. Applying this method we were able to annotate less than 3% of the detected masses. The main drawback of this strategy is the incompleteness of the databases used, which can reduce the annotation efficiency by overlooking metabolites that are known, but not yet integrated into the database. For instance, even though we have strong evidence that TDA was produced during the stationary phase under −P~i~ conditions, and the annotation performed using the DNP identified one mass consistent with being TDA, we could not identify it during the annotation processes using KEGG. The reason is the absence of the biosynthetic pathway for TDA among the annotated ones in *Pseudovibrio*. Databases such as KEGG are mostly restricted to genome-reconstruction pathways. Wrongly annotated genes and absence of compounds for which the biosynthetic routs have not been completely elucidated yet can decrease the number of identified molecules in metabolomic studies, and limit the capabilities of techniques such as FT-ICR-MS. This underlines the lack of knowledge we have about the biosynthetic ability of marine bacteria and also the necessity to create more comprehensive databases, containing information about both primary and secondary metabolites.
Conclusions {#s5}
===========
In this study we investigated in detail the exo-metabolome of a marine heterotrophic bacterium using ultra-high resolution mass spectrometry. Our work shows that HRAM instruments represent promising tools to unravel the complexity of the metabolites secreted from microorganisms. We show that the exo-metabolome is unexpectedly large and diverse, it is characterized by a dynamic recycling of compounds, and it is drastically affected by the physiological state of the strain. Our data clearly illustrate that phosphate limitation triggered a pronounced increase in the secretion of DOC and at the same time greatly affected its composition, leading to an increased production of functionalized phenols and polyphenols. A Part of the molecular formulae discovered in the exo-metabolome was also detected in natural marine DOM. Therefore, future studies on the exo-metabolomes of different strains and DOM from different locations might help to understand to what extent the compounds secreted by heterotrophic bacteria influence the oceanic DOM composition. The discrepancy between the number of measured masses and the number of annotated molecules obtained using different databases underlines the gap in our knowledge concerning the biosynthetic ability of marine bacteria, indicating the necessity of further work directed to chemically characterize the secreted metabolites. However, the integrated metabolic annotation we performed using multiple databases gave us a first glimpse of the composition of the secreted compounds, suggesting that the large bacterial exo-metabolome can represent a "chemical reservoir" for the discovery of new molecules of biotechnological interest. Our data underline the great biosynthetic ability of heterotrophic bacteria and suggest that, using the words of Traxler and Kolter [@pone.0096038-Traxler1], ''*the chemical landscape inhabited and manipulated by bacteria is vastly more complex and sophisticated than previously thought*''.
Supporting Information {#s6}
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**Bootstrap analyses performed on the dendrograms obtained using the paired group algorithm and the Bray-Curtis similarity index calculated for the FT-ICR-MS samples analyzed in ESI-negative mode.** Since the cophenetic correlation coefficients were \> 90%, the dendrograms can be considered a reliable representation of the similarity matrices. 1000 reiterations were allowed for the bootstrap analyses. Dendrograms were constructed using the data of the unfiltered **(A)** and filtered **(B)** datasets. All biological triplicates of +P~i~ and −P~i~ conditions are shown.
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**Similarity among the FT-ICR-MS samples analyzed in ESI-positive mode during bacterial growth under +P~i~ and −P~i~ conditions.** Non metrical multidimensional scaling (NMDS) was performed by employing the Bray-Curtis similarity index and using the data of the unfiltered (**A**) and filtered (**B**) datasets. All biological triplicates of +P~i~ (filled circles) and −P~i~ (empty circles) conditions are shown. Nearest neighbor samples (i.e. most similar) are connected to visualize pairwise sample similarities. The stress value for **A** is 0.07 and for **B** is 0.08.
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**Bootstrap analyses performed on the dendrograms obtained using the paired group algorithm and the Bray-Curtis similarity index calculated for the FT-ICR-MS samples analyzed in ESI-positive mode.** Since the cophenetic correlation coefficients were \> 95%, the dendrograms can be considered a reliable representation of the similarity matrices. 1000 reiterations were allowed for the bootstrap analyses. Dendrograms were constructed using the data of the unfiltered **(A)** and filtered **(B)** datasets. All biological triplicates of +P~i~ and −P~i~ conditions are shown.
(TIF)
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**Venn diagram showing unique and shared masses detected in ESI-positive mode in all biological triplicates of the different samples.** Only masses detected in all biological triplicates for each time point were considered.
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**Number of metabolites annotated in the metabolic pathways of** ***Pseudovibrio*** **sp. FO-BEG1 collected in the KEGG database.** The masses obtained from the ESI-negative FT-ICR-MS analysis were annotated using the MI-Pack package. The bars of each color, representing the different time points, indicate the absolute number of metabolites annotated in the respective pathways reported in the KEGG database.
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**Metabolites annotated using as target the metabolic pathways of** ***Pseudovibrio*** **sp. FO-BEG1 reported in the KEGG database.**
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**Metabolic pathways present in the KEGG database, which contain the annotated metabolites.**
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**Metabolites annotated using the Drug Bank as target.**
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**Metabolites annotated using the Human Metabolome Database as target.**
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**Metabolites annotated using the Dictionary of Natural Products as target.**
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**List of molecular formulae shared between the exo-metabolome of** ***Pseudovibrio*** **sp. FO-BEG1 and the North Sea DOM.**
(XLS)
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Click here for additional data file.
We are very grateful to K. Klaproth for technical support with the FT-ICR-MS analyses and I. Ulber and M. Friebe for help in the DOM extraction and DOC measurements. We are indebted to Dr. J. Niggemann for sharing DOM data of the North Sea. We acknowledge Dr. A. Ramette and G. Jessen for the constructive discussions concerning the statistical analysis.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: SR HNSV TD. Performed the experiments: SR VB. Analyzed the data: SR TD. Contributed reagents/materials/analysis tools: SR TD RJMW MRV HNSV. Wrote the paper: SR.
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The Monastery of the Holy Virgin of
Bishoi (Wadi Natrun, Egypt) was founded in the 6th century, but
since Syrian monks joined the Coptic community around 800AD it was better known
as Deir al-Surian, the Syrian monastery. For centuries monks of the two denominations
lived together, creating an environment where material and immaterial heritage
of the Coptic and Syriac Orthodox Churches were brought together, resulting in
a unique library and a church with extraordinary wall paintings. Most of these
paintings vanished out of sight when they
were covered by plaster in the 18 th
century. Since 1996 these paintings are gradually uncovered and give us an
impression of the Syrian influence on Egyptian Christianity. In this lecture
special attention will be given to the most recent discoveries of the past two
years.
About Me
I have studied Theology at the National and Kapodistrian University of Athens, School of Theology, International Relations at the University of London (Queen Mary). My Master's Thesis was published as a book: 'The Aegean Sea Dispute Between Greece and Turkey - The Consequences for NATO and the EU'. For more information see: http://www.akakia.net/el/the-aegean-sea-dispute-between-greece-and-turkey
I have also studied Byzantine Music in Athens and I am currently undertaking a research on the “Fellowship of St. Alban and St. Sergius and its contribution towards Anglican – Orthodox Relations”, at the University of Winchester.
I also represent the Greek Orthodox Archdiocese of Thyateira and Great Britain at the A.E.C.A. If you wish to contact me you can email me: [email protected]
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Enter the Dragon
Why “state capitalism” is China’s biggest knockoff.
Like China, the West has long used state resources to bolster industry.Credit Illustration by JOHN RITTER
Currency wars, trade battles, threats of economic sanctions—these days, it’s hard to open a newspaper without encountering at least one story about rising tensions between China and the United States. With all the talk of how China could displace the U.S. as the leading financial superpower, it is easy to forget that economic disputes between Beijing and Western capitals are nothing new, and that in the past they sometimes went well beyond diplomatic démarches. In the late eighteenth century, for example, the inhabitants of Great Britain were drinking so much tea from imperial China that a big trade deficit had arisen between the two countries. China demanded payment in silver, which was putting pressure on the Exchequer and the pound sterling. Eager to find a product that the Chinese would import, the English settled on opium, which was produced in parts of India that they controlled. In 1773, the governor-general of Bengal broke up the local opium-smuggling cartel and granted the London-based East India Company a monopoly, which endured for more than fifty years. During the next five decades, China’s annual imports of British-supplied opium went from seventy-five tons to nine hundred.
The rulers of imperial China took exception to this development, which was turning millions of their subjects into shiftless dope fiends. They tried banning the import of opium, to little effect. Finally, in 1839, a commissioner of the Canton region clamped down on the illegal trade, forcing British merchants to hand over thousands of chests of opium, and sent Queen Victoria a letter declaring, “We mean to cut off this harmful drug forever.” In London, there was outrage. Rather than negotiate with China, Lord Palmerston, the Foreign Secretary, dispatched a naval flotilla.
Confronted with iron-hulled steamships and powerful cannons, the Chinese military was hopelessly outmatched; the British seized control of Canton and the surrounding areas, killing thousands. Palmerston and his allies loftily insisted that the intervention was in the service of broader British interests and of the principle of free trade, which London was promoting throughout the Empire. The Times of London nonetheless dubbed the conflict the Opium War, and the young William Ewart Gladstone, in one of his early parliamentary speeches, said that the British flag “is become a pirate flag, to protect an infamous traffic.”
In 1842, the Chinese government was forced to sign the Treaty of Nanking, promising Britain more than twenty million silver dollars in reparations (around half a billion dollars in today’s currency), minimal tariffs on its goods, docking rights at five Chinese ports, and sovereignty over Hong Kong. Fifteen years later, complaining of trade impediments, France, Russia, and the United States, all of which had growing business interests in the Far East, joined the British in a second Opium War. Under the terms of the Convention of Peking, in 1860, China agreed to open up more of its ports to foreign exporters, to pay more in reparations, to allow British ships to transport indentured Chinese laborers (“coolies”) to the United States, and to legalize the opium trade. The country’s economic subjugation—the Chinese Communists later referred to the period as the “century of humiliation”—may have ultimately helped bring down the Qing dynasty and usher in civil war and revolution. But it certainly cleared up Britain’s trade deficit.
Today, of course, China can’t be pushed around as easily. It is now the world’s second-largest economy. Two of the four biggest banks in the world are state-controlled Chinese corporations, as are two of the ten biggest oil companies. With economic might comes strategic influence, evident in China’s increasingly active role in Africa and Latin America. Many observers foresee a coming clash of civilizations between an economically vibrant yet politically illiberal developing world, led by China, and a slow-growing democratic West. “China poses the most serious challenge to the United States since the half-century Cold War struggle with the Soviet Union,” Stefan Halper, a veteran foreign-policy expert, writes in his book “The Beijing Consensus: How China’s Authoritarian Model Will Dominate the Twenty-first Century” (Basic; $28.95).
Note the word “model” in the subtitle of Halper’s book. The elements of this model, in most accounts, include keeping key areas of the economy under state ownership or state control; using government subsidies and currency manipulation to promote exports; setting up sovereign wealth funds to buy companies and influence in the West; and making backdoor deals with equally autocratic states to insure access to oil and other natural resources. In all of this, the unifying theme is a reliance on the guiding hand of the state rather than on private decisions made in the marketplace. In “The End of the Free Market: Who Wins the War Between States and Corporations?” (Portfolio; $26.95), Ian Bremmer, the president of the economic consultancy firm Eurasia Group, writes that, until recently, “private wealth, private investment, and private enterprise appeared to have carried the day. But as the sun sets on the first decade of the twenty-first century, that story has already become ancient history. The power of the state is back”—and back in a way that “threatens free markets and the future of the global economy.” Picking up the same theme, the Wall Street Journal recently published a front-page story about how the rise of Chinese “state capitalism” was provoking a global backlash. “Since the end of the Cold War, the world’s powers have generally agreed on the wisdom of letting market competition—more than government planning—shape economic outcomes,” the Journal reported. “China’s national economic strategy is disrupting that consensus.”
Things could hardly be otherwise. The astonishing transformation since China adopted, in 1978, what Deng Xiaoping described as “socialism with Chinese characteristics” poses a big challenge to Western ideas about politics and about economics—but it is important to distinguish between the two. Within the political realm, what remains of the reassuring notion, popularized by Francis Fukuyama, that liberal democracy is the only system compatible with modernity? In promoting the development of a dynamic, competitive economy within the confines of a one-party state, the descendants of Chairman Mao seem to have arrived at a new social contract that says to the governed: Go and engage with the global economy, set up businesses, invest, make as much money as you can, but leave the politics to us. Russia, Cambodia, and other rapidly developing countries, too, have shifted in an authoritarian direction.
From a Jeffersonian perspective, what’s going on may look like repressive regimes foisting unpopular policies on peoples striving to be free. But, as Halper, who served in the Nixon, Ford, and Reagan Administrations, points out, these policies enjoy a good deal of popular support. “Given a choice between market democracy and its freedoms and market authoritarianism and its high growth, stability, improved living standards, and limits on expression—a majority in the developing world and in many middle-sized, non-Western powers prefer the authoritarian model,” Halper writes. If this unsettling trend isn’t arrested in the coming decades, he adds, “the United States will be left in a world unsympathetic to the democratic values and principles that have guided Western progress for more than two centuries.”
That may be overstating the case: once you look beyond Hu Jintao and Vladimir Putin, the political futures of China and Russia are far from clear. History suggests that “market authoritarianism” is often a transitional stage of development. During the nineteen-seventies and eighties, a number of Southeast Asian countries employed it to drag themselves out of poverty. Today, South Korea, Thailand, and Indonesia are democracies, of sorts. Singapore, on the other hand, remains essentially a one-party city-state. Who can say for sure which of these paths Russia (already a democracy, albeit a distinctly curtailed one) and China will end up following? Despite recent developments, China, in particular, is still a pretty poor place, with a per-capita G.D.P. of about three thousand dollars in 2008. By 2050, according to a recent study from the Carnegie Endowment, this figure will rise to about thirty-three thousand dollars, which would place China roughly where Spain is today. It is hard to disagree with George Magnus, an economic adviser to UBS Investment Bank, when he says, in his new book, “Uprising: Will Emerging Markets Shape or Shake the World Economy” (Wiley; $34.95), that “some sort of change in China is inevitable”— that, “sooner or later, rising living standards and the spread of modernity through the country are going to generate a growing public clamor for political participation and institutional reform.”
But it’s in the economic realm that Halper, Bremmer, and many other analysts have fundamentally misrepresented the Western pattern of development. Apart from economics textbooks and reports from the International Monetary Fund, the free-market model of capitalism that China, Russia, and other fast-growing nations are supposedly supplanting has never actually existed. The closer you look at how countries such as Britain and the United States became prosperous, the less you see of laissez-faire and the more you see of government intervention. Lord Palmerston’s “gunboat diplomacy” in defense of the opium trade may have been an especially bald version of state capitalism, but the basic strategy of enlisting state power in pursuit of commercial advantage, and vice versa, has been anything but the exception. Far from subverting the Western way of doing business, the developing world is, at last, stealing some of its tricks.
Take free trade. From Lord Palmerston to Secretary of State Hillary Clinton, Western officials have long demanded that countries open their domestic markets to foreign competition. But Britain and the United States embraced free trade as an ideal only after they had built up manufacturing industries that could dominate those of foreign rivals. The rise of Britain as an economic power can be dated to the fourteenth century, when King Edward III banned the import of woollen cloths from Belgium and Holland, the market leaders in textiles. In the centuries that followed, Britain’s trade policy was geared toward promoting its wool and cotton industries; duties were placed on exports of raw wool, to encourage British merchants to move into the more lucrative business of assembling finished cloths.
It wasn’t just the textile industry that received favorable treatment. In 1721, the government of Robert Walpole placed a range of tariffs on all manufactured imports, erecting a protective wall around businesses that created the Industrial Revolution. A century later, while the heirs of Adam Smith were expounding the theoretical virtues of free trade, Britain retained some of the highest import tariffs in the world: more than fifty per cent on many manufactured goods. Those levies stayed high until the eighteen-sixties, when the country’s competitive advantage in textiles, steel, and other industries was firmly established. As the late economic historian Paul Bairoch stressed, the idea that Britain rose to economic dominance through free trade is nonsense.
The same is true of the United States. Even before 1791, when Alexander Hamilton published his famous “Report on the Subject of Manufactures,” Congress used tariffs to protect favored industries. During the War of 1812, which was precipitated in part by trade disputes, it doubled import duties on manufactured goods, to twenty-five per cent. A few years later, the levies were raised to an average of forty per cent. Then Abraham Lincoln raised them again, to roughly fifty per cent. Ha-Joon Chang, an economist at the University of Cambridge, has observed that Lincoln, revered as the Great Emancipator, “might equally be labeled the great protector—of American manufacturing.”
During the half century after Lincoln’s Presidency, the business-backed Republican Party was in power for most of the time, and tariffs on manufactured goods remained at forty to fifty per cent, the highest levels anywhere. It was during these years that the U.S. economy grew to rival the economies of Britain and Germany in industries such as iron and steel and chemicals—all of which benefitted from protection. Even during and after the First World War, when President Woodrow Wilson, in his Fourteen Points, elevated free trade to a goal of U.S. policy, hefty tariffs remained in place. (For some agricultural products, such as sugar and ethanol, they still exist—greatly to the advantage of big U.S. agribusinesses such as Cargill and Archer Daniels Midland, which otherwise would struggle to compete with foreign suppliers. And that’s aside from the billions that these companies have received in government subsidies.) The fact is that not one of today’s economic powers practiced free trade during its developmental stage. “Free trade economists have to explain how free trade can be an explanation for the economic success of today’s rich countries,” Chang notes, “when it simply had not been practiced very much before they became rich.”
China, Korea, and other rising economies are often reproached for using government money and influence to bolster home industries to the disadvantage of foreign competitors, a practice that is known as “industrial policy,” and is frowned on by international trade law. Such discriminatory policies are also referred to as dirigisme—a clue that the concept didn’t originate in the Far East. After the Second World War, the government of France’s Fifth Republic created “national champions” in strategic areas of the French economy, such as transportation, energy, and aerospace. The policy gave rise to big companies like Air France, the French railway operator S.N.C.F., the utility company E.D.F., and the aeronautics contractor EADS, all of which are partly or wholly owned by the French government. It continues under President Nicolas Sarkozy, a devoted interventionist, who made his reputation as finance minister by bailing out Alstom, a large engineering firm, and encouraging a merger between the drug companies Aventis and Sanofi in order to avert a threat of foreign takeover.
But industrial policy long predates General de Gaulle. It was Walpole who wrote, “Nothing so much contributes to promote the public well-being as the exportation of manufactured goods,” and the British government long subsidized exporters while imposing tight quality standards on them to safeguard the British brand. “These policies are strikingly similar to those used with success by the ‘miracle’ economies of East Asia, such as Japan, Korea, and Taiwan, after the Second World War,” Chang has argued in “Bad Samaritans: The Myth of Free Trade and the Secret History of Capitalism” (2008). “Policies that many believe, as I myself used to, to have been invented by Japanese policy-makers in the 1950s . . . were actually early British inventions.”
Here, too, the United States was quick to follow Britain’s lead. In 1791, Hamilton proposed a series of policies to help transform America into an industrial economy, including export subsidies, prizes for industrial inventions, and public investment in infrastructure. During and after the Civil War, the federal government, by providing generous land grants and cheap financing, was instrumental in opening up the Great Plains and directing the expansion westward. The Central Pacific and Union Pacific railroads were both government-chartered companies that benefitted from large land grants, not to mention vast sums in government loans.
U.S. industrial policy may be less visible these days, but it still plays a key role in maintaining our competitive edge. Much of this assistance comes through the Pentagon, which, by paying for research-and-development projects that private investors would be reluctant to finance, has helped to create three of our biggest export industries: commercial aircraft, military aircraft, and computers. The Boeing 747 and many other modern jetliners were developed from designs for military aircraft. Fairchild Semiconductor, which helped pioneer the development of the silicon transistor, in the nineteen-fifties, was a military contractor, as were many other technology firms that helped launch the modern computer industry, such as Texas Instruments. And, famously, the Internet was created by the Pentagon’s Defense Advanced Research Projects Agency (DARPA), which continued to operate it until 1990.
The Department of Defense remains the indispensable client for many major U.S. manufacturers, such as Boeing, Lockheed, and Honeywell. Despite laws that direct federal officials to obtain the best terms available for taxpayers, personal links still appear to influence dealings between the Pentagon and big defense contractors, as a steady stream of procurement scandals suggests. On those rare occasions when foreign firms manage to secure large military contracts, Congress often objects. (Witness the continuing row over Airbus’s bid for an order of refuelling tankers.) To be sure, domestic favoritism exists in many other countries, perhaps to an even greater extent. But since the United States spends almost as much on its military as the rest of the world combined, no other government department comes close to exerting the commercial sway of the Pentagon.
Then, there’s the financial sector. Developing Asian countries have been criticized for propping up struggling banks rather than allowing market forces to operate. During the recent financial crisis, of course, the United States—along with other prominent members of the World Trade Organization, like Britain and Germany—found itself doing precisely the same thing. As for the U.S. bailouts of General Motors and Chrysler, a case can be made that they violated W.T.O. rules, not that anybody is going to call Washington to account. Compared with these naked exercises in industrial policy, some of the Chinese infractions that have most exercised the W.T.O. seem relatively minor. Last year, China was criticized for preventing foreign firms from selling books, movies, and music directly to Chinese customers; instead, Western entertainment companies like Disney and Time Warner have had to work with local distributors. Such measures are plainly discriminatory and protectionist, but they have hardly prevented big U.S. corporations from making inroads into the Chinese markets. In the first half of this year, General Motors’ Chinese subsidiary, which is a joint venture with Chinese manufacturers, sold more than a million cars and trucks, a jump of almost fifty per cent over last year. For the first time, the Detroit automaker sold more cars in China than it did in the United States.
Ian Bremmer concedes that many American firms are doing well in China, and he relegates the prospect of a trade showdown with China to a distant threat. In a chapter about the challenges ahead, he asks what will happen if and when “Chinese companies use their growing influence to lobby for greater support against foreign competition. . . . In other words, what happens if China closes the door?” The likely answer is that China would get burned and quickly turn back. A trade confrontation with the U.S. and other Western countries—an Opium War in reverse—would undermine the very basis of China’s success: the export of cheap manufactured goods.
Both Bremmer and Halper devote considerable space to China’s recent efforts to secure its future supplies of energy and natural resources, which have involved cozying up to repressive regimes in places like Zimbabwe, Sudan, and Iran. Halper, in particular, emphasizes the threat posed by China’s dealings among aid-dependent countries. He considers China’s rise in Africa to be a “tragedy,” though his alarmism doesn’t do justice to the complexity of the topic, or, indeed, of the region. Part of the evidence he presents for China’s malign influence is the fact that it helped build a hospital, an irrigation project, and a vocational-training center in Ghana—a multiparty democracy that, mystifyingly, is on his list of repressive African regimes. If this is a strange example to have chosen, it’s not the only one. Bemoaning China’s success in limiting American influence, he describes China’s transactions with oil-producing Angola, which has been left ravaged by three decades of a failed but spectacularly bloody insurgency. He points out that China offered development loans with none of the good-government strictures that the I.M.F. sought to impose. He doesn’t point out that the United States was, together with apartheid-era South Africa, a chief sponsor of the insurgency, which cost the lives of three hundred thousand people and displaced nearly a quarter of the country’s population. (In fact, much of the support came during the Reagan Administration, in which Halper served as a member of the State Department’s Bureau of Political-Military Affairs.) As for whether I.M.F.-style conditions on foreign assistance really have a positive effect on freedom and democracy, the evidence is far from clear.
“The use of oil, gas, and other commodities as political tools and strategic assets,” Bremmer writes, “can be an essential part of state capitalism,” and he notes that three-quarters of the world’s oil reserves are owned by national oil companies. There is no disputing that securing access to natural resources is a major strategic objective in foreign policy, but that’s equally true of Western countries and their developing rivals. During the late nineteenth century, tales of diamonds, gold, and other precious minerals in limitless quantities helped spark the Scramble for Africa. After the First World War, Britain and the United States, conscious of the future importance of the Persian Gulf ’s vast oil reserves, helped install a series of pliable desert monarchs who granted access to Western oil companies on favorable terms. In 1953, two years after Mohammad Mosaddegh, the democratically elected Prime Minister of Iran, nationalized the British-owned Anglo-Iranian Oil Company, the C.I.A. helped organize a successful coup against him. Needless to say, the United States continues to stabilize “friendly” oil-exporting governments, like the Kingdom of Saudi Arabia and the Sultanate of Oman, with extensive military and technical assistance.
The heavy hand of American domestic and foreign policy in shaping economic outcomes tends to get ignored in current policy debates, in which economic issues are usually cast in ideological terms, devoid of their historical context. Yet our amnesia comes with a cost: the idealized free-market model that is supposedly under threat offers only the harshest of solutions to the energy problem. If demand for oil continues to soar in the face of limited supply, prices will eventually skyrocket, forcing the United States and other oil importers to cut drastically their consumption of gasoline. There’s an alternative to this market-administered shock therapy: a vigorous energy policy that raises taxes on carbon fuels, subsidizes energy conservation, and provides cheap financing for the development of renewable energy sources. But a major obstacle to such a policy is the popular misconception about the role of the state in economic development.
There are some encouraging signs of revisionism. In a recent book, “The Great Betrayal,” Clyde Prestowitz, a former U.S. trade negotiator who founded the Washington-based Economic Strategy Institute, includes a chapter on “How America Really Got Rich.” And in the book “Losing Control: The Emerging Threats to Western Prosperity,” Stephen D. King, the chief economist at HSBC, the London-based global bank, notes, “Western governments have used the methods of state capitalism for hundreds of years in their bid to shape the world around them. . . . The idea that market forces alone led to the West’s success is nonsense.”
Unfortunately, in policy circles—and among much of the general public—the old mantras about the free market and private enterprise continue to dominate. In seeking to broaden access to private health insurance, the Obama Administration was accused of plotting a takeover of the entire health-care industry. In cutting taxes and boosting federal spending to avert a depression, it was accused of embracing socialism. Even supposedly serious economists lend support to these views, arguing that the dysfunctional health-care industry is best left to its own devices, or that the eight-hundred-billion-dollar stimulus program has had virtually no impact on jobs and on G.D.P. This is what comes of forgetting the critical role that states have played in nurturing, protecting, and financing their industries, as well as in taxing and taming them. The greatest danger that Western prosperity now faces isn’t posed by any Beijing consensus; it’s posed by the myth of the free market. ♦
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Grinding operations on structural materials (e.g. metallic and ceramic workpieces) typically involves contacting the structural material workpiece with an abrasive article (e.g. grinding wheel) to remove material from and shape the workpiece. Such grinding operations generally involve the input of large amounts of energy (i.e. grinding energy) into the removal of material from the workpiece and often employ high rotating speeds for the abrasive article (e.g. grinding wheel) and/or the workpiece. In some grinding operations it is known to rotate both the grinding wheel and the workpiece. Where high material removal rates, workpieces that are especially tough or hard, high grinding wheel speeds and deep cuts are employed the amount of energy applied to the grinding operation can be and often is very high. This energy in large measure translates into heat that is mostly applied to the workpiece and grinding wheel. The heat often has a detrimental effect on both the grinding wheel and the workpiece. Excessive heat generated during grinding can and often does result in burning of metallic workpieces (ie the formation of a yellow brownish or dark brown to black discoloration on the ground surface of the workpiece). Burning of the metallic workpiece results in a scrapped part. Often the effects of excessive heat generated during grinding can be distortion of the workpiece, out of tolerance parts, changes in the surface appearance and properties of the ground part (e.g. surface hardening effects), excessive break down of the grinding wheel, loss of grinding performance and efficiency, loss of productivity and increase costs.
Creep feed, snagging and cut off grinding operations are high heat generating processes because of the desire for high metal removal rates (i.e. cubic inches of metal removed per unit of time). In snagging and cut off grinding operations the burning of the metal part due to the high generation of heat is not critical because the metal part is in a rough condition after the snagging and cut off operations and is subject to subsequent shaping and finishing steps. The creep feed grinding operation also generates large amounts of heat because of the desire for high metal removal rates in the shaping of the metallic workpiece. However burning of the metallic piece (i.e. the formation of a yellow brown, brownish or brownish black discoloration on the surface) during creep feed grinding operations is a very undesirable condition resulting in the scrapping of the workpiece or article. Additionally, excessive heat generated in a creep feed grinding operation can cause distortion of the part, alteration of the surface appearance and surface properties of the part (e.g. change the surface hardness of the part) and cause the production of an out of tolerance part. Typically in the creep feed grinding operation the metallic workpiece, article or part is fed into a rotating grinding wheel which remains in one location. The rate at which the workpiece is fed into the grinding wheel and the depth of cut are established to maximize the metal removal rate consistent with the desires to produce quality parts, reduce scrap, achieve high grinding efficiency and lower grinding operation costs. Thus the higher the metal removal rate, the greater the G-ratio (i.e. amount of metal removed per unit of grinding wheel lost) without burning the part the greater the efficiency and productivity and the lower the cost of the creep feed grinding operations. Creep feed grinding is used for example in the production of gears. In the production of gears, formed grinding wheels (i.e. wheels having a particular shape) are often used in the creep feed grinding process. It is therefore important that such shaped wheels retain their shape for as long as possible consistent with the other desirable conditions of the creep feed grinding operation (e.g. high metal removal rate, high G-ratio, low heat production and non-burning of workpiece). Although the burning of metallic workpieces and excessive heat generation are of major concern in creep feed grinding operations they are also important concerns in other grinding operations for shaping metallic workpieces to produce useful articles. Such other grinding operations include, for example, surface, internal, plunge and roll grinding operations. Thus it is important and highly desirable to have grinding wheels which produce or contribute to low heat generation during grinding and reduce or eliminate part burn or the risk of part burn while providing high grinding efficiencies and performance, long wheel life and high productivity to reduce grinding operation costs.
It is known to employ metalworking fluids (e.g. water based or oils) in grinding operations to improve grinding performance and efficiency. These fluids are, in many cases, known to reduce friction and remove heat during the grinding operation. Reduction of friction by the fluids can reduce the heat generated during grinding. The ability of these fluids to reduce friction (i.e. friction between the workpiece and the grinding wheel and/or components thereof) and remove heat during grinding can depend upon such factors as the composition of the fluid and the ability of the fluid to penetrate into the grinding zone or interface (i.e. the area of contact between the grinding wheel and the workpiece during grinding). Many metalworking fluids are known to be effective in many grinding operations and have been found to be of value in mild (i.e. low heat generating) grinding operations to improve grinding efficiency or performance. However in severe (i.e. high heat producing) grinding operations (e.g. creep feed grinding) they are often found to be of limited, if any, effectiveness in reducing or preventing part burn when high metal removal rates are sought. In such severe grinding operations it has been found that the metalworking fluids often exhibit poor penetration into the grinding interface, i.e., the region within which material removal occurs, to reduce friction and remove heat.
In the art it is known that different grinding operations (e.g. surface vs internal vs roll vs plunge vs snagging vs cut off vs creep feed grinding) involve different conditions. Such operations therefore often employ for example different forces, speeds, temperatures, infeed rates, metal removal rates and workpiece materials. Some grinding operations (e.g. finish grinding or surface grinding) may employ mild physical conditions involving low forces, low feed rates and low metal removal rates etc. Other grinding operations (e.g. creep feed, plunge and cut off grinding) may employ severe physical conditions involving high forces, high feed rates and high metal removal rates etc. Thus it is known to produce grinding wheels tailored to particular grinding operations and/or workpiece materials. Such wheels may differ in composition (i.e. amount and kind of abrasive grit, bonding material binding together the abrasive grit and additives) and/or structure depending upon their end use. The wheel structure may vary in the amount and type of porosity it contains. The porosity of a grinding wheel, particularly a vitreous bonded grinding wheel, can be of an open and/or closed cell structure. In the open cell porosity the cells or pores are interconnected much like the pores of a sponge or open celled foam. In the closed cell porosity the cells or pores are not interconnected and remain as separated totally enclosed voids much like closed cell foam. Closed cell, rather than open cell, porosity is generally found in resin bonded grinding wheels. The pore structure of a vitreous bonded grinding wheel can serve a number of functions including, for example, controlling the physical strength of the wheel, controlling the breakdown of the wheel to present fresh cutting edges, the elimination of swarf and providing means for getting metalworking fluid to the grinding zone. In a vitreous bonded grinding wheel having an open pore structure it is known to have an essentially random distribution of pore or cell sizes (i.e. some pores being large and other pores being small) and in some cases a random distribution of pores. Thus vitreous bonded grinding wheels can have a heterogeneous open pore structure with respect to pore size and in some cases pore distribution. Pore sizes larger than the abrasive grain average size may be found. Grinding wheels, particularly resin bonded grinding wheels, are known in the art to include thermally conducting particles (e.g. metal particles) to act as heat sinks and improve the dissipation of heat from the grinding wheel. In the case of resin bonded grinding wheels the dissipation of heat from the wheel by such thermally conducting particles serves to protect the poor thermally conducting resin bond from thermally induced breakdown and thus helps protect (i.e. preserve) the strength of the wheel during grinding.
In the grinding process and in particular a grinding operation under severe physical conditions, as are encountered in creep feed grinding operations, using an open cell porosity vitreous bonded grinding wheel, the open pore structure of the wheel can serve as a significant avenue or means by which metalworking fluid can penetrate into the grinding zone or interface and by which metalworking fluid can be captured by the wheel during grinding to reduce friction and remove heat generated during grinding. Such reduction in friction and dissipation of heat are significant factors in reducing or preventing grinding burn of the metallic workpiece, increasing performance and efficiency and lowering the power or energy needed for the grinding operation. These improvements in turn can lead to higher metal removal rates, increased productivity and lower grinding operation costs
Vitreous bonded grinding wheels in the prior art are known to be less than desirable in preventing or reducing grinding burn of metallic workpieces under severe physical grinding (e.g. high metal removal rate) conditions even when the grinding operation is carried out in the presence of a metalworking fluid. Thus grinding burn obtained with prior art vitreous bonded grinding wheels under severe physical conditions is known in the art. In many cases, in the art, grinding burn is overcome by reducing the severity of the physical grinding conditions (e.g. reducing metal removal rate and/or infeed rate and/or wheel speed etc.) leading to a loss of productivity and increased grinding costs. Additionally the excessive heat generated during grinding under severe physical conditions with prior art vitreous bonded grinding wheels is often known to lead to scrapped metal parts because of out of tolerance conditions and/or adverse changes in surface appearance and/or properties (e.g. reduction or increase in surface hardness) of the parts. Improvements in vitreous bonded grinding wheels, particularly for use under severe physical grinding conditions, which reduce or prevent grinding burn of metallic workpieces, reduce power or energy consumption during grinding, improve grinding performance and efficiency and increase grinding productivity therefore are needed and desirable. This invention seeks to overcome these and other problems of prior art vitreous bonded grinding wheels, particularly those vitreous bonded grinding wheels used under severe physical conditions in a grinding operation and provide vitreous bonded grinding wheels with improved grinding performance, and improved penetration of metalworking fluids into the grinding zone for reducing or preventing grinding burn of metal workpieces and in reducing the energy or power used in the grinding operation.
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Q:
Dynamic if-then Code
I'm using Decision Tree algorithm and I get if-then rules (returned as text) for example:
if(Parameter1 > 10) then
if(Parameter2< 5) then do A
else do B
else do C
I want to use these rules in order to get decisions for few items: item(Parameter1, Parameter2) example: item1(15, 5), item2(10, 20), ...
Problem is, the if-then rules are dynamic and I want to write a code capable of reading this rules and apply them on items.
A:
You can flip this around and use Predicates to implement your tests. For example,
public class GreaterThan implements Predicate<Integer> {
private final int point;
public GreaterThan(final int point) {
this.point = point;
}
public boolean test(final Integer incoming) {
return incoming > point;
}
}
and
public class LessThan implements Predicate<Integer> {
private final int point;
public LessThan(final int point) {
this.point = point;
}
public boolean test(final Integer incoming) {
return incoming < point;
}
}
And so on. You can then use this to dynamically construct logical checks because your tests are now functional.
Predicate<Integer> gt10 = new GreaterThan(10);
Predicate<Integer> lt5 = new LessThan(5);
if(gt10.test(Parameter1)) then
if(lt5.test(Parameter2)) then do A
else do B
else do C
Wrap the execution of A, B and C in functions and you're on your way to a flexible system. Now you're dealing with functional objects, you can structure things dynamically - instead of the fixed test shown above, you can compose tests and consequences as required.
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Anxiety disorders are the most common mental illness among adolescents. Behavioral inhibition (Bl), an early-life predisposition to withdraw from novel, social stimuli, predicts high risk for adolescent anxiety, particularly social phobia (SP). This association persists over decades, yet the neurophysiologicai mechanisms of this association are unclear. The current study examines whether adolescents with SP or a history of temperamental riskfor SP (i.e., Bl) share anomalies in striatal circuitry reflecting hypersensivity to motivationally salient nonsocial and social stimuli. The current aims examine neural response to: (1) nonsocial incentives in adolescents with SP vs. healthy adolescents; (2) social incentives in adolescents with SP vs. healthy adolescents; and (3) social incentives in adolescents with and without temperamental risk for SP. To accomplish these aims, I will use fMRl tasks involving response to (a) anticipated monetary rewards and (b) anticipated peer evaluation. My career goal is to become an independent scientist conducting translationally-oriented work integrating developmental psychology and clinical neuroscience. I have obtained a tenure-track Assistant Professor position at the University of California, Davis (UCD) to begin September 2009.1 am confident that this position, in conjunction with my K99/R00 grant awarded September 2007, will support my goal of becoming an independent researcher whose work focuses on developmental clinical neuroscience. I am seeking to transition this grant from NIMH to the Centerfor Mind and Brain at UCD in accordance with the design ofthe K99/R00 (NIH Pathway to Independence Career Development Award). The intramural component of my grant and accompanying Research Fellowship ends August 2009. The grant is designed for the PI to transition to an extramural institution, i.e. my Assistant Professor position at UCD. I will continue collaboration with NIMH to insure successful completion of my grant, as stipulated in my proposal to achieve my research and career development aims. In particular, I will maintain my strong collaboration with Dr. Daniel Pine (grant sponsor), Chief of the Emotion and Development Branch at NIMH, as well as Dr. Nathan Fox (grant co-sponsor), Distinguished University Professor at U Maryland.
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**Core tip:** The purpose of this article was to investigate the role of sirtuin 1 (SIRT1) in intestinal epithelial cells (IECs) in ulcerative colitis (UC) in a UC coculture model and in mice with dextran sodium sulfate (DSS)-induced colitis. It was found that SIRT1 activation contributes to enhanced intestinal barrier and reduced apoptosis of IECs via the suppression of endoplasmic reticulum stress-mediated apoptosis-associated molecules CCAAT/enhancer-binding protein homologous protein and caspase-12. SIRT1 activation may be a potential therapeutic strategy for UC.
INTRODUCTION
============
Ulcerative colitis (UC), the main subtype of inflammatory bowel disease (IBD), is a chronic relapsing inflammatory disorder of the large intestine. The incidence and prevalence of UC have increased in recent years\[[@B1]\]. The etiology of UC remains obscure and involves a combination of genetics, environment, microbiota, and the immune system\[[@B2],[@B3]\]. It is widely believed that dysregulation of cytokines (*e.g*., tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-10, and IL-21), oxidative stress, and abnormal immune responses are key players in the progression of UC\[[@B4]\]. Recent data demonstrate that the intestinal epithelium, which plays a crucial role in the occurrence and persistence of UC, is a highly dynamic tissue rather than a simple physical barrier\[[@B5]\]. Although a large number of therapeutic agents, including 5-ASA drugs, immunosuppressants, steroids, and emerging biological agents, have appeared in the past few years, most patients still experience severe complications or recurrence of the disease, which greatly reduces their quality of life\[[@B6]\]. Therefore, it is imperative to develop effective treatments for UC.
The endoplasmic reticulum (ER) is a principal compartment in eukaryotic cells for protein folding and trafficking. Cellular stresses such as perturbations of Ca^2+^ homeostasis and oxidative stress disrupt ER homeostasis, resulting in the accumulation of unfolded and misfolded proteins in the ER lumen, which initiates the unfolded protein response (UPR)\[[@B7]\]. The UPR reduces protein synthesis, accelerates protein folding, and activates ER-associated degradation to orchestrate the recovery of ER function. However, if ER stress is too severe or persistent, intrinsic apoptotic pathways are eventually triggered, leading to cell death\[[@B8],[@B9]\]. Recently, increasing evidence suggests that ER stress and UPR are involved in the pathogenesis of UC by regulating apoptosis, autophagy, and inflammatory responses\[[@B6],[@B10]\].
Sirtuin 1 (SIRT1), a member of the mammalian sirtuin family of proteins, is a nicotinamide adenine dinucleotide (NAD^+^)-dependent protein deacetylase and plays an essential role in caloric restriction, life span modulation, and cell fate de-termination\[[@B11],[@B12]\]. Recently, a number of studies have demonstrated that SIRT1 plays a protective role in colitis\[[@B13]-[@B16]\]. In particular, a report by Melhem et al\[[@B14]\] illustrated that SIRT1 relieves experimental colitis by modulating ER stress and reducing the UPR. However, the mechanism underlying the regulatory effect of SIRT1 on ER stress-mediated apoptosis in colitis is still unclear.
In the present study, we aimed to investigate the role of SIRT1 in the intestinal barrier in a UC coculture model and in mice with dextran sodium sulfate (DSS)-induced colitis. The mechanisms underlying the effect of SIRT1 on ER stress-mediated apoptotic pathways within intestinal epithelial cells (IECs) were further explored.
MATERIALS AND METHODS
=====================
Reagents
--------
The SIRT1 activator SRT1720 and inhibitor nicotinamide (NAM) were obtained from Selleck Chemicals (Houston, TX, United States). DSS was purchased from MP Biomedical (Santa Ana, CA, United States). Anti-occludin, anti-zona occludens 1 (ZO-1), and anti-caspase-3 primary antibodies were purchased from Proteintech (Wuhan, China), anti-caspase-12 and anti-caspase-9 antibodies were obtained from LSBio (Seattle, WA, United States), and anti-SIRT1, anti-glucose-regulated protein 78 (GRP78), anti-CCAAT/enhancer-binding protein homologous protein (CHOP), and anti-β-actin antibodies were obtained from Abcam (Cambridge, UK).
Cell culture and coculture
--------------------------
We established an *in vitro* coculture model of Caco-2 and THP-1 cells based on previous studies\[[@B17]-[@B19]\].The human colon carcinoma Caco-2 and monocyte THP-1 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, United States), cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, United States) and RPMI-1640 cell culture medium (Gibco), respectively, supplemented with 10% fetal bovine serum (FBS; Gibco), and incubated at 37 °C in a 5% CO~2~ atmosphere. To establish the coculture model, Caco-2 cells were cultured in 6-well culture inserts (Transwell inserts; Corning Costar, NY, United States) at a density of 2 × 10^5^ cells/insert for 17-20 d to obtain an integrated monolayer. THP-1 cells were cultured in 6-well plates at a density of 1.5 × 10^6^ cells/well and treated with serum-free RPMI-1640 medium containing 100 ng/mL phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, United States) and 0.3% bovine serum albumin (BSA; Sigma-Aldrich) for 48 h. After confirming that THP-1 cells had differentiated into macrophages, the Transwell insert on which Caco-2 cells had been cultured for 17-20 d was placed in the culture well in which human macrophage-like THP-1 cells were cultivated, then lipopolysaccharide (LPS; Sigma-Aldrich) was added to the lower chamber at a final concentration of 10 ng/ml. Ultimately, the two cell lines were cocultured for 24 h. Once the coculture model was established, the SIRT1 activator SRT1720 or inhibitor NAM was added to the upper chamber medium at a final concentration of 10 µM and 5 mM, respectively.
Enzyme-linked immunosorbent assay (ELISA)
-----------------------------------------
The levels of secreted inflammatory cytokines IL-1β and TNF-α in the coculture model as well as in the colon tissues of treated mice were assayed using ELISA kits (Boster, Wuhan, China) according to the manufacturer's instructions. Cell-free supernatants from the upper chamber after coculture for 24 h and colon homogenate supernatants of mice were collected. The absorbance at 450 nm was detected with a microplate reader (Thermo Fisher Scientific, Waltham, MA, United States).
Annexin V-APC/7-AAD assays
--------------------------
Caco-2 apoptosis was evaluated with an Annexin V-APC/7-AAD Apoptosis Detection kit (Keygen Biotech, Nanjing, China). After SRT1720 or NAM treatment for 48 h, Caco-2 cells were harvested with EDTA-free trypsin, washed twice with cold phosphate-buffered saline, and resuspended in 500 µL of 1 × binding buffer. The resuspended cells were incubated with 5 µL of Annexin V-APC and 5 µL of 7-AAD for 5 min in the dark prior to being analyzed with a CytoFLEX flow cytometer (Beckman Coulter, CA, United States).
Animals
-------
Twenty-four female C57BL/6 mice (6-8 wk old, weighing 18-22 g) were obtained from SIPPR-BK Lab Animal Co. Ltd. (Shanghai, China) and kept at room temperature (22-23 °C), with a light/dark cycle of 12/12 h, and free access to food and water. All experimental protocols were designed to minimize pain or discomfort to the animals and were approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University.
Female mice were randomly divided into four groups of six per group: The control group had free access to drinking water; the UC group was fed 3% DSS (w/v) for 7 consecutive days; and the UC + SRT1720 and UC + NAM groups received 3% DSS (w/v) for 7 consecutive days, followed by treatment with SRT1720 (100 mg/kg·d, intraperitoneal injection) or NAM (500 mg/kg·d, intraperitoneal injection) for another 7 d, respectively. The disease activity index (DAI) was measured daily after successful induction of acute colitis, as previously described\[[@B20]\].
All mice were sacrificed by decapitation. Distal colon samples were harvested for subsequent studies. The histological score (HS) of colon sections stained with hematoxylin and eosin was evaluated as described previously\[[@B20]\].
Transferase-mediated dUTP nick-end labelling (TUNEL) assay
----------------------------------------------------------
Apoptosis of cells in the colon tissue was assessed using a commercially available TUNEL assay kit (In Situ Cell Death Detection kit; Roche Applied Science, Basel, Switzerland) according to the manufacturer\'s instructions. In brief, tissue sections were incubated with proteinase K solution at 37 °C for 15 min. Afterwards, the enzyme solution and label solution were mixed (1:9) and added to the samples. The addition of 50 µL of converter-POD for 30 min was performed sequentially. Ten fields per section were assayed randomly in each experiment, and the percentage of positive cells was calculated.
Quantitative real-time PCR (qRT-PCR)
------------------------------------
Total RNA was extracted from human macrophage-like THP-1 and Caco-2 cells using TRIzol reagent (TaKaRa, Shiga, Japan) and reverse transcribed using a PrimeScript^TM^ RT reagent kit with gDNA Eraser (TaKaRa). qRT-PCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System using the SYBER Green Premix Ex Taq kit (TaKaRa) according to the manufacturer's protocol. The primer sequences are listed in Table [1](#T1){ref-type="table"}. The relative mRNA expression was analyzed by the 2^-△△Ct^ method.
######
Primer sequences for quantitative real-time polymerase chain reaction
**Gene name** **Primer sequence**
--------------- --------- -------------------------------
*IL-1β* Forward 5'-ATGGCTTATTACAGTGGCA-3'
Reverse 5'-TGTAGTGGTGGTCGGAGA-3'
*TNF-α* Forward 5'-TCAGAGGGCCTGTACCTCAT-3'
Reverse 5'-GGAAGACCCCTCCCAGATAG-3'
*GRP78* Forward 5'-GGAACCATCCCGTGGCATAA-3'
Reverse 5'-CTTGGTAGGCACCACTGTGT-3'
*CHOP* Forward 5'-CACCACTCTTGACCCTGCTTCTC-3'
Reverse 5'-TGACCACTCTGTTTCCGTTTCC-3'
*β-actin* Forward 5'-AGCGAGCATCCCCCAAAGTT-3'
Reverse 5'-GGGCACGAAGGCTCATCATT-3'
GRP78: Glucose-regulated protein 78; CHOP: CCAAT/enhancer-binding protein homologous protein.
Western blot analysis
---------------------
Total protein from Caco-2 cells and colon segments of mice was isolated with RIPA buffer (Beyotime Biotechnology, Shanghai, China). Protein was quantified by BCA assay, separated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). The membranes were blocked with 5% BSA diluted in TBS containing 5% Tween-20 for 2 h at room temperature, and incubated with primary antibodies at 4 °C overnight. Finally, the membranes were treated with ECL reagent and exposed to X-ray film. β-actin was used as an internal control.
Statistical analysis
--------------------
Data are shown as the mean ± standard deviation (SD). All statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software, San Diego, United States) using unpaired Student's *t*-test or one-way analysis of variance followed by Tukey's test for multiple comparisons. *P* \< 0.05 indicated a statistically significant difference.
RESULTS
=======
Establishment of a coculture model in vitro
-------------------------------------------
Cell-free supernatants from the upper chamber, human macrophage-like THP-1 cells, and Caco-2 cells were collected for the evaluation of IL-1β and TNF-α levels by ELISA or qRT-PCR. In the present study, the levels of secreted IL-1β and TNF-α in the upper chamber supernatants were dramatically increased upon LPS stimulation (Figure [1A](#F1){ref-type="fig"} and [B](#F1){ref-type="fig"}; *P* \< 0.001 *vs* coculture). In addition, the mRNA expression levels of inflammatory cytokines in macrophages and Caco-2 cells were significantly increased compared with the cells cocultured without added LPS (Figure [1C](#F1){ref-type="fig"}; *P* \< 0.01 for IL-1β in Caco-2 *vs* coculture, *P* \< 0.001 for IL-1β and TNF-α in macrophages and TNF-α in Caco-2 *vs* coculture). Herein, LPS administration for 24 h significantly increased IL-1β and TNF-α levels in the coculture model, which is in accordance with precious studies\[[@B17]-[@B19]\], suggesting that it is a suitable model to mimic acute colitis *in vitro*.
{#F1}
SIRT1 activation enhances tight junctions in Caco-2 monolayers
--------------------------------------------------------------
To assess the integrity of the intestinal barrier, we detected the expression levels of tight junction (TJ) proteins occludin and ZO-1. After coculturing with SRT1720 (10 µM) or NAM (5 mM), Caco-2 cells were collected, and the protein was extracted for Western blot analysis. As expected, compared with the UC group, SRT1720 administration significantly upregulated the expression of the TJ proteins occludin and ZO-1, while NAM treatment suppressed the expression of occludin and ZO-1 (Figure [2](#F2){ref-type="fig"}). Clearly, our data indicate that drug treatment for 48 h results in the strongest protective and damaging effect on Caco-2 monolayers, respectively. Therefore, we chose 48 h as the time point for drug treatment in subsequent experiments.
{#F2}
SIRT1 inhibits Caco-2 apoptosis
-------------------------------
Annexin V-APC/7-AAD staining assays were applied to assess the apoptosis of Caco-2 cells treated with SRT1720 or NAM for 48 h. As shown in Figure [3A](#F3){ref-type="fig"}, the induction of colitis caused significant damage to the Caco-2 monolayers, with the apoptosis rate (Annexin V-APC+/7-AAD+ quadrant and Annexin V-APC+/7-AAD- quadrant) reaching 10.21%. Though administration of SRT1720 reduced the apoptosis rate of Caco-2 cells to some extent, there were no significant differences between the UC + SRT1720 group and the UC group (Figure [3B](#F3){ref-type="fig"}). However, NAM treatment led to a significant increase in the rate of apoptosis (Figure [3B](#F3){ref-type="fig"}; *P* \< 0.001 *vs* UC).
{#F3}
SIRT1 negatively regulates ER stress-mediated apoptotic pathways in Caco-2 monolayers
-------------------------------------------------------------------------------------
As shown in Figure [4A](#F4){ref-type="fig"}, SRT1720 administration increased the protein level of SIRT1, while NAM treatment downregulated its expression. To explore the molecular mechanisms underlying the protective role of SIRT1, we detected the mRNA levels of the ER stress chaperone GRP78 and the ER stress-induced apoptosis marker CHOP in Caco-2 cells. Exposure to SRT1720 for 48 h resulted in significantly decreased mRNA expression levels of GRP78 (Figure [4B](#F4){ref-type="fig"}; *P* \< 0.001 *vs* UC) and CHOP (Figure [4C](#F4){ref-type="fig"}; *P* \< 0.01 *vs* UC), whereas NAM treatment increased the expression of GRP78 and CHOP (Figure [4B](#F4){ref-type="fig"} and [C](#F4){ref-type="fig"}; *P* \< 0.01 *vs* UC). Western blot was also performed to verify the protein levels of ER stress- and apoptosis-related molecules. Consistent with the mRNA levels, we found that the level of GRP78 was significantly decreased in the SRT1720-treated group compared with the UC group (Figure [4D](#F4){ref-type="fig"}). In addition, the levels of CHOP and cleaved caspase-12, which play important roles in ER stress-induced apoptosis, were also decreased after SRT1720 treatment (Figure [4D](#F4){ref-type="fig"}). Moreover, the expression of downstream molecules, such as caspase-9 and caspase-3, was also suppressed (Figure [4D](#F4){ref-type="fig"}). In contrast, NAM treatment increased the expression of GRP78 and CHOP and upregulated the levels of the activated forms of caspase-12, caspase-9, and caspase-3 (Figure [4D](#F4){ref-type="fig"}).
{#F4}
SIRT1 clinically and histologically ameliorates DSS-induced colitis
-------------------------------------------------------------------
Symptoms of acute colitis, including weight loss, diarrhea, and rectal bleeding, were observed daily after 7 d of DSS exposure. As expected, the administration of DSS successfully induced colitis, as the DAI dramatically increased in the UC group compared with the control group (Figure [5A](#F5){ref-type="fig"}; *P* \< 0.001 *vs* control). The DAI score was higher in the UC + NAM group than in the UC group (Figure [5A](#F5){ref-type="fig"}; *P* \< 0.01 *vs* UC), and SRT1720 treatment markedly reduced the DAI score (Figure [5A](#F5){ref-type="fig"}; *P* \< 0.01 *vs* UC, *P* \< 0.001 *vs* UC). Histologically, integrity loss, goblet cell damage, and inflammatory cell infiltration were observed in the DSS group compared with the control group (Figure [5B](#F5){ref-type="fig"} and [C](#F5){ref-type="fig"}; *P* \< 0.001 *vs* control). Compared with those in the UC group, the above changes were ameliorated in the SRT1720-treated group and aggravated in the NAM-treated group (Figure [5B](#F5){ref-type="fig"}). The HS of the UC + SRT1720 group was higher than that of the UC group (Figure [5C](#F5){ref-type="fig"}; *P* \< 0.001 *vs* UC), while the UC + NAM group did not show significantly aggravated colitis (Figure [5C](#F5){ref-type="fig"}). Taken together, these data show that SIRT1 activation reduces susceptibility to DSS-induced acute colitis both clinically and histologically.
{#F5}
SIRT1 decreases inflammatory cytokine expression in DSS-induced colitis
-----------------------------------------------------------------------
The expression levels of IL-1β and TNF-α in colon tissues were detected by ELISA to assess the inflammatory response. The data indicated that DSS treatment increased the levels of IL-1β and TNF-α significantly (Figure [6A](#F6){ref-type="fig"} and [B](#F6){ref-type="fig"}; *P* \< 0.001 *vs* control). Reduced expression levels of inflammatory cytokines were observed in the UC + SRT1720 group compared with the UC group (Figure [6A](#F6){ref-type="fig"} and [B](#F6){ref-type="fig"}; *P* \< 0.01 for IL-1β and *P* \< 0.001 for TNF-α *vs* UC). In addition, the UC + NAM group showed increased expression levels of IL-1β and TNF-α (Figure [6A](#F6){ref-type="fig"} and [B](#F6){ref-type="fig"}; *P* \< 0.05 *vs* UC).
{#F6}
SIRT1 activation reduces the apoptotic cell rate in DSS-induced colitis
-----------------------------------------------------------------------
To estimate apoptosis in the colon tissue, TUNEL assays were performed, and positively stained cells were counted. The DSS group presented a dramatically larger number of apoptotic cells than the control group (Figure [7A](#F7){ref-type="fig"} and [B](#F7){ref-type="fig"}; *P* \< 0.001 *vs* control). There were fewer apoptotic cells in the UC + SRT1720 group than in the UC group (Figure [7A](#F7){ref-type="fig"} and [B](#F7){ref-type="fig"}; *P* \< 0.01 *vs* UC). Moreover, NAM administration decreased the percentage of apoptotic cells (Figure [7A](#F7){ref-type="fig"} and [B](#F7){ref-type="fig"}; *P* \< 0.001 *vs* UC).
{#F7}
SIRT1 activation suppresses ER stress-mediated apoptotic pathways during DSS-induced colitis
--------------------------------------------------------------------------------------------
Protein levels in colon tissues of treated mice were assessed by Western blot. DSS administration caused a lower protein level of SIRT1. Besides, SRT1720 and NAM treatment upregulated and downregulated SIRT1 expression levels, respectively (Figure [8A](#F8){ref-type="fig"}). As shown in Figure [8B](#F8){ref-type="fig"}, compared with the control group, the DSS-treated group showed significantly elevated protein levels of GRP78, CHOP, and cleaved caspase-12, suggesting that ER stress and the UPR were activated. Consistent with the *in vitro* results, SRT1720 administration downregulated the protein levels of GRP78, CHOP, cleaved caspase-12, cleaved caspase-9, and cleaved caspase-3 (Figure [8B](#F8){ref-type="fig"}). Additionally, NAM administration caused the opposite effect (Figure [8B](#F8){ref-type="fig"}). Altogether, these results show that SIRT1 activation inhibits ER stress-mediated apoptotic pathways in DSS-induced colitis mice.
{#F8}
DISCUSSION
==========
UC is characterized by chronic colonic mucosal inflammation and is a known risk for colorectal cancer. Although novel pharmacological therapies have been emerging recently, the existing treatments for UC are still not satisfactory\[[@B21]\]. Thus, developing effective drugs is essential. As a well-known modulator of lifespan, SIRT1 is involved in various diseases, including cancers, metabolic diseases, and inflammation-associated diseases\[[@B22]-[@B25]\]. However, the role of SIRT1 in the intestinal barrier and intestinal inflammation is still obscure. Here, we applied the SIRT1 activator SRT1720 and inhibitor NAM to investigate the potential effects of SIRT1 on the intestinal barrier in a UC coculture model and in mice with DSS-induced colitis. Our results demonstrate that the pharmacological activation of SIRT1 enhances TJ integrity of the intestinal barrier and reduces apoptosis of IECs by downregulating the expression of CHOP and suppressing the activation of caspase-12, which are key molecules in ER stress-mediated apoptotic pathways.
SIRT1 was found to be downregulated in colonic epithelium and lamina propria mononuclear cells (LPMCs) of IBD patients and elevated after successful infliximab treatment, suggesting that SIRT1 is involved in the development of IBD\[[@B13],[@B14]\]. Furthermore, previous studies have identified the protective role of SIRT1 in intestinal inflammation, the molecular mechanisms of which include intestinal microbiota alteration and nuclear factor kappa B (NF-κB) pathway suppression\[[@B13],[@B16],[@B26]\]. In our study, SIRT1 activation significantly alleviated DSS-induced experimental colitis both clinically and histologically; this alleviation of colitis was accompanied by the downregulation of the levels of the inflammatory cytokines IL-1β and TNF-α in colon tissues, which is consistent with previous studies. Nevertheless, further studies are required to illuminate the underlying mechanisms.
The intestinal epithelial barrier isolates the internal milieu from the external environment and plays a crucial role in intestinal homeostasis. Intestinal inflammation is associated with increased permeability of the intestinal mucosa caused by intestinal barrier damage\[[@B27]\]. Occludin and ZO-1 are important TJ proteins and are essential for the maintenance of intestinal mucosal barrier integrity\[[@B28]\]. Occludin interacts directly with claudins and actin and takes part in the regulation of the intestinal barrier\[[@B29]\]. ZO-1 is a peripheral membrane protein that is essential for TJ assembly and maintenance\[[@B29]\]. A recent study revealed that occludin and ZO-1 expression in UC patients was not only significantly decreased compared with that of healthy controls but also positively related to intestinal mucosal healing\[[@B30]\]. Moreover, previous studies have demonstrated that SIRT1 enhances TJs in other physical barriers\[[@B31],[@B32]\]. Herein, we found that SIRT1 activation significantly increased the expression of occludin and ZO-1 in Caco-2 monolayers, suggesting that SIRT1 may exert protective effects on colitis by promoting intestinal barrier integrity.
IECs, which comprise enterocytes, goblet cells, and Paneth cells, have well-developed ER structures for the biosynthesis of large amounts of proteins. Increasing evidence suggests that ER stress and the UPR in IECs are involved in the pathogenesis of UC\[[@B10],[@B33],[@B34]\]. Unresolved ER stress is a common character of the UC epithelium and results in the activation of the UPR to restore ER homeostasis or the induction of cell apoptosis if ER stress is too severe to be rescued\[[@B35]\]. Additionally, ER stress in IECs is related to intestinal dysbiosis and dysregulated immune response, leading to cell dysfunction, mucosal barrier damage, and intestinal inflammation\[[@B36]\]. In the present study, exposure to DSS increased the expression of the ER stress marker GRP78, which was accompanied by an increased rate of apoptosis in the colon and the activation of apoptosis-related proteases caspase-9 and caspase-3. GRP78 is an ER chaperone that binds to transmembrane ER stress sensors (inositol-requiring enzyme 1α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor 6α (ATF6α)) in non-stressed cells\[[@B37]\]. It is released and contributes to the apoptosis of IECs upon ER stress\[[@B6]\]. Moreover, we found that SIRT1 activation reduced apoptosis in Caco-2 monolayers and colon tissues of DSS-induced colitis mice, indicating that SIRT1 may play a protective role in ER stress-induced injury.
To further confirm the protective effect of SIRT1, we detected the protein levels of CHOP and caspase-12. As a downstream transcriptional factor of PERK/eukaryotic translation initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4), CHOP plays a critical role in ER stress-induced apoptosis through suppression of the antiapoptotic protein Bcl-2 and induction of the proapoptotic molecules Bim, death receptor 5 (DR5), and telomere repeat binding factor 3 (TRB3)\[[@B38]\]. The intestinal epithelium of IBD patients shows higher expression of CHOP compared with normal people\[[@B39]\]. Furthermore, CHOP overexpression increases susceptibility to intestinal inflammation and mucosal tissue injury in mice, whereas knockdown of CHOP alleviates IEC apoptosis\[[@B40],[@B41]\]. Previous studies have revealed that SIRT1 alleviates ER stress-mediated cell apoptosis through the downregulation of the PERK-eIF2α-CHOP axis in the UPR pathway in cardiac cells and chondrocytes\[[@B42],[@B43]\]. Caspase-12 is another marker of ER stress-mediated apoptosis, which is separated from the ER membrane and cleaved into active fragments upon ER stress, resulting in caspase-3 cleavage and apoptosis\[[@B44]\]. Guo et al\[[@B45]\] reported that SIRT1 may alleviate ER stress-mediated apoptosis of cardiomyocytes *via* reduced expression levels of CHOP and cleaved caspase-12. Here, we show that SIRT1 activation decreases the expression of CHOP and suppresses the activation of caspase-12 in Caco-2 cells as well as in DSS-induced colitis mice, while treatment with the SIRT1 inhibitor NAM induced the opposite effect, which indicates that SIRT1 protects IECs from ER stress-induced apoptosis by suppressing CHOP, caspase-12, and their downstream signaling cascades.
In conclusion, we discovered that SIRT1 activation contributes to enhanced intestinal barrier integrity and reduced IEC apoptosis *via* the suppression of ER stress-mediated apoptotic proteins such as CHOP and caspase-12. SIRT1 may serve as a novel drug target, and SIRT1 activation is a promising therapeutic strategy for UC.
ARTICLE HIGHLIGHTS
==================
Research background
-------------------
Ulcerative colitis (UC), the main subtype of inflammatory bowel disease (IBD), is a chronic relapsing inflammatory disorder of the large intestine. The incidence and prevalence of UC have increased in recent years. Sirtuin 1 (SIRT1), a member of the mammalian sirtuin family of proteins, is a nicotinamide adenine dinucleotide (NAD^+^)-dependent protein deacetylase and plays an essential role in caloric restriction, life span modulation, and cell fate determination. Recently, a number of studies have demonstrated that SIRT1 plays a protective role in colitis.
Research motivation
-------------------
Although a large number of therapeutic agents, including 5-ASA drugs, immunosuppressants, steroids, and emerging biological agents, have appeared in the past few years, most patients still experience severe complications or recurrence of the disease, which greatly reduces their quality of life.
Research objectives
-------------------
To investigate the role of SIRT1 in intestinal epithelial cells in UC and further explore the underlying mechanisms.
Research methods
----------------
We developed a coculture model using macrophages and Caco-2 cells. After treatment with the SIRT1 activator SRT1720 or inhibitor nicotinamide (NAM), the expression of occludin and zona occludens 1 (ZO-1) was assessed by Western blot. Annexin V-APC/7-AAD assays were performed to evaluate Caco-2 apoptosis. DSS-induced colitis mice was exposed to SRT1720 or NAM for 7 d. Transferase-mediated dUTP nick-end labeling (TUNEL) assays were conducted to assess apoptosis in colon tissues. The expression levels of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, caspase-9, and caspase-3 in Caco-2 cells and the colon tissues of treated mice were examined by quantitative real-time PCR and Western blot.
Research results
----------------
SRT1720 treatment increased the protein levels of occludin and ZO-1 and inhibited Caco-2 apoptosis, whereas NAM administration caused the opposite effects. DSS-induced colitis mice treated with SRT1720 had a lower disease activity index (*P* \< 0.01), histological score (*P* \< 0.001), inflammatory cytokine levels (*P* \< 0.01), and apoptotic cell rates (*P* \< 0.01), while exposure to NAM caused the opposite effects. Moreover, SIRT1 activation reduced the expression levels of GRP78, CHOP, cleaved caspase-12, cleaved caspase-9, and cleaved caspase-3 in Caco-2 cells and the colon tissues of treated mice.
Research conclusions
--------------------
SIRT1 activation contributes to enhanced intestinal barrier integrity and reduced apoptosis of intestinal epithelial cells *via* the suppression of endoplasmic reticulum (ER) stress-mediated apoptosis-associated molecules CHOP and caspase-12.
Research perspectives
---------------------
SIRT1 may serve as a novel drug target, and SIRT1 activation is a promising therapeutic strategy for UC.
ACKNOWLEDGEMENTS
================
We are grateful to Dr. Wei-Xiang Zhong for excellent assistance in pathology.
Manuscript source: Unsolicited manuscript
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report classification
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Institutional review board statement: This study was reviewed and approved by the Institutional Review Board of the First Affiliated Hospital, College of Medicine, Zhejiang University.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University.
Conflict-of-interest statement: No potential conflicts of interest exist.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Peer-review started: July 16, 2019
First decision: August 2, 2019
Article in press: September 13, 2019
P-Reviewer: Chuang SM, Chen YK, Touil-Boukoffa C, Tanabe S S-Editor: Wang J L-Editor: Wang TQ E-Editor: Zhang YL
[^1]: Author contributions: Zhou XX designed the experiments; Ren MT, Gu ML, Yu MS, and Pan HH performed the experiments and analyzed the data; Ren MT drafted the manuscript; Zhou XX and Gu ML critically revised the manuscript; Ji F and Ding CY offered help during the experiments; all authors have read and approved the final manuscript.
Supported by the National Nature Science Foundation of China, No. 81600414; the Natural Science Foundation of Zhejiang Province, No. LQ16H030001; and Zhejiang TCM Science and Technology Project, No. 2016ZA123 and No. 2018ZA013.
Corresponding author: Xin-Xin Zhou, MD, Attending Doctor, Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, Zhejiang Province, China. <[email protected]>
Telephone: +86-571-87236863 Fax: +86-571-87236611
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Traction for low-back pain with or without sciatica.
Traction is used to treat low-back pain (LBP), often with other treatments. To determine traction's effectiveness, compared to reference treatments, placebo, sham traction or no treatment for LBP. We searched CENTRAL (The Cochrane Library 2006, issue 4), MEDLINE, EMBASE, and CINAHL to October 2006, references in relevant reviews and personal files. Randomized controlled trials (RCTs) involving traction to treat acute (less than four weeks duration), sub-acute (four to 12 weeks) or chronic (more than 12 weeks) non-specific LBP with or without sciatica. Study selection, methodological quality assessment and data extraction were done independently by two authors. As there were insufficient data for statistical pooling, we performed a qualitative analysis. We included 25 RCTs (2206 patients; 1045 receiving traction). Five trials were considered high quality. For patients with mixed symptom patterns (acute, sub-acute and chronic LBP with and without sciatica) there is: strong evidence of no statistically significant difference in outcomes between traction as a single treatment and placebo, sham or no treatment; moderate evidence that traction as a single treatment is no more effective than other treatments; limited evidence of no significant difference in outcomes between a standard physical therapy program with or without continuous traction. For LBP patients with sciatica (with acute, sub-acute or chronic pain), there is conflicting evidence in several comparisons: autotraction compared to placebo, sham or no treatment; other forms of traction compared to other treatments; different forms of traction. In other comparisons, there were no statistically significant differences; the evidence is moderate for continuous or intermittent traction compared to placebo, sham or no treatment, and limited for light versus normal force traction. The results of the available studies involving mixed groups of acute, sub-acute and chronic patients with LBP with and without sciatica were quite consistent, indicating that continuous or intermittent traction as a single treatment for LBP is not likely effective for this group. Traction for patients with sciatica cannot be judged effective at present either, due to inconsistent results and methodological problems in most studies. We conclude that traction as a single treatment for LBP is probably not effective. Any future research on traction for patients with LBP should distinguish between symptom pattern and duration, and should be carried out according to the highest methodological standards.
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Ernie Manouse
Ernie Manouse (born September 1, 1969, in Binghamton, New York) is an American television host, radio personality, writer and producer. He currently hosts the interview show InnerVIEWS with Ernie Manouse, produced by HoustonPBS. His work with HoustonPBS has met critical acclaim in the southern United States, earning him numerous KATIE awards and regional Emmy Awards
Early life
Manouse was born Ernest David Manouse in Binghamton, New York. He is of Greek descent. After graduating from high school, and despite being accepted to Massachusetts Institute of Technology with a perfect math SAT score, he attended Loyola University Chicago and studied to be a music video director. While in college, he guest-hosted Outlook, a Chicago-based radio show, with a classmate. Their radio presentation received such a positive reaction that Outlook hired them permanently.
Career
Manouse began his career in television with NBC Network News, then moved into radio with WLS and WLUW in Chicago, and then moved back to TV at HoustonPBS. Ernie has since worked his way through many aspects of talk shows, from screening calls on the call-in radio shows Sex Talk and The Phyllis Levy Show to hosting his own brand of chat and magazine programs. He can also be seen on PBS Stations across the country hosting numerous pledge and entertainment specials, including three of public television's most successful pledge events with financial guru Suze Orman.
In 1996, Ernie moved to Houston, Texas, and spent six years hosting and producing the daily magazine program WeekNight Edition, which evolved into WeekDAY and became Houston's most celebrated local television program, earning multiple Emmy and Houston Press Club awards. Manouse shared with Matthew Brawley the 2006 Katie Award for "Outstanding Interview/Talk Show" for the southern region.
In October 2002, Manouse helped to create and produce the local primetime magazine show The Connection, which he hosted for two years.
In 2004, Manouse launched the syndicated series InnerVIEWS with Ernie Manouse. This award-winning series is distributed nationally to PBS stations across the country and airs in more than 100 cities in the U.S. and the Virgin Islands. The show features Manouse in unedited, one-on-one interviews with noted personalities such as Patti LuPone and Jamie Foxx. Manouse thoroughly researches his guests before interviewing them and arranges an informal setting to encourage spontaneous discussion.
Another area of broadcasting that Manouse has explored is late night talk, and on February 9, 2005, Manouse launched The After Party, combining arts coverage and light-hearted interviews reminiscent of Johnny Carson and Jack Paar. The show received positive reviews from both critics and audiences alike, garnering the coveted Emmy nomination for "Best Entertainment/Variety program" in its first season. The program ended its run on November 15, 2006, after over 50 episodes.
In 2006, Manouse produced and anchored A Conversation on RACE for HoustonPBS. He also produced the political Red, White & Blue and moderated the 2002 Houston Mayoral Debates, the 2008 Texas Supreme Court Judicial Debate, and the 2008 Texas US Senate Debate. In 2009, Manouse became the anchor and producer of Houston 8, a weekly current events discussion series. He also hosted the 2009 HoustonPBS Spelling Bee, the largest regional qualifying spelling bee for the national Scripps Spelling Bee.
After seven nominations for the Lone Star (Texas) regional chapter of the Emmy Awards, Manouse won three Emmys in 2009. He won for Best Information Series and Best On-Air Talent. He also won an Emmy for his work on the Houston Spelling Bee in the category of Best Event Coverage. He has won an additional two awards over the last few years, bringing his Emmy total to five.
Manouse is also a voice actor. He has done the English voiceovers for over a dozen Japanese anime videos produced by ADV Films, including Gilgamesh, Le Chevalier D'Eon, and Cromartie High School.
Community involvement
Manouse is an active member of the Houston LGBT community. Manouse has hosted community events such as the U.S. Military Ball and the Cattle Baron's Ball and served as a conversationalist at the University of Houston Honors College Great Conversation. He serves on the advisory board of both Stages Repertory Theatre and Dominic Walsh Dance Theater. He served as Vice President of Public Affairs for the Greater Houston United Services Organization (USO). He was also vice president of the Houston Gay and Lesbian Film Festival.
References
External links
USO Houston
InnerVIEWS with Ernie Manouse
Houston Gay and Lesbian Film Festival
Ernie Manouse's website
Category:1969 births
Category:Living people
Category:American people of Greek descent
Category:American television talk show hosts
Category:LGBT producers
Category:Loyola University Chicago alumni
Category:Writers from Binghamton, New York
Category:LGBT broadcasters from the United States
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Nucleic acid is formed from nucleotide bases, e.g., uracil (U) cytidine (C), adenine (A), thymine (T) and guanine (G), formed as a single stranded linear molecule. Such a linear molecule can form a hybrid complex, or double stranded molecule (also called a duplex), with another linear molecule by forming specific base pairs, e.g., between A and T, A and U, or G and C. Such paired molecules are called complementary molecules.
Nucleic acid hybridization is a method in which two single stranded complementary molecules form a double stranded molecule. This method is commonly used to detect the presence of one single stranded molecule in a sample (the target nucleic acid) by use of a labelled probe formed of a complementary single stranded molecule. For example, Muran et al., WO 87/04165, published Jul. 16, 1987, describe a probe having a single stranded region complementary to the nucleic acid to be detected, and a double stranded region having a non-radioactive label. The general uses and design of nucleic acid probes are well known in the art, see e.g., Mifflin 35 Clin. Chem. 1819, 1989, and Matthews and Kricka, 169 Anal. Blochem. 1, 1988.
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When I found out yesterday that Rick Warren was speaking at Obamas inauguration, I was pretty grossed out. Grossed out, and confused. Obama and Warren appear to be polar opposites… but whatever. Its Obamas party, he can invite whoever he wants.
I didnt care.
Until I read how Obama was justifying this decision:
Pastor Rick Warren has a long history of activism on behalf of the disadvantaged and the downtrodden. He’s devoted his life to performing good works for the poor and leads the evangelical movement in addressing the global HIV/AIDS crisis. In fact, the President-elect recently addressed Rick Warren’s Saddleback Civil Forum on Global Health to salute Warren’s leadership in the struggle against HIV/AIDS and pledge his support to the effort in the years ahead.
…
…
…waaaaaaaaat?
Warren started ‘caring’ about HIV in 2005. December 2005. A good 25 years after the HIV epidemic began. For fucks sake, Ive been a full-time HIV researcher longer than Warren has given a crap about HIV.
Starting a website on how evangelicals should deal with the homoplague HIV/AIDS three years ago doesnt mean you have ‘devoted your life’ to shit, nor are you a ‘leader’ of shit.
Warren has ‘devoted his life’ to a radical branch of Christianity. He is a ‘leader’ of a radical branch of Christianity.
The ‘good’ Warren does for HIV is the same ‘good’ all radical theists provide: misinformation and proselytization.How to S.T.O.P AIDS
While condoms are easily accessible in the United States, it is reported that the average African man will see no more than 5 or 6 condoms in his life. The solution to the AIDS pandemic is not to “condomize” the world. Besides giving a false sense of security to the user, they have a limited shelf-life due to heat, poor manufacturing, etc., and they require a man’s cooperation. But the main reason not to depend on condoms as the ultimate solution is because of the possibility of behavior change.
Supplying condoms indiscriminately does not promote the positive behavior changes that only the church has the moral authority to suggest. AIDS is nearly a 100 percent behaviorally-driven illness; we know how it is spread and we know how to prevent it, and while condoms have a place in prevention, we believe a more biblical approach is to encourage sexual purity before marriage and mutual faithfulness in marriage afterwards.
From 2005:
Ministering to those poor African women and babies. Saving the souls of those sinnin’ colored men.
Obama, your religious beliefs are your own. As long as you arent saying Baby Jesus came to you in a vision and told you to invade Iraq, or forcing women to wear burkhas cause Mohameds got a raging boner, I dont care.
Seriously though, I don’t know anything about Warren and I haven’t read the Purpose Driven Life (why would I?). The fact that he even admitted that condoms have a place in the fight against HIV/AIDS is a lot more then you’d get out of most other traditional religious leaders. It’s just not realistic to expect everybody to be a secular humanist all of a sudden. If he is helping to move the evangelical base towards a more pragmatic (and Christian) attitude (in other words making them a little less base), good for him. Isn’t this the sort of thing that Christians are supposed to be doing, helping those in need? Anyway, about Warren as a speaker, to me it seems like a pragmatic move. If Obama needs to get shit that needs to be done, done then he needs to have the support of a large majority of Americans, even ‘Murcans. It’s the whole political capital thing. So if he finds someone who the evangelical right listens to, and who is less distasteful than the other options, then maybe that is the best move. It’s not like he’s giving the guy a job. It’s just a speech.
The other thing is that much of Africa is already very religious (most of sub-Saharan Africa is very Christian). I don’t think that Warren et al will change that situation much. I’m not going to try to say that I know what the best strategy there is. I will say I’m sure that it includes condoms. Nevertheless, a little abstinence never hurt anyone and there is plenty of work that needs to be done that evangelicals could do (and fund) that wouldn’t conflict with their anti-condom nonsense (care for AIDS orphans, funding schools and hospitals, funding treatment etc). Someone else can hand out the condoms. As long as their tactics do not undermine the broader strategy then there is probably more good then harm to come of it.
I do agree though that 2 years as an HIV activist (=oportunistic preacher) does not a world HIV superfighter make. Yeah, Obama’s pilin’ the manure on this. Still, I say let the evangelicals do some good for once.
Basically, I agree that it seems sketch but I don’t think it’s as bad as it looks.
I can think of all kinds of ways HIV/AIDS and gay rights organizations need to smear themselves all over Obama for this. He’s going to owe them big time for this screw-up, and they need to hold him to it.
He’s devoted his life to performing good works for the poor and leads the evangelical movement in addressing the global HIV/AIDS crisis
“For fucks sake” as you say so well, Obama’s statement separated devoting his “life” to doing good works for the poor, NOT for AIDS activism, that was stated in the present tense.
Starting a website on how evangelicals should deal with the homoplague HIV/AIDS three years ago doesnt mean you have ‘devoted your life’ to shit, nor are you a ‘leader’ of shit
Again oh shitter thrower, your aim is off on that language, which I think Obama chose carefully and didn’t say what you imply.
In any event, although your panties are in a bunch over this and for the most part I support bunchen’em up over faith head stupidity, I’d say he wasn’t mis-characterized about how LONG he “cared” about HIV/AIDS by Obama.
No, he couldn’t. There aren’t any that are both committed political christians and not totally homophobic, and none that come remotely close to having both qualities that have any sway in among the more religiously deluded of our compatriots. In fact, it seems remarkable to me that he managed to find a preacher that thinks political christianity should include anything aside from homophobia and general sexual repression and even more remarkable that they are things like social justice and environmentalism. Do we have any reason to believe at this point that the plan is anything other than to have warren get up in front of all his followers on the national stage and speak politely to and about a guy who’s getting ready to repeal DADT, etc instead of flinging shit and molotov cocktails, and then have Obama get up and say something along the lines that we disagree vehemently about a lot of things, but we agree about a few, so we ought sit down like big people and advance those causes.
I came up in Arkansas so I got more than my fair share of evangelical bullshit and I hate these fucking people more than anybody, but I gotta say the reaction on the left seems pretty stupid. The point is that they found a main stream christian politician with any redeeming qualities whatsoever. Either run with that and try to encourage any larval good habits the opposition may have or do away with the invocation altogether.
i dont mind the choice. i see it as just a way to bridge gaps towards the right by way of some prayer, which basically doesnt do anything anyway. calm them down a little- earlier better than later. as i saw in excerpt from a creationist propaganda from the amazing “why do people laugh at creationists” series on youtube, “you have to get behind them before you can stab them in the back,” or another favorite phrase “you can do more damage from the inside.” I’m not exactly sure how obama truly feels about many social issues so i guess we’ll just have to see how he goes about actually addressing gay rights, hiv/aids research, etc… i do hope this isnt an actual omen but more of a carrot for the crazies
I am also from AR originally. But I am also a gay man with HIV, and this really roasts my chestnuts. Calling Warren mainstream betrays the AR mindset. Warren is NOT mainstream. (Though it’s all batshit crazy to me.) He is a fake-smiling Fundamentalist who has admitted that he and James Dobson have similar views, but a different “tone”.
I should probably buy my ticket for Canada now. I can feel my disappointment in the administration already. Sad.
As much as I think this is a completely retarded choice, I just wanted to say some of your reasoning is wrong for hating on his reply.
“He’s devoted his life to performing good works for the poor and leads the evangelical movement in addressing the global HIV/AIDS crisis.”
He compounded that sentence, in a grammatical sense Rick Warren didn’t devote his life to addressing the global HIV/AIDS crisis, he devoted his life to performing good works for the poor. He simply LEADS the evangelical movement, which I suppose is true (not saying much because the evangelical movement isn’t addressing shit in this crisis).
If you can persuade men that routinely using condoms is the thing to do, isn’t that behavior change? in and of itself?
Yes, but it’s not the behavior they want; they want you to remain perfectly pure (i.e. a virgin) until you get married, because after all marriage fixes everything. What’s better than two virgins in bed trying to figure out what goes where and if it goes there, do you need to get permission from her first?
It actually reminded me of some lingerie I saw several years ago. It was a black g-string, but on the back at th top there was a condom holder. I thought it was pretty good in that it mixed sexy with safe-sex; the message was somewhat similar to “if you’re seeing this, you’re gonna need this”. So behaviors can change in that regards to make people safer, but the church has moral objections to it.
And yet, when Rick Warren’s cholesterol-hardened arteries seal off completely and he collapses face-down in his third pie of the night, his flailing arms scattering a plate (atop which sat a T-bone, once a scaffolding for 16 ounces of medium-rare red meat, stripped completely clean) and a half dozen empty martini glasses…
…the heart attack will, of course, just be “God’s will”, and not a “consequence of sinful behavior”.
I prefer to think that we now have a chip on our table when the fundies try to critique PEBO’s policies:
“Oh yeah? Well, if Obama is sooooo wrong, why does your beloved pastor Warren support him? If condoms/gay marriage/CO2 sequestration is good enough for this man of God, what does that make you? An agent of Satan?”
Obama said that Warren “devoted his life to performing good works for the poor and leads the evangelical movement in addressing the global HIV/AIDS crisis.” “And” is a conjunction that joins two discrete verbs–“devoted” and “leads”– and their respectiv objects–“good works for the poor” and “evangelical movement in addressing the global HIV/AIDS crisis.”
Obama did not say that Warren has devoted his life to “giving a crap” about HIV/AIDS.
Pastor Rick Warren has a long history of activism on behalf of the disadvantaged and the downtrodden. He’s devoted his life to performing good works for the poor and leads the evangelical movement in addressing the global HIV/AIDS crisis. In fact, the President-elect recently addressed Rick Warren’s Saddleback Civil Forum on Global Health to salute Warren’s leadership in the struggle against HIV/AIDS and pledge his support to the effort in the years ahead.
Independent of the Obama invocation controversy, it saddens me–no, it *angers* me–that such a large demographic will look towards someone so notoriously backwards as Rick Warren and hail him as a revolutionary in the field of HIV/AIDS prevention and eradication research…
…while not only ignoring, but forcefully blocking the actions of biomedical researchers, healthcare workers, and sociologists who actually have a prayer in hell of making significant progress.
It’s just sick, sick, sick how people put their faith and trust in the ramblings of money-hungry megalomaniacs while slicing the tongues of the proven experts.
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Worker sues group over complaint of flipping homes
A loan officer for a mortgage company accused of financing property flipping with government-backed mortgages has sued a community organization for defamation and loss of income.
Fred Vanover, the loan officer for American Skycorp Inc., a Timonium company, filed the suit last week in Baltimore County Circuit Court against the Association of Community Organizations for Reform Now (ACORN), a citizen group that has been highly critical of the company. Mitch Klein, an ACORN organizer, is also a defendant.
The 14-count suit seeks $280 million in compensatory and punitive damages. It is similar to a suit filed in federal court in May by American Skycorp against ACORN, accusing it of defamation, causing a loss of business and retribution for the company's refusal to pay the group $200,000 to counsel borrowers.
The suits come at a time when American Skycorp faces problems on several fronts.
The U.S. Department of Housing and Urban Development is trying to bar the company's main office from issuing mortgages backed by the Federal Housing Administration because of the high default rate on its FHA loans, particularly those made in 1998, its first year of business. The company is challenging that action in federal court and has won a temporary reprieve.
Sources say the FBI and the Maryland attorney general's office are investigating the company.
Norma Washington, head of ACORN's Baltimore chapter, said the firm would fight the suit. "We've got documents to prove everything we're saying," she said.
In his suit, Vanover alleges that ACORN had accused him wrongly in a publicly circulated "position paper" of falsifying the mortgage application of a borrower and had demanded that American Skycorp fire him.
The 14-count suit also accuses ACORN of unfair and deceptive trade practices in seeking a contribution of $40,000 a year for five years from American Skycorp to finance the organization's loan counseling program. And, in an accusation joined by his wife, Vanover claimed that his "marital relationship has suffered."
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<?xml version="1.0" encoding="UTF-8"?>
<RenoiseScriptingTool doc_version="0">
<ApiVersion>5</ApiVersion>
<AutoUpgraded>false</AutoUpgraded>
<Id>com.renoise.VoiceRunner</Id>
<Version>1.03</Version>
<Author>danoise [[email protected]]</Author>
<Name>VoiceRunner</Name>
<Category>Pattern</Category>
<Description>A tool for advanced sorting of notes in pattern-tracks and phrases</Description>
<Homepage></Homepage>
</RenoiseScriptingTool>
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Q:
Can't run project anymore
My project used to be called "TextFieldDemo", then I renamed it to "ShiftCipher". I cannot launch the project anymore. This is the message I'm getting. Can so please help?
GNU gdb 6.3.50-20050815 (Apple version gdb-1708) (Mon Aug 8 20:32:45
UTC 2011) Copyright 2004 Free Software Foundation, Inc. GDB is free
software, covered by the GNU General Public License, and you are
welcome to change it and/or distribute copies of it under certain
conditions. Type "show copying" to see the conditions. There is
absolutely no warranty for GDB. Type "show warranty" for details.
This GDB was configured as "x86_64-apple-darwin".sharedlibrary
apply-load-rules all Attaching to process 2285. 2011-10-30
19:35:49.914 ShiftCipher[2285:f803] * Terminating app due to
uncaught exception 'NSInternalInconsistencyException', reason: 'Could
not load NIB in bundle: 'NSBundle (loaded)' with name 'TextFieldDemoViewController''
* First throw call stack: (0x14ce052 0x11b9d0a 0x1476a78 0x14769e9 0x585838 0x42ce2c 0x42d3a9 0x42d5cb 0x38da73 0x38e0b8 0x230b 0x3659d6
0x3668a6 0x375743 0x3761f8 0x369aa9 0x1bc4fa9 0x14a21c5 0x1407022
0x140590a 0x1404db4 0x1404ccb 0x3662a7 0x367a9b 0x225d 0x21d5)
terminate called throwing an exceptionCurrent language: auto;
currently objective-c (gdb)
TextFieldDemoViewController is now called ShiftCipherViewController. I don't know why it's looking for it with the old name.
A:
You have a nib that use to be named TextFieldDemoViewController.xib. The file was probably renamed to something like ShiftCipherViewController.xib. You are likely still referencing TextFieldDemoViewController in your initWithNibName.
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Q:
Wordpress paginated comments and comment_reply_link issue
So I have comments via wp_list_comments with pagination. The comments display correctly and pagination works. The issue I'm having is that when you click the comment reply link it navigates back to page 1 of the paginated pages. So on any paginated page I'm unable to reply to a comment on that page.
Obviously the comment_reply_link doesn't care that I'm on page 2, page 3, etc. It just wants to go the original post page to comment.
I'm thinking I should hook into comment_reply_link and tell it to use the current_page url as a base but not sure where to start. Not seeing a hook in the codex.
edit:
Should have added that I'm working with nested comments...many levels deep.
Using standard reply with $args
<?php echo comment_reply_link(array_merge( $args, array('depth' => $depth,
'max_depth' => $args['max_depth']))); ?>
A:
please add this code into your wp_enqueue_scriptshooks
if ( is_singular() && comments_open() && get_option( 'thread_comments' ) ) {
wp_enqueue_script( 'comment-reply' );
}
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The Bromley Boys
The Bromley Boys is a 2018 British, coming-of-age, warm-hearted, humorous film. Based on an eponymous autographical book by author Dave Roberts, the film is set in north Kent, in the suburbs of London, during the late 1960s and early 1970s. The story is about the teenaged David "Dave" Roberts, who becomes a fan of his local football club, Bromley F.C., a club who at the time were, "the worst football team in Britain". The film touches briefly on the euphoria caused by England winning the World Cup in 1966, but mainly recounts the events in the protagonist's life during the 1969/1970 season of the Bromley team.
Plot summary
In the late 1960s, a young British teenager, David (Dave) Roberts (Brenock O'Connor), is living in his parents' house in Sevenoaks. He wishes to follow a major football (soccer) team, but because of his father's strong disapproval, he is forced into secretly following his local club, Bromley FC, who at that time were losing almost every game they played. Nonetheless, David instantly becomes a devoted fan. He attends, and carefully analyses every match, and keeps a scrapbook of every press mention they get, no matter how negative. His favorite player is the team's star, center forward Alan "Stoney" Stonefield (Ross Anderson).
David meets and become close friends with three adult Bromley FC fans (TJ Herbert, Mark Dymond, Ewen MacIntosh) who encourage and support him. Dave also meets, and rapidly falls in love with, Ruby McQueen (Savannah Baker), the pretty and bright teenage daughter of Charlie McQueen (Jamie Foreman), the tough scary Chairman of the football club.
Having sneaked into Charlie McQueen's office, and noticed some notes on player's files, Dave believes that McQueen has received large cash offers from both Manchester United and Leeds United to sell Stoney away from Bromley. It seems to Dave that McQueen is planning to accept, in order to pay off his massive gambling debts, which have rendered the club bankrupt.
Dave happens to accidentally meet Stoney, who turns out to be very kind, and strikes up a friendship with him.
The news of the supposed offer to buy Stoney is leaked to the press. The Chairman see this news on television, and now imagines he will be able to pay off all his gambling debts and come out ahead. He announces the good news about Stoney at a party, and explains he can now afford to send his daughter to university to become a doctor, her dream.
But Dave suddenly understands that he misinterpreted what he read: the notes he saw were not about cash offers from leading football clubs, instead they were offers to Ruby from Manchester University and Leeds University. There will be no money coming in.
In order to save Bromley, Dave browbeats the Chairman into selling his expensive sports car, and betting all of the cash on Bromley FC to win their final game of the season, at odds of 10 to 1 against. Dave also demands that the Chairman allow him to manage the team for this one final game.
Despite Dave's attempts to suggest a new game plan, the first half of the game goes poorly, with Bromley scoring an own goal. But then Dave accidentally finds out that his father was originally a brilliant athlete who played youth soccer for England before being crippled in an accident on the field. Before the Bromley team goes out for the second half, Dave gives an impassioned speech, which Stoney endorses, and which causes the team to play better than anyone would have thought possible. First a tie goal is scored, and then Stoney manages to score a very challenging goal on a free kick, and Bromley FC wins 2-1. Dave is carried off the field in triumph, and joy all round.
References
External links
Category:British films
Category:2018 films
Category:Bromley F.C.
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Introduction
============
Corneal cross-linking (CXL) has been employed for many years as a means of stabilizing cornea ectasia.[@b1-opth-13-581]--[@b6-opth-13-581] It has been introduced and reported for the use of higher fluence UV light for accelerated CXL in keratoconus.[@b7-opth-13-581],[@b8-opth-13-581] Among the multitude of treatments and technique variations applied, CXL has almost invariably documented results in central anterior corneal flattening. This flattening has often been attributed as "disease regression".
It has also been introduced and reported extensively on combining a partial topography-guided normalization surface ablation with CXL as a means to not only stabilize ectasia but also significantly reshape the irregular corneal surface and improve visual function in a technique named "The Athens Protocol".[@b9-opth-13-581]--[@b15-opth-13-581]
Other investigators have also reported on combining a customized partial refraction surface ablation and CXL in the management of keratoconus and ectasia.[@b16-opth-13-581]--[@b19-opth-13-581]
One of the limitations of the Athens Protocol for ectasia has been the removal of stromal tissue in already thinned cornea. Several investigators alongside us have introduced and reported subsequently the predictable refractive effects that customized variable pattern and variable fluence topography customized CXL can have -- refractive CXL.[@b20-opth-13-581]--[@b24-opth-13-581]
A custom application of staged refractive CXL is presented herein, enabling the topographically customizable administration of very high fluence CXL applied in a specific toric pattern, in order to achieve, beside ectasia stabilization, additional specific corneal change to the topography-guided partial surface ablation. This can acchieve more refractive normalization by "flattening" the cone, with less tissue removal.
Methods
=======
This study received approval by the Ethics Committee of our Institution (Laservision.gr Clinical and Research Eye Institute) and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained at the time of the first study visit. All equipment, techniques, and materials had already been approved for clinical use within the EU (CE mark), thus the combination technique was novel and not the technologies used and its applications.
Patient enrolment: 25 eyes of 18 consecutive progressive keratoconus patients were enrolled. The patients who underwent the enhanced Athens Protocol reported cases of 36 months postoperative evaluation of a novel customized toric application of CXL.
A topography-guided partial photorefractive keratectomy (PRK) treatment of maximum 30 µm was applied initially followed by a 7 mm, 50 µm phototherapeutic keratectomy (PTK) treatment to address epithelial removal. 0.02% Mitomycin C was applied for 20 seconds and then the exposed stroma was soaked with 0.1% riboflavin solution for 5 minutes.[@b25-opth-13-581]--[@b27-opth-13-581] The standard Athens Protocol technique has been reported in extent in previous studies.[@b8-opth-13-581]--[@b11-opth-13-581]
The cornea was then treated with a customized pattern as follows: 20 mW/cm^2^ of UV light fluence were delivered uninterrupted in 3 different concentric patterns: the inner-smaller curved trapezoid pattern received a total of 15 J of energy over, the intermediate 10 J and the outer 5 J. The inner curved trapezoid pattern and the other two as well, were centered to the thinnest part of the cornea, as defined by the corneal pachymetry map provided by the anterior segment 9 mm OCT map (Optovue, CA, USA) and illustrated in [Figure 1](#f1-opth-13-581){ref-type="fig"}. The refractive CXL technique has been introduced and reported previously.[@b19-opth-13-581]
Visual acuity, refractive error, cornea clarity, keratometry, topography, and pachymetry with a multitude of modalities, as well as endothelial cell density, were evaluated over 36 months (36--42 months, mean: 38 months).
Results
=======
Keratoconus was stabilized in all cases. The keratoconus stage by Amsler--Krumeich criteria improved from an average of 3.2 (1--4) to 1.8 (0--3). Uncorrected distance visual acuity (UDVA) changed from preoperative 20/80 to 20/25 at 6 months. A maximum astigmatic reduction of 7.8 D (5.3--15.6) and a significant cornea surface normalization (an index of height decentration \[IHD\] improvement from 0.155 \[±0.065\] to 0.045 \[±0.042\]) were achieved by 1 month and remained relatively stable for 36 months postoperatively ([Table 1](#t1-opth-13-581){ref-type="table"}). Two cases delayed full reepithelialization for up to 9 days.
Example case
------------
A 28-year-old male with progressive keratoconus changes and corneal thinning over 1 year with the following parameters: (uncorrected distance visual acuity) UDVA: CF ("counting fingers"), corrected distance visual acuity (CDVA): 20/60 with refraction −2.00/−4.00 @ 105, thinnest cornea by Scheimpflug tomography (413 µm), keratometries 47.10 and 51.70 @ 6, steepest K: 60.90 D. The enhanced Athens Protocol was performed in the following sequence. First, the partial topography-guided PRK of 27 µm planned ablation over the thinnest and steepest apex of the cone: the actual topography-guided PRK treatment was −1.50/−1.50 @ 82, with a 5 mm optical zone and a 2.00 mm transition zone using the Wavelight EX500 excimer laser (Alcon Laboratories, Inc., Fort Worth, TX, USA), followed by the 50 µm depth, 7 mm diameter PTK to account for epithelium removal, 20 seconds soaking with 0.02% Mitomycin C, followed by 3 minutes soaking with 0.1% riboflavin solution administered on the corneal surface every 30 seconds active tracker, and the customized CXL treatment took place, while riboflavin drops were administered every 1-minute for KXL-II device was engaged over the cornea to activate the (Vibex rapid, Avedro, Waltham, MA, USA). Finally, the the duration. The variable pattern of 20 mW/cm^2^ fluence applied is illustrated in [Figure 1](#f1-opth-13-581){ref-type="fig"}. Of significance is the orientation of the central curned trapezoid treatment area that received a total of 15 J of energy; the intermediate-outer to that, 10 J, and the circular outer area of 7 mm received 6 J of total energy and was centered at the pupillary center. The customized CXL treatment with the KXL-II lasted for a total of 12 minutes and 30 seconds.
At the end of the CXL treatment, a bandage contact lens (+0.50 D, BC: 9.2 and 14 mm diameter, Acuvue, Johnson-Johnson) was placed on the cornea along with one drop of Moxifloxacin drops (Vigamox, Alcon Ft. Worth, TX, USA) and one drop of a combination 1% dexamethasone and 0.3% chloramphenicol (Dispersadron-C, Alcon).
The postoperative regimen was continued four times a day for 1 week, and then the dexamethasone/chloramphenicol combination drop for up to 1 month, replaced by loteprednol etabonate drops used twice-a-day for the duration of the second postoperative month.
Follow-up visits were 1--5 days, daily, and after that at 1, 2, 3, 6, 12, 18, 24, 36, and 42 months. The cornea was re-epithelialized by day 4 and the bandage contact was removed.
The cases with delayed epithelialization were managed with autologous serum drops, occasional bandage contact lens use for a few days, and/or abstinence for the corticosteroid drops for 1--2 days until re-epithelialization was complete. The Scheimpflug tomography pre-to postcomparison demonstrates the significant flattening of the cone achieved with the combination enhanced Athens Protocol treatment (12.3 D on the difference map, [Figure 2](#f2-opth-13-581){ref-type="fig"}) at month 6, The topographic correlation of the corneal flattening seen on the right map of [figure 2](#f2-opth-13-581){ref-type="fig"} compared to the actual cone curvature illustrated in the left image of [Figure 2](#f2-opth-13-581){ref-type="fig"}, underline impressively the effective accuracy of the topographic normalization achieved with the combination technique.[@b38-opth-13-581]--[@b40-opth-13-581]
At month 1, the UDVA was 20/50, and CDVA 20/25 with a −2 D soft contact lens, at month 6, the UDVA was 20/40 and with refraction of −1.50--0.50 @ 105° the CDVA was 20/20−. There were no significant changes noted in the follow-up time from month 6 till 42 months, with UDVA at 20/30-- and CDVA 20/20−; keratometry 45.00 and 47.30 @ 18° and steepest K 49.10 D. These impressive changes are also illustrated in the topometric indices comparison (a significant visual function normalization metric in keratoconus as it has been reported[@b37-opth-13-581]),with a dramatic improvement of the IHD (index of height decentration) from 0.241 (preoperative) to 0.046 at 6 months, ISV (index of surface variance) from 142 to 65 and a keratoconus stage from 3--4 on the Amsler-Krumeich scale to 2, respectively, according to the Scheimpflug tomography (Pentacam HR, Oculus, Germany) illustrated in [Figure 3](#f3-opth-13-581){ref-type="fig"}. Finally, as shown in [Figure 4](#f4-opth-13-581){ref-type="fig"}, on anterior segment optical coherence tomography (OCT) section image of the treated cornea at month 3 (Optovue RTVue device, CA, USA), there is a clear demonstration of a very deep "CXL line", supporting evidence for the depth and width of the CXL effect accomplished, as it has been introduced as a clinical hallmark.[@b41-opth-13-581]
Discussion
==========
There are significant refractive changes documented with classic CXL[@b1-opth-13-581]--[@b6-opth-13-581] when applying the Dresden Protocol (3 mW/cm^2^ for 30 minutes), as well as with higher fluence accelerated CXL that was initially introduced and reported both in vivo and ex vivo.[@b7-opth-13-581],[@b8-opth-13-581]
Similar effects were accomplished and reported with alternate CXL techniques such as in-situ (within the corneal stroma) riboflavin administration, placed within a femtosecond laser-created pocket or later within intrastromal corneal ring segment channels.[@b28-opth-13-581]--[@b30-opth-13-581]
Such flattening, appears to constitute a refractive effect resulting from differential crosslinking-induced stiffening effects within the corneal stroma of the same cornea, has been described in the past by a number of clinicians as "disease regression".
The use of higher fluence CXL has been reported for refractive stabilization in high-myopic and hyperopic LASIK[@b31-opth-13-581]--[@b35-opth-13-581] and has also reported significant refractive changes in astigmatic keratotomy, when "flash" CXL was used just on the incision margins.[@b36-opth-13-581]
The development of a device that offers pupillary intra-operative tracking and delivery of a variable pattern, variable customized energy, customized on tomographic data has made refractive CXL more predictable and accurate.[@b20-opth-13-581]--[@b24-opth-13-581]
Topographically regular and predictable central cornea flattening effects have been introduced and reported in vivo, consistently with a correction of myopia of about 2.5 D.[@b20-opth-13-581],[@b21-opth-13-581] The available interim data appear promising with regard to the potential of correcting low myopic refractive errors without tissue removal in an excimer-like fashion or other previously described thermal techniques combined with CXL. Myopic, hyperopic, and astigmatic corrections are other novel applications with this concept and are currently under study with this technology by other investigators as well.
The combination of this concept, refractive CXL in eyes treated with the Athens Protocol, aims to enhance the normalization and refractive effects, while reducing the amount of tissue planned to be removed with the partial topography-guided PRK. In some mild cases, the PTK alone may suffice, or in combination with same session refractive CXL, in significant ectasia normalization and effective visual rehabilitation, another theoretical concept that may necessitate further studies to be validated, especially in cases where contact lens use is not tolerated by the patients, and this represents the majority in the practiced geographic area.
When compared with our previously reported data on applying the standard Athens Protocol in ectasia and keratoconus,[@b9-opth-13-581],[@b10-opth-13-581] the data in this small case series suggest a significantly enhanced refractive effect, with higher levels of corneal flattening despite less tissue removal (30 µm instead of 50 µm) and improved UDVA and CDVA outcomes while offering in what appears sufficient CXL evidence such as the deep CXL demarcation line on the anterior segment OCT imaging. The refractive and normalization advantage of the enhanced Athens Protocol appears to be compelling and studied within for a significant amount of time (\>3 years).
Further studies may further validate the data presented in this preliminary feasibility study.
Conclusion
==========
The paper herein introduces a novel technique based on the combination of refractive customized CXL along with a more conservative (up to 30 µm), partial topography-guided PRK (Athens Protocol) for safe and effective management of progressive keratoconus in young adult patients.
This study was presented in part at the refractive subspecialty day (ISRS Annual Meeting) pre-AAO Annual Meeting in Chicago, Illinois, on October 26, 2018.
**Disclosure**
The author reports no conflicts of interest in this work.
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Pre-and postoperative visual and keratometric data
------------------------------------------------------------- -------------- -------------- --------------- --------------- ---------------- ---------------- ---------------- ---------------- ------------------------------------------------------
(N=eyes) Preop (25) 1 month (25) 3 months (25) 6 months (25) 12 months (25) 24 months (25) 36 months (23) 42 months (23) *P*-value[a](#tfn1-opth-13-581){ref-type="table-fn"}
**Visual acuity data**
UDVA
Average±SD 0.27±0.21 0.51±0.14 0.75±0.10 0.82±0.10 0.86±0.08 0.85±0.10 0.86±0.08 0.85±0.10 0.0008[\*](#tfn3-opth-13-581){ref-type="table-fn"}
Range 0.10--0.60 0.40--0.80 0.60--1.00 0.80--1.00 0.80--1.00 0.80--1.00 0.80--1.00 0.80--1.00
CDVA
Average±SD 0.55±0.32 0.74±0.18 0.82±0.1 0.95±0.10 1.02±0.07 1.02±0.07 1.03±0.08 1.05±0.09 \<0.0001[\*](#tfn3-opth-13-581){ref-type="table-fn"}
Range 0.10--1.00 0.50--1.00 0.80--1.20 0.80--1.00 1.00--1.20 1.00--1.20 1.00--1.20 1.00--1.20
**Anterior keratometry (K) measured by Scheimpflug device**
K1 (flat)[b](#tfn2-opth-13-581){ref-type="table-fn"}
Average±SD 47.67±3.78 45.02±4.82 44.01±3.76 43.46±2.91 42.28±3.37 42.12±2.97 42.21±3.73 42.17±3.88 \<0.0001[\*](#tfn3-opth-13-581){ref-type="table-fn"}
Range 40.30--62.90 40.80--55.10 38.40--54.90 40.81--53.88 39.12--52.10 40.17--51.97 39.98--51.99 39.97--51.87
K2 (steep)[b](#tfn2-opth-13-581){ref-type="table-fn"}
Average±SD 54.18±4.17 51.23±3.65 48.04±3.79 46.10±4.12 45.21±3.38 45.11±4.97 45.07±4.24 45.04±4.31 \<0.0001[\*](#tfn3-opth-13-581){ref-type="table-fn"}
Range 45.70--75.84 44.10--66.30 41.99--61.22 40.18--60.18 39.52--59.44 39.52--60.03 39.54--59.71 39.51--59.67
**Anterior surface topometric indices**
ISV
Average±SD 119.66±47.29 79.50±40.07 73.21±39.21 70.11±37.24 68.97±38.17 67.31±37.30 65.91±39.42 65.47±39.27 0.0001[\*](#tfn3-opth-13-581){ref-type="table-fn"}
Range 18--204 19--187 19--185 19--185 17--185 17--185 16--185 16--185
IHD
Average±SD 0.155±0.065 0.045±0.042 0.041±0.042 0.041±0.042 0.040±0.042 0.040±0.042 0.040±0.042 0.040±0.042 \<0.0001[\*](#tfn3-opth-13-581){ref-type="table-fn"}
Range 0.049--0.262 0.035--0.198 0.025--0.187 0.025--0.187 0.025--0.185 0.025--0.185 0.025--0.185 0.025--0.185
------------------------------------------------------------- -------------- -------------- --------------- --------------- ---------------- ---------------- ---------------- ---------------- ------------------------------------------------------
**Notes:**
*P*-value at 1 year compared to preoperative values;
All units in keratometric diopters (D);
Statistically significant, *P*\<0.05.
**Abbreviations:** CDVA, corrected distance visual acuity; IHD, index of height decentration; ISV, index of surface variance; Preop, preoperative; UDVA, uncorrected distance visual acuity (decimal).
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Chemical Burn in Fruit TreesNot all pests are classified as insects, fungi, bacteria, or virus. Sometimes poor cultural practices will cause fruit trees to suffer from disease. Remember, a disease is anything that interrupts an organism's normal functions. Take this for example. Black...
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"Bravo Lizard." "Iwo doesnt have the strength in the market anymore." "Iwo Andonof is furious." "The Market needs a new boss." "Soon and this will be done." "If we find the Lizard will find and Iwo." "They Want exchange." "What are we doing ?" "We call Djaro." "Kuka !" "Give me the keys." "Lets go, lets go." "Why there is no progress in my legal issues?" "That's all." "Put it back." "The Evidences against Djaro we need them just in case treason." "Get out Eh!" "Hey bitch ... what did u talk about me, eh?" "That You are a traitor Rosene." "Fucking traitor." "Sit down!" "Stay calm." "Say it again!" "You're a traitor Rosen, were with Djaro in the car." "Hey boy ..." "Don't show with the finger eh." "Rosene !" "Stand up, garbage." "Stand up, I will not spank u so eh." "Stand up eh." "Don't Touch me eh." "Come Here eh gangster." "Continue your meal otherwise will be cold." "This Here is from the cameras in the Mall." "What can you tell me about this here?" "What can I tell you;" "Everything." "If you explain what you mean, I can answer you." "I Want to speak the truth." "And to say the straight." "The truth;" "Or that u would like to hear?" "I want to tell you that I work for Djaro." "Perfect." "Great." "This is nonsense." "Nonsense is something that has little to do with reality." "These here are facts." "Facts?" "Fuck to these facts." "How Dare you talk to me so?" "Come here." "Where Is the Lizard now?" "In the jail." "How you see that I do something against the police?" "He killed a child." "Do not forget it." "And Djaro ..." "No one expects that appears, we are not prepared." "Yes And Kukata like Batman flew and picked him." "Hey Martine ..." "For who do you work?" "What do you want from me?" "The Simplest thing." "The truth." "... according to the situation and the tempo of Ali," "The packed will be in their hands in 2-3 weeks." "I hope it will not last any longer." "There is something else ..." 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"Zori, I searched for her, but isnt with her mother, I called in London..." "And in the university did not have seen ..." "What are you laughing?" "You know something?" " Zornitsa is at my home." "When Will you tell me?" "This Is not a concern of yours." "It's big girl." "Alone can decide with whom and where to stay." "I Am the father and I have the right to know at least if she is well." "It's OK." "I take care, do not worry." "See, happened misunderstanding." "I want to see her, will you bring?" "Should I need facilities?" "Because the only way is to bring her by force." "How about handcuffs?" "Martine, you have no idea how serious the matter." "If Zori was so important for you, you would have talked some time with her." "With normal way." "Explanations as to the man what happens in your family." "The least to meet with your girlfriend." "And not now telling me to bring in as if you want to to nobody questioning." "Go away." "Kukata is here." "13." "Card?" 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"You Are right, I agree with you." "All Will think, that is us!" "The most Djaro not count anyone." "You My absolutely right, son." "We must take measures." "Understood, Dad." "This is mine." "I'm sorry I have not told you never something as nickname." "My baby." "Babe." "Your father sends you greetings." "And What did you tell him?" "He is worried." "Parents do it sometimes." "The Parents maybe yes The commissioner is concerned only for that swab." "Bullshit." "You know that it is not so." "Clear." "You are too, the part of my father." "I Am with part noone." "Where is the problem to be in care?" "I Did not say that I do not care." "Just cares more for her." "Zori!" "Martine!" "We want to change theme, huh?" "Ok." "What you watched on YouTube today?" "This Was silly." "Ok." "Tell me how you imagine the things." "We can not continue so we must speak with your father." "To decide what to do." "See Now u talk like him." "Now its only remains to say that I have to grow up." "Yes Needs to grow." "You must begin to assume responsibility for your acts." "Perfect." "You have to write it and to send it." "Where Is eh?" "What?" "Where Is the DNA of Elitsa Vlanteva?" "How To know what will be within." "Are you kidding me?" "I do not know what you are talking about?" "Here Missing data." "Nothing I have not mattered." "Because is not your job to make ends, maybe someone has not put." "Where is?" "Where is eh?" "Where is?" "That's all." "This Is not all retro." "Where is eh?" "Where is eh?" "That's all." "Where Is eh?" "That's all." "Where Is eh?" "Nothing." "All WAS COMPLETE." "Yes, He showed us the papers and the connector in the office." "It's logical." "We must catch them with another way." "They are Waited and prepared." "If Continue the research can reveal the plan with drug dealers." "Well." "Nab drug traffickers." "This does not tell us anything." "We can not do otherwise and leave them." "After beating and bus." "And no matter if the laboratory has instruments will only find seats from the time when the ticket was 10 cents." "It's easy to Andonof to hire the first unemployed moral manageable chemist." "To give the formula and to restart production." "Just change the part." "Research is a slow and methodical process." "Where are we going." "At the airport." "The Patience is the most important thing in the job of the police." "And I will get down to the Andonof's spot." "But so also disclose information we have about the plans of the Andonof will remain free." "You know I believe that is right." "It is hard to say however I need a little time." "Wait." "Do not interrupt." "This is not you like?" "Responsibility." "But this is something new to me." "I hope you understand." "Now I have to leave." "When I am ready I will call." "I love you." "What Was this speech?" "You shouldnt care." "By the Bus what to do?" "Slowly and methodically or ..." "Act As we know." "Finally had Zaref right." "They cook the material just right there." "Lets descend to put the bracelets because I have an appointment.OK ?" "Come on because ..." "Who Is that eh?" " Can be a drug dealer who has been late." "What going on here eh?" "I do not know. - How Do not you know, do not you see it Marto?" "Here." "Down, down, down." "Lets go." "There Will be done another day." "Arise." "Get up I tell you boy eh." "Freeze!" "Freeze!" "Marto." "I dont care For the fucking bus.I Want Spas." "Because only the fucking Spas knows how to make the fucking pill." "Couldnt find another Chemist that ..." "No we can not find any other Chemist." "Any other chemist will made another pill." "We want the pill of Spas." "And what you lain there, maybe you wanna fetch and blanket?" "Disappear eh!" "Stay there." "Since when u work as funeral agent?" "I have always been, but only undercover." "The serial magic pill in the history of mankind." "Isnt some kind of placebo?" "Its work." "What?" "Works." "What works?" "The pill." "Well." "I am satisfied." "Where is the lab?" "Fuck The laboratory." "This is the important." "Yes but I liked and the laboratory." "Djaro 15 years working on your amphetamine." "I will make him a new laboratory." "Stop, stop!" "Is this the famous Chemist?" "Yes." "Faruk said to tell you that you were up here with the bullshit." "And hopes were quite clear." "And All this was done although I warned you." "Expressly." "Who does it after explicit warning not to do?" "Who?" "And besides to do it as shit." "This is not act, it is a farce." "Prank." "Look something crafty." "What did you do, huh?" "Smelled the dust ... of a bus, which in the middle of the chase suddenly stops." "And you" "Allow drug dealers to do whatever they want." "And steal hearse." "The Situation required ..." "We could stop them." "I could Rumble Gatsof for ... - There were innocent people." "I Could shoot him." "And What you have endanger innocent people?" "So not even ..." "I have no words." "Boss ..." "Get away." " Please disappear." "Was there Innocent man, huh?" "How Is the new job?" "Its ok." "There are some warped colleagues, but you know how it is, in every office is so." "I Want u to become the employee of the month." "See pal, shitty situation became with that chemist.With Spas." "But I promise you ... will compensate and for him and for the laboratory." "There is a time for everything, Rosene." "This is only the beginning."
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Q:
Cleanly shutting down a warp server without exiting the process
I am looking at using wai/warp for some custom interprocess communications. This would have the unusual requirement that I would need to start/stop warp instances dynamically within a process. The main issue seems to be cleanly stopping warp. This question is addressed here:
How do I implement a shutdown command in a WAI server?
but the suggested solution end up exiting the process by returning from main. Can I just throw a ThreadKill exception to the warp thread?
This thread:
https://groups.google.com/forum/#!topic/yesodweb/VoenrabRUBQ
suggests that an IORef could be passed on startup, which could trigger a shutdown externally. This sounds ideal, but isn't part of the current API.
A:
You can run Warp in a separate thread via forkIO and then kill that thread only.
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The role of an amino acid triad at the entrance of the heme pocket in human serum albumin for O(2) and CO binding to iron protoporphyrin IX.
Complexation of iron(II) protoporphyrin IX (Fe(2+)PP) into a genetically engineered heme pocket on recombinant human serum albumin (rHSA) creates an artificial hemoprotein which can bind O(2) reversibly at room temperature. Here we highlight a crucial role of a basic amino acid triad the entrance of the heme pocket in rHSA (Arg-114, His-146, Lys-190) for O(2) and CO binding to the prosthetic Fe(2+)PP group. Replacing His-146 and/or Lys-190 with Arg resolved the structured heterogeneity of the possible two complexing modes of the porphyrin and afforded a single O(2) and CO binding affinity. Resonance Raman spectra show only one geometry of the axial His coordination to the central ferrous ion of the Fe(2+)PP.
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DIAMETER INCHES
A patented winding of bronze wire is tightly wrapped around the lock twist of the ball end. String slippage and breakage are minimized at the ball end where these most often occur. RPS
Ò strings last longer and stay in tune better than conventional plain strings. Used for electric and acoustic guitars (except classics).
Electric Strings
Wound strings sound the lower pitch notes. They consist of wrap wire lightly coiled around a core wire. Nickel plated steel, the most popular, produces a well balanced all around good sound. Stainless steel provides a brighter sound. Pure nickel, which some believe gives a richer, fuller sound.
Plain strings sound the higher pitch notes. They are made of a specially tempered high carbon steel wire. They are used as first, second, and often third strings.
Core Wire
Core wire is the center of a wound string. It is of specially tempered high carbon steel. The crosscut end view reveals a shape that is not round, but hex shaped. The points of the hex shape help hold the wrap wire securely in place. Hex core wire is used for the wound strings of both electric and acoustic guitars, banjos, and mandolins - but not classic guitars.
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stila - Smudge Stick Waterproof Eye Liner Deep Burgundy
Now:
$22.00
(You save)
Qty
+DESCRIPTION
Deep Burgundy (Matte Black Plum)
Smudge that won’t budge!
Our bestselling Smudge Sticks are available in an array of matte and shimmer formulas that stay—not stray—for reliable color payoff that’s second to none. With a range of gorgeous shades to choose from, they feature a cocktail of moisturizing ingredients that ensures color glides on smooth (no tugging or pulling) and the waterproof formula won’t budge or fade.
What’s more, our Smudge Sticks can be used as a classic pencil liner or a smudgy eye shadow - simply smudge with your fingers or a brush. The color sets after you’ve had time to get your desired effect, and then stays put all day.
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Q:
AngularJS composite view with the same directive
I have a question, about composite view in AngularJS, that is close to that one : AngularJS Composite Components
However, I would like to know if it is possible to have a directive that includes a list of the same directive, like this :
<mydirective name="thecontainer">
<mydirective name="a"/>
<mydirective name="b"/>
<mydirective name="c"/>
</mydirective>
Thanks,
David.
Edit
Answering blesh, I will be more precise. I'm trying to display boxes (a simple square) that can have one or many boxes, themselves can have many boxes, etc.
Here is the directive
myApp.directive('box', function () {
return {
restrict:'E',
replace:true,
templateUrl:"partials/box.html",
scope: {
name : '@'
},
link:function (scope, element, attrs, ctrl) {
console.log("trying to log " + attrs.name);
}
}
});
Here is the template :
<div class="box">
<div class="box-header">
<div>{{ name }}</div>
</div>
<div class="box-container">
<!-- display other boxes here-->
</div>
</div>
Here is the interesting code in the view :
<box name="TOP" >
<box name="SUB" >
</box>
<box name="SUB" >
</box>
</box>
Obviously something is missing to tell the sub-boxes "hey please display in the right place into your parent and please, parent, adapt your size to the number of children you have"
David.
A:
The answer was really simple, without ng-transclude, angular has no way to interpret correctly the content of the innner HTML tags. So the correct way to do it is to correct the template by adding a ng-transclude like this:
<div class="box">
<div class="box-header">
<div>{{ name }}</div>
</div>
<div class="box-container" ng-transclude>
<!-- display other boxes here-->
</div>
</div>
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The present invention relates to clock signal generators suitable for use in an integrated circuit memory device. More particularly, the present invention relates to an internal clock signal generator for synchronizing an internal clock with an external clock signal.
Integrated circuit devices such as an integrated circuit memory or central processing units, which operate in synchronization with an external clock signal, typically generate an internal clock signal using a clock buffer and a clock driver. As a result, the internal clock signal may be delayed compared to the external clock signal. Such a delay may cause deterioration in the performance of the device during high frequency operation. In particular, at high frequencies of operation, the access time tac (i.e., the time required for outputting data after an external clock signal is input) may become longer than the time required for generating an internal clock signal from the received external clock signal. Deterioration of the performance of a semiconductor device at higher frequencies may be reduced by synchronizing the internal clock signal with the external clock signal. Conventionally, this synchronization may be accomplished with a delay locked loop (DLL) or a phase locked loop (PLL) which are used as the internal clock signal generator.
FIG. 1 is a schematic diagram of a conventional DLL. As seen in FIG. 1, the DLL includes a phase detector 1, a low pass filter (LFF) 2, and a voltage control delay line 3. The phase detector 1 compares the phases of an external clock signal Ext.CLK and an internal clock signal Int.CLK and detects the difference between the phases. The LPF 2 is connected to the output of the phase detector 1 and generates a control voltage vcont for controlling the delay time of the voltage control delay line 3. The voltage control delay line 3 typically includes a plurality of inverters which are serially connected and outputs the internal clock signal Int.CLK by delaying the external clock signal by a predetermined time specified by the voltage control input. Unfortunately, however, synchronizing the internal clock signal with the external clock signal may take hundreds of cycles of the external clock signal. Furthermore, the DLL circuit may require several tens of milliamps of current during operation. Therefore, it may be difficult to utilize the DLL in an integrated circuit device.
FIG. 2 is a schematic diagram of a conventional PLL. As seen in FIG. 2, the PLL includes a phase-frequency detector 11, a LPF 12, and a voltage control delay line 13. The phase-frequency detector 11 compares the phases and frequencies of the external clock signal Ext.CLK and the internal clock signal Int.CLK, and detects the differences in phases and frequencies. The LPF 12 is connected to the output of the phase detector 11 and generates a control voltage vcont for controlling the delay time of the voltage control delay line 13. The voltage control delay line 13 outputs the internal clock signal Int.CLK in response to the control voltage vcont and the internal clock signal Int.CLK which is fed back to the input of the voltage control delay line 13. Thus, the voltage control delay line 13 acts as a ring oscillator.
The PLL of FIG. 2, however, may have the same problems as the DLL of FIG. 1. Recently, the PLL and the DLL have been coupled together to utilize the quick locking time of the PLL and the wide locking range of the DLL. However, this combination may not solve all of the problems with the DLL and the PLL.
As a result of the shortcomings of the PLL and the DLL, a synchronized delay circuit has been suggested which uses simple delay means to match the phase of the internal clock signal with the phase of the external clock signal. This phase matching may be accomplished by making the delay time of the internal clock signal an integer multiple of the period of the external clock signal. In such a system, a synchronous delay line (SDL), a synchronous mirror delay (SMD), and a hierarchical phase lock delay (HPLD) may be utilized as the synchronized delay circuit.
FIG. 3 is a schematic diagram of a conventional synchronous delay circuit. As seen in FIG. 3, the synchronous delay line includes a clock buffer 21, a dummy clock delay 22, a first clock delay portion 23, a comparing portion 24, a second clock delay portion 25, and a clock driver 26. In the circuit of FIG. 3, the clock buffer 21 receives an external clock signal Ext.CLK and outputs a first clock signal CLK1 in which the external clock signal is delayed by a first delay time d1. As is further seen in FIG. 3, tCK represents the period of the external clock signal. The dummy clock delay 22 controls the phase difference between the external clock signal and internal clock signal Int.CLK such that the phase difference is an integer multiple of the period tCK. The dummy clock delay 22 delays the first clock signal CLK1 by the sum of the first delay time d1 and a second delay time d2 to provide a second clock signal CLK2.
The first clock delay portion 23 includes first unit delays 27 which are serially connected and which output a third clock signal CLK3. The output CLK3 is a delayed version of the second clock signal CLK2 and may be delayed by different times. The comparing portion 24 includes a plurality of comparators 28 which compare the first clock signal CLK1 with the third clock signal CLK3 delayed by the period tCK from the first clock signal CLK1. Thus, a fourth clock signal is delayed by the difference between the period tCK and the sum of the first and second delay times d1 and d2 compared with the second clock signal CLK2.
The second clock delay portion 25 includes second unit delays 29 which are connected in series and output a fifth clock signal CLK5 by delaying the first clock signal CLK1 by the difference between the period tCK and the sum of the first and second delay times d1 and d2. The clock driver 26 outputs the internal clock signal Int.CLK delayed by the second delay time d2 by receiving the fifth clock signal CLK5. FIG. 4 is a timing diagram illustrating the operational state of the synchronous delay line of FIG. 3. As seen in FIG. 4, the first clock signal CLK1 is delayed by a first time d1 when compared with the external clock signal Ext.CLK. The second clock signal CLK2 is delayed by the sum of the first delay time d1 and a second delay time d2 compared with the first clock signal CLK1. The third clock signal CLK3 is the second clock signal CLK2 delayed by an integer multiple of the delay time of the first unit delay 27. The fourth clock signal CLK4 is one of the third clock signal CLK3 which was delayed by an integer multiple of the period tCK of the external clock Ext.CLK. In the embodiment illustrated in FIG. 3, the fourth clock signal CLK4 corresponds to the clock signal CLK1 delayed by one period of the external clock signal.
The fifth clock signal CLK5 corresponds to the fourth clock signal CLK4 delayed by the difference tCK minus (d1+d2) between the period tCK of the external clock signal Ext.CLK and the sum of the first delay time d1 and the second delay time d2. The Internal clock signal Int.CLK then corresponds to the fifth clock signal CLK5 delayed by the second delay time d2. Thus, the internal clock signal Int.CLK is delayed from the external clock signal Ext.CLK by twice the period tCK of the external clock signal. Thus, the internal clock signal Int.CLK is synchronized with the external clock signal Ext.CLK.
The conventional SDL circuit described above is an open loop circuit unlike the PLL and DLL circuits which are closed loop circuits. Thus, the locking time of the SDL circuit is an integer multiple of the period tCK of the external clock signal. Accordingly, the locking time of the SDL circuit may be shorter than the locking time of the PLL and DLL circuits. However, the degree of locking accuracy of the SDL circuit may be lower than the PLL or DLL circuits because the SDL circuits may have a narrow margin of its locking range.
In view of the above discussion, it is an object of the present invention to provide for the synchronization of an internal clock to an external clock with less delay in locking to the external clock signal than may be experienced in using a phase locked loop or a delay locked loop.
Another object of the present invention is to provide an internal clock which can be more closely synchronized to an external clock than is provided in a synchronized delay circuit.
These and other objects of the present invention are provided by an internal clock signal generator which utilizes a synchronized delay circuit to provide a coarsely synchronized clock signal which is then more finely synchronized to the external clock by either a delay locked loop or a phase locked loop. By first synchronizing the signal to the external clock with a synchronized delay circuit the coarse clock signal may be rapidly generated. By utilizing the coarse clock signal with either the delay locked loop or the phase locked loop, these loops may more quickly lock on the external clock signal to provide the finely synchronized signal. Thus, the advantages of both the synchronized delay line and the delay or phase locked loops may be obtained through use of the present invention.
In a particular embodiment of the present invention, an internal clock signal generator is provided which includes a synchronized delay circuit which receives an external clock signal and outputs a clock signal which is coarsely synchronized with the external clock signal. A delay locked loop (DLL) receives the coarsely synchronized clock signal and generates an internal clock signal which is more finely synchronized with the external clock signal.
In an alternative embodiment, a phase locked loop (DLL) receives a coarsely synchronized clock signal and generates an internal clock signal which is more finely synchronized with the external clock signal.
In particular embodiments of the present invention, the synchronized delay circuit is one of synchronous a delay line (SDL), a synchronous mirror delay (SMD) and a hierarchical phase locking delay (HPLD). Accordingly, the synchronized delay circuit may include a plurality of serially connected unit delays which provide a corresponding plurality of delayed clock signals. A controller selects one of the plurality of clock signals which is coarsely synchronized with the external clock signal and generates a flag signal for enabling the DLL or the PLL. The DLL or PLL is enabled by the flag signal and generates the internal clock signal which corresponds to the external clock signal delayed by an integer multiple of the period of the external clock signal.
The synchronized delay circuit may also include a clock buffer which receives the external clock signal and outputs a delayed first clock signal corresponding to the external clock signal delayed by a first delay time. A first dummy clock delay receives the delayed first clock signal and outputs a second delayed clock signal corresponding to the first delayed clock signal delayed by a second delay time. A first variable clock delay circuit receives the second delayed clock and outputs a plurality of third delayed clock signals corresponding to the second delayed clock delayed by integer multiples of a unit delay time. A plurality of comparators receive the third delayed clock signals and the first delayed clock signal and compare the phases of each third clock signal and the first clock signal to generate first control signals and the flag signal.
In a particular embodiment, the DLL includes a second variable clock delay circuit which receives the first delayed clock and which generates a plurality of fourth delayed clock signals corresponding to the first delayed clock delayed by integer multiples of a second unit delay time. The second variable clock delay further includes switching means responsive to the first control signals for selectively outputting one of the plurality of fourth delayed clock signals where the duration of the second unit delay time is controlled by a second control signal. A clock driver receives the selected fourth clock signals and outputs the fourth clock signal delayed by a predetermined time so as to provide an internal clock signal. A second dummy clock delay receives the internal clock signal and outputs the fourth clock signal delayed by a fourth delay time so as to provide a fifth delayed clock signal. A phase detector receives the fifth delayed clock signal and the first delayed clock signal and detects the phase difference therebetween. A low pass filter (LPF) connected to the output of the phase detector outputs the second control voltage so as to control the second unit delay time.
In further embodiments of the present invention, the second delay time is the sum of the delay time of the clock buffer and the delay time of the clock driver. The second unit delay time may also vary between the unit delay time and twice the unit delay time based on the second control voltage. The fourth delay time may also be equal to the delay time of the clock buffer. The second variable clock delay circuit may also be a voltage-controlled delay line in which the second unit delays are controlled by the second control voltage and the flag signal. The second variable clock delay circuit may also output one of the plurality of third clock signals whose phase coincides with that of the first clock signal.
In yet another embodiment of the present invention, each comparator includes a first latch which latches one of the plurality of third clock signals when the first clock signal is in a one logic state. A second latch latches the output of the first latch when the first clock signal transitions to a zero logic state. The output of the second latch then generates a first control signal which controls the switching means corresponding to the comparator. The flag signal may also be generated by a combination of first control signals generated by the comparators. A plurality of comparators receive the third delayed clock signals and the first delayed clock signal and compare the phases of each third clock signal and the first clock signal to generate first control signals and the flag signal.
In a particular embodiment of the present invention, the PLL includes a second variable clock delay circuit which receives the first delayed clock and which generates a plurality of fourth delayed clock signals corresponding to the first delayed clock delayed by integer multiples of a second unit delay time. The second variable clock delay further includes switching means responsive to the first control signals for selectively outputting one of the plurality of fourth delayed clock signals and wherein the duration of the second unit delay time is controlled by a second control signal. A clock driver receives the selected fourth clock signals and outputs the fourth clock signal delayed by a predetermined time so as to provide an internal clock signal. A second dummy clock delay receives the internal clock signal and outputs the fourth clock signal delayed by a fourth delay time so as to provide a fifth delayed clock signal. An inverter for inverting the fifth delayed clock signal output from the second dummy clock delay provides an inverted fifth delayed clock signal. A phase detector receives the inverted fifth delayed clock signal and the first delayed clock signal and detects the phase difference therebetween. A low pass filter (LPF) connected to the output of the phase detector outputs the second control voltage so as to control the second unit delay time.
In another embodiment, the second variable clock delay circuit is controlled by the second control voltage and forms an oscillator together with the clock buffer, the second dummy clock delay and the inverter.
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PHOTOSHOP - Bug within "Slice Options" edit window on MacOS 10.10
Found a bug in CC version 2014.2.2, running on Mac OS X Yosemite version 10.10.2
When editing in the "Slice Options" window, hitting control + Z crashes the window and erased the data you entered, giving the error: "undo: NSCellUndoManager 0x60800048e20 is in invalid state, undo was called with too many nested undo groups". I have recreated this problem every time, without fail.
Additionally, there also seems to be an issue in the "Slice Options" window where when you paste text data into the fields you have copied from somewhere else (e.g. TextEdit), the pasted text does not appear until you click into another data field.
Not major problems, but majorly annoying when you work with a lot of slices and are used to hitting control + Z when you make a mistake. :)
I was able to reproduce this in MacOS 10.9.5 as well.
It is something specific to that dialog.
The good news is that I cannot reproduce the problem in our current builds - so this should be fixed in the next release.
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Angara-5 takes to the sky On Dec. 23, 2014, at 08:57 Moscow Time, Russia's new-generation Angara-A5 launch vehicle blasted off from a snow-buried launch pad at Site 35 in Plesetsk in the north of Russia on its maiden voyage. Previous chapter: Flight scenario for the first Angara-5 launch Above: The first Angara-A5 rocket lifts off on Dec. 23, 2014. From the publisher: Please help to keep this site open and current! The pace of our development depends primarily on the level of support from our readers. Final preparations for launch On December 15, 2014, the fully assembled Angara-A5 was rolled out from the assembly building to a fueling facility, known as KZ BND, for loading low-pressure propellant tanks of the Briz-M upper stage with toxic fuel and oxidizer. The hazardous facility was located near the perimeter of Angara's launch complex in Plesetsk, just 700 meters from the rocket's launch pad. Based on similar operations with Proton rockets in Baikonur, the fueling at the KZ BND site was expected to take up to two days. On December 16, the official TASS news agency quoted Designer General of the Angara project Vladimir Nesterov as saying that the State Commission overseeing the launch would convene on the evening of December 17 to make a decision to proceed with the rollout of the rocket to the launch pad. Around the same time, the launch schedule was apparently moved back by 24 hours. On December 18, TASS reported that the liftoff would take place on Dec. 23, 2014, at 08:57 Moscow Time. The State Commission made a decision to proceed with the fueling of the Angara's Briz-M upper stage, which was underway at the KZ BND site during December 18. The short transfer of the rocket from the fueling station to the launch pad took place on December 20. On December 22, after a series of electrical checks on the pad, the State Commission overseeing the launch cleared the rocket for fueling and launch. Pre-launch countdown Preparations for the launch of the first Angara-A5 rocket were conducted largely in secret and no live coverage of the historic launch was provided, however postings on the online forum of the Novosti Kosmonavtiki magazine based on the information from the launch of the Angara-1.2PP rocket in July 2014, allowed to assume following details from the final countdown preceding a scheduled liftoff on Dec. 23, 2014, at 08:57:25 Moscow Time (12:57 a.m. EST): December 22 23:57 Moscow Time (T-9 hours): Preparations of the launch complex' hardware for the fueling of the launch vehicle and the retraction of service access bridges to the vehicle. The Pre-launch Preparation Program, PSP, is activated. December 23 00:02 Moscow Time (T-8 hours 55 minutes): Beginning of preparation for the propellant loading into the launch vehicle's main tanks. Around 05:00 (T-4 hours): Beginning of the cooling of the liquid oxygen fueling system for loading cryogenic oxidizer of the launch vehicle's third stage. Beginning of the kerosene fuel loading into the main tanks and the engine's bottles; filling of pneumatic systems with nitrogen and helium. Around 06:00 Moscow Time (T-3 hours): Beginning of the cooling of the launch vehicle's oxidizer tanks, filling of the helium bottles inside the tanks. The completion of fuel loading onboard the rocket. Around 07:00 Moscow Time (T-2 hours): The completion of oxidizer loading onboard the rocket. Around 08:00 Moscow Time (T-1 hour): Refilling of the oxidizer tank on the third stage. 08:42: Pre-launch operations with the flight control and propulsion system of the launch vehicle. 08:54: Drainage of the oxidizer from launch vehicle's supply lines. Pre-launch pressurization of the first and second stages. 08:54:40: Undocking and retraction of oxidizer umbilicals. 08:57:25 LIFTOFF! Above: Angara-5 during fueling on the eve of its launch on Dec. 23, 2014. Launch: 2014 December 23 The liftoff of the Angara-A5 rocket took place as scheduled on Dec. 23, 2014, at 08:57:00 Moscow Time from Site 35 in Plesetsk. Shortly after the launch, the Russian space agency, Roskosmos, announced that the payload section of the rocket had reached an initial parking orbit 12 minutes after leaving the pad (09:09 Moscow Time). The launch vehicle had an official mass of 763,621 kilograms at liftoff, as opposed to 773 tons in the official specifications of the Angara-A5 rocket. No TV broadcasts or any other live coverage of the mission had been provided. According to visualizations of the mission emerged in official post-launch TV reports, the first Angara-A5 rocket flew a following profile during its ascent to an initial parking orbit: Milestone Elapsed time Altitude Range Velocity Liftoff T+0 sec 0 0 0 Stage I engine cutoff T+211.320 sec 90.435 km 181.226 km 3,048.820 m/s Stage I separation T+213.700 sec ? ? 3,054.100 m/s Stage II engine cutoff T+327.909 sec 161.695 km 617.399 km 4,807.454 m/s Stage II separation T+330.924 sec 161.695 km 617.399 km 4,807.454 m/s Stage III ignition T+332.924 sec 164.807 km 640.714 km 4,823.323 m/s Payload fairing separation T+345.075 sec 170.730 km 687.596 km 4,856.782 m/s Stage III separation T+738.412 sec 215.867 km 2,936.759 km 7,144,316 m/s The complete launch timeline, which surfaced on the online forum of the Novosti Kosmonavtiki magazine in January 2015 looked as following: Milestone Elapsed time Liftoff 00:00:00 Stage I (URM-1) separation 00:03:29 Stage II (URM-1) separation 00:05:26 Payload fairing separation 00:05:41 Stage III (URM-2) final thrust mode, KST 00:12:11 Stage III (URM-2) engine cutoff 00:12:13 Stage III (URM-2) separation 00:12:15 Briz-M engine firing 1 begins 00:13:50 Briz-M engine firing 1 ends 00:22:38 Briz-M engine firing 2 begins 01:06:04 Briz-M engine firing 2 ends 01:21:04 Briz-M engine firing 3 begins 03:28:48 Briz-M engine firing 3 ends 03:44:11 Briz-M external tank separation 03:45:32 Briz-M engine firing 4 begins 08:47:13 Briz-M engine firing 4 ends 08:59:27 Simulated payload release (GMM dummy cargo remains attached) 09:00:37 Briz-M low-thrust burial orbit insertion maneuver 1 begins 11:03:50 Briz-M low-thrust burial orbit insertion maneuver 1 ends 11:04:05 Briz-M low-thrust burial orbit insertion maneuver 2 begins 12:15:00 Briz-M low-thrust burial orbit insertion maneuver 2 ends 12:16:40 According to industry sources, at the moment of separation from the third stage, the payload section had a mass of 25,766 kilograms, including a 2,042-kilogram dummy satellite, known as GVM or GMM, a Russian abbreviation for Gabaritno-Massovy Maket or Size and Mass Mockup. Other sources identified the payload as NVP PM for Naturny Vesovoy Maket Poleznoy Nagruzki or Full-scale Mass Mockup of Payload. The first engine firing initiated by the Briz-M upper stage at 09:11 Moscow Time inserted the stack into a 215-kilometer circular orbit and additional three maneuvers where expected to carry the dummy cargo into a geostationary orbit 36,000 kilometers above the Earth surface during a nine-hour mission. The second firing was initiated at 10:03 Moscow Time, the third at 12:26 Moscow Time and the fourth at 17:44 Moscow Time. In the hours following the launch, the official Russian media confirmed that the two-ton dummy cargo would remain attached to the Briz-M stage during the entire mission. Russian tracking stations monitoring the launch confirmed a nominal insertion of the stack into the transfer orbit toward the geostationary altitude and the on-time separation of the donut-shaped external tank of the Briz-M stage. Western radar showed an object associated with the launch in a 442- by 35,801-kilometer transfer orbit with an inclination 60.60 degrees toward the Equator. Around ten hours after the liftoff, Roskosmos confirmed that the Briz-M upper stage had successfully delivered its cargo to the geostationary orbit 35.8 thousand kilometers above the Earth's surface. According to the Ministry of Defense, the Briz-M and a GMM dummy cargo reached its targeted geostationary orbit at 17:58 Moscow Time. According to the official announcement of the Russian Ministry of Defense, the Defense Minister Sergei Shoigu reported to President Putin about the successful completion of the Russian military space launch program in 2014, including the first phase of testing of the Angara rocket. Post-launch revelations The target orbit in the first Angara mission was expected to have an altitude of 35,793 kilometers and an inclination of zero degrees, matching the plane of the Equator. However, as it transpired in the first week of 2015, no true geostationary orbit was actually achieved during the launch. According to industry sources, instead, Briz-M entered a 35,625 by 36,946-kilometer orbit with an inclination 0.49 degrees toward the Equator and an orbital period of 1,461.6 minutes or 24.36 hours. Still, in an interview with the official Interfax-AVN news agency in January 2015, Designer General of the project Vladimir Nesterov claimed that the payload spent an hour and a half in the geostationary orbit. Briz-M then successfully completed two maneuvers designed to take it away from a busy traffic of the geostationary orbit, carrying the dummy satellite with it. According to preliminary information gathered by Russian tracking services, Briz-M entered a 35,841- by 38,680-kilometer orbit with an inclination of 0.3 degrees toward the Equator. In this orbit, the object had a rotation period of 1,528 minutes, or 25.5 hours, instead of 24 hours required to complete a single orbit in the geostationary orbit. As of December 24, tracking data indicated that the Briz-M had an orbital period of 1,512 minutes and drifted westward. It reappeared over the Eastern hemisphere by the end of the first week of January 2015. At the time, the stage was yet to complete dumping of onboard pressurized gas and propellant in order to avoid their overheating and potential explosion of the stage. The ejection of pressurized gas could cause further changes in the object's orbital parameters. In the first half of January, NORAD finally released its radar data on the Angara's cargo. The ground radar tracked the object, as it was beginning its second full circle caused by its drifting relative to the Earth's rotation at 67 degrees West longitude. According to NORAD, the vehicle was in a 36,158 by 39,086-kilometer orbit. Its orbital period was 1,529.1 minutes or around 25.5 hours. Credit: Russian Ministry of Defense Above: The moment of ignition as seen by a camera in Plesetsk and on screen of the National Center of Defense Control on Dec. 23, 2014, at 08:56:57 Moscow Time. Credit: Russian Ministry of Defense Above: From a brand-new National Center of Defense Control, Russian officials including Minister of Defense Sergei Shoigu monitored the first launch of the Angara rocket on Dec. 23, 2014. President Vladimir Putin also joined in via a video link and even gave a ceremonial permission to fire the rocket. Credit: Russian Ministry of Defense Debris recovery Between June 22 and June 26, 2015, search crews were finally able to search the 150 by 50-kilometer drop zone of the first stage URM-1 boosters dropped during the Angara-5 launch on December 23, 2014. According to the local media, the debris field was concentrated within a 25 by 15-kilometer area in the Vuktylsk District of Komi Republic. More than 30 fragments were reportedly recovered from the site including identifiable components of an RD-191 engine and tank walls. Next chapter: Second launch of the Angara-5 rocket Read (and see) much more about Angara rockets and many other space projects in Russia
in a richly illustrated, large-format glossy edition: Components of the Angara-A5 rocket for the first flight: Component Manufacturing designations, series Angara-A5 (a.k.a. Angara-A5.1L or 1LM) vehicle 14A127, Series No. 71751 Briz-M upper stage 14S43 No. 88801 Payload simulator GVM, TPS2 DT-24 No. 1210483 Five RD-191 engines D013, D014, D015 (core), D018, D019 Four strap-on boosters BB1, BB2, BB3, BB4 Writing and illustrations by Anatoly Zak; Last update: July 2, 2015 All rights reserved
BACK IN PRINT ! IMAGE ARCHIVE Rollout of the Angara rocket from the assembly building to a fueling site on Dec. 15, 2014. Credit: Russian Ministry of Defense The first Angara-5 leaves the fueling site on its way to the launch pad on Dec. 20, 2014. Click to enlarge. Credit: Russian Ministry of Defense Angara-5 arrives to its launch pad. Click to enlarge. Credit: Russian Ministry of Defense Angara-5 is erected on the launch pad on Dec. 20, 2014. Click to enlarge. Credit: Russian Ministry of Defense Short winter day in Plesetsk was coming to an end when the rocket took vertical position on its launch pad. Click to enlarge. Credit: Russian Ministry of Defense Angara-5 on the launch pad in Plesetsk probably on the morning of Dec. 21, 2014. Click to enlarge. Credit: Russian Ministry of Defense Weather apparently cleared on December 22. Credit: Russian Ministry of Defense Click to enlarge. Credit: Russian Ministry of Defense Angara-5 during fueling on the eve of the launch on Dec. 23, 2014. Click to enlarge. Credit: Russian Ministry of Defense Click to enlarge. Credit: Russian Ministry of Defense Click to enlarge. Credit: Russian Ministry of Defense Angara-A5 lifts off on Dec. 23, 2014. Click to enlarge. Credit: Russian Ministry of Defense Russian military officials including Minister of Defense Sergei Shoigu monitor the first launch of the Angara rocket on Dec. 23, 2014 from a brand-new National Center of Defense Control. Credit: Russian Ministry of Defense
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Pulmonary complications following supraglottic laryngectomy.
Supraglottic laryngectomy has been generally assumed to protect the patient from aspiration changes in the lungs. This paper details a retrospective survey, the results of which suggest that respiratory infections are common after supraglottic laryngectomy and that pneumonia is a fairly common cause of death. The need for prolonged follow-up after this operation is emphasized.
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Follow us on
EpicDuel is Artix Entertainment’s most PvP oriented RPG to date. The game is set in the distant future on a world known as Delta V. The art style and turn based combat are in the same fashion as previous BattleOn games, but this time PvP takes center stake. Players will be able to jump into 1v1 or 2v2 duels using the game’s auto arrange system from any map. There are some NPCs scattered around the world which players can challenge, but this servers as a minor distraction. Achievements, player run factions (guilds), and player housing add some replay value, but this is definitely one of those games best enjoyed in short busts.
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Q:
Getting sbt-assembly working
So thus far I've been compiling my Scala project with SBT (via Typesafe stack). I want to run the code across several machines now, via sbt-assembly. Following directions, the only one change I made was in my project/Build.scala file. Here is the related part:
resolvers += "Typesafe Releases" at "http://repo.typesafe.com/typesafe/releases",
resolvers += "artifactory" at "http://scalasbt.artifactoryonline.com/scalasbt/sbt-plugin-releases",
libraryDependencies += "com.eed3si9n" % "sbt-assembly" % "0.8.3"
When I run sbt compile however, I get this error:
sbt.ResolveException: unresolved dependency: com.eed3si9n#sbt-assembly/scala_2.9.1/sbt_0.11.2;0.8.3: not found.
What am I doing wrong?
Thanks!
EDIT
Created a build.sbt file in the same folder as Build.scala (folder is /project/) and have these two lines in it:
Seq[Setting[_]](resolvers += "artifactory" at "http://scalasbt.artifactoryonline.com/scalasbt/sbt-plugin-releases",
addSbtPlugin("com.eed3si9n" % "sbt-assembly" % "0.8.3"))
Now the error is:
[warn] ::::::::::::::::::::::::::::::::::::::::::::::
[warn] :: UNRESOLVED DEPENDENCIES ::
[warn] ::::::::::::::::::::::::::::::::::::::::::::::
[warn] :: com.eed3si9n#sbt-assembly;0.8.3: not found
[warn] ::::::::::::::::::::::::::::::::::::::::::::::
[warn]
[warn] Note: Some unresolved dependencies have extra attributes. Check that these dependencies exist with the requested attributes.
[warn] com.eed3si9n:sbt-assembly:0.8.3 (sbtVersion=0.11.2, scalaVersion=2.9.1)
[warn]
[error] {file:/Users/myname/current/projectname/project/}default-d7da9a/*:update: sbt.ResolveException: unresolved dependency: com.eed3si9n#sbt-assembly;0.8.3: not found
EDIT 2
Hm, after I do a successful sbt compile, should I just be able to enter the sbt console and type in assembly?
> assembly
[error] Not a valid command: assembly
[error] Not a valid project ID: assembly
[error] Not a valid configuration: assembly
[error] Not a valid key: assembly
[error] assembly
[error]
EDIT 3 JK got it. Had to add the build.sbt info as specified in the GitHub README.
A:
There are two points here. One is that SBT plugins are not just library dependencies -- in particular, they use the current SBT version in a similar way that other Scala libraries use the Scala version. The other is that libraryDependencies in project/Build.scala affects the dependencies for the project, not for the build.
An SBT full build is itself an SBT project, just located one level down the directory tree, and so can have a build of its own configured the same way a normal build is. Unlike a normal build, where going for a "full build" is necessary under a handful of circumstances, there is almost never a reason to use a full build for a build, so using .sbt files located in project/ is almost always sufficient.
The other issue is the versioning. SBT has a utility function called addSbtPlugin that handles everything for you. It takes a moduleID and adds all the necessary SBT and Scala versioning information.
So, to get sbt-assembly working in a full build, you create a .sbt file in under project/ (conventionally either project/build.sbt or project/plugins.sbt) and place your build's resolvers and dependencies there:
resolvers += Resolver.url("artifactory", url("http://scalasbt.artifactoryonline.com/scalasbt/sbt-plugin-releases"))(Resolver.ivyStylePatterns)
addSbtPlugin("com.eed3si9n" % "sbt-assembly" % "0.8.3")
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Oncodermatology of the Head and Neck.
The European Academy of Facial Plastic Surgery celebrates its 40th anniversary. We aimed to describe innovations in the diagnostics and treatment in head and neck skin cancer over the past 40 years as well as future perspectives. Landmark events, developments, and highlights over the past decades for basal cell carcinoma, cutaneous squamous cell carcinoma, and melanoma are discussed.
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Q:
Chrome Custom Tabs change the default close button not working
I am trying to change the default close button on the actionbar of the custom chrome tabs. I have tried to set using setCloseButtonIcon() However, the default close button still shows. I want to change the close to an arrow.
My code below:
public void openHomePage() {
final CustomTabsIntent.Builder builder = new CustomTabsIntent.Builder();
builder.setToolbarColor(ContextCompat.getColor(getActivity(), R.color.primary));
final Bitmap backButton = BitmapFactory.decodeResource(getResources(), R.drawable.ic_arrow_back_black_48dp);
builder.setCloseButtonIcon(backButton);
builder.setShowTitle(true);
final CustomTabsIntent customTabsIntent = builder.build();
customTabsIntent.launchUrl(getActivity(), Uri.parse(mTvHomepage.getText().toString()));
}
A:
I have an observation. Last month, when searching through SO for various chrome custom tab issues, I found this answer suggesting to use 24dp size icon and also found this question saying that it is working fine with PNG.
I have checked your code with using back arrow icon from here.
When I used "ic_arrow_back_black_48dp", it didn't change the default close button to an arrow (see left image).
final Bitmap backButton = BitmapFactory.decodeResource(getResources(), R.drawable.ic_arrow_back_black_48dp);
But when I used "ic_arrow_back_black_24dp", it perfectly changed the default close button to an arrow (see right image).
final Bitmap backButton = BitmapFactory.decodeResource(getResources(), R.drawable.ic_arrow_back_black_24dp);
As it has worked perfectly for me, you should also try with "24dp" size back arrow icon from here instead of "48dp" size back arrow icon.
Screenshot : [ Device: ASUS_Z00UD; OS: 6.0.1 ]
A:
Presuming you are using the Google library and not on of the related ones the icons size should be 24dp as documented here.
This can be achieved with BitmapFactory Options:
BitmapFactory.Options options = new BitmapFactory.Options();
options.outWidth = 24;
options.outHeight = 24;
options.inScaled = true; //already default, just for illustration - ie scale to screen density (dp)
... = BitmapFactory.decodeResource(getResources(), R.drawable.ic_arrow_back_black_48dp, opts);
A:
You can directly get BitmapDrawable from Drawable but not from VectorDrawable as setCloseButtonIcon requires @NonNull Bitmap icon
You can also use svg as follows. Download the svg from here ic_arrow_back_black_24px
Below methods are self explanatory:
private static Bitmap getBitmapFromDrawable(Context context, int drawableId) {
Drawable drawable = ContextCompat.getDrawable(context, drawableId);
if (drawable instanceof BitmapDrawable) {
return ((BitmapDrawable) drawable).getBitmap();
} else if (drawable instanceof VectorDrawable) {
return getBitmapFromVectorDrawable((VectorDrawable) drawable);
} else {
throw new IllegalArgumentException("Unable to convert to bitmap");
}
}
@TargetApi(Build.VERSION_CODES.LOLLIPOP)
private static Bitmap getBitmapFromVectorDrawable(VectorDrawable vectorDrawable) {
Bitmap bitmap = Bitmap.createBitmap(vectorDrawable.getIntrinsicWidth(),
vectorDrawable.getIntrinsicHeight(), Bitmap.Config.ARGB_8888);
Canvas canvas = new Canvas(bitmap);
vectorDrawable.setBounds(0, 0, canvas.getWidth(), canvas.getHeight());
vectorDrawable.draw(canvas);
return bitmap;
}
You can use the above as:
builder.setCloseButtonIcon(getBitmapFromDrawable(this, R.drawable.ic_arrow_back_black_24px));
Ref from SO
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Hugh Pavletich, one of the report’s authors, said housing had become more affordable in Australia over the past year as prices fell amid tightening credit. He said that Ms. Ardern’s government, which took office in October 2017, had been “messing around” by focusing too much on the hotly debated plan to build 100,000 more houses — called KiwiBuild — instead of freeing up more land for construction, which he said could have kept prices in check.
“If they’d got out of the starting blocks with structural reforms centered around land supply and infrastructure financing soon after the election, it would have sent a far clearer signal to the market and subdued it significantly as these changes were put in place,” he said.
But the center-left Labour Party that Ms. Ardern leads has now conceded that its flagship policy will not ease the housing crisis as rapidly as it had hoped. The prime minister told reporters Wednesday that the government would still build 100,000 housing units in a decade, but that its interim targets would be scrapped.
The government now expects to have 300 new homes built under the plan by July, rather than the original plan for 1,000.
Phil Twyford, the housing minister, said Wednesday that there would be a “recalibration” of the policy, noting that demand for the new homes in some areas had been weaker than expected. KiwiBuild has faced criticism from political opponents that even its cheapest houses are too expensive for first-time buyers who had been shut out of the market.
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Recovery of herpes simplex virus genetic information from human trigeminal ganglion cells following superinfection with herpes simplex virus type 2 temperature-sensitive mutants.
Explant cultures of human trigeminal ganglia were derived from 36 individuals. Those cultures which failed to release herpes simplex virus (HSV) spontaneously were superinfected at various times after establishment in vitro with a range of HSV-2 temperature-sensitive (ts) mutants. Eight cultures from six individuals contained HSV-specific genetic information which could be detected or rescued following superinfection. Restriction enzyme analysis of ts+ virus recovered from the ganglia of two individuals following superinfection was identical to that of endogenous HSV-1 spontaneously released from parallel cultures. Retrieval of ts+ virus by this technique suggests products of the superinfecting virus activated expression of whole genomes or that spontaneous virus expression occurred unrelated to the act of superinfection.
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A conventional device and method for processing a carcass part of slaughtered poultry in a processing line are known from e.g., EP-B-2 606 737; EP-B-2 289 340 and EP 2 449 886. U.S. Pat. No. 4,951,354; U.S. Pat. No. 5,372,539 and in particular EP 0 800 768 represent examples of other types of devices and methods in which cutting and/or removing of the wishbone from the carcass part is avoided. The general idea of not cutting or removing the wishbone is to avoid bone splinters. Embodiments of the present invention, however, relate to cutting and/or removing of the wishbone as opposed to the teachings of U.S. Pat. No. 4,951,354; U.S. Pat. No. 5,372,539 and EP 0 800 768.
In conventional devices and methods, cutting and/or removing of the wishbone from the carcass part inevitably involves cutting and/or removing of (minute) fractions of the naturally present meat on the carcass part. It is a standing challenge for the skilled person operational in this field to reduce the fractions of naturally present meat that are taken from the carcass part during removal of the wishbone. These challenges and problems occur irrespective of the manner in which the cutting and/or removing of the wishbone is executed.
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Passive immunization against hepatitis A.
Administration of human serum immune globulin (Ig) is an effective means of protecting individuals against hepatitis A virus (HAV) infection and disease. Several large field studies have demonstrated that if given before exposure, Ig will prevent infection with HAV. Furthermore, if Ig is given during the incubation period of hepatitis A, the severity of infection may be reduced and potentially clinical infections may be converted into subclinical ones. Although uncommon, infection which occurs in the presence of circulating antibody may occasionally lead to passive-active immunity. Unfortunately, the duration of Ig protection is dose dependent, and high dose administration provides less than six months protection. Ig preparations contain HAV antibodies at levels detectable by commercial immunoassays; however, recipients of Ig do not have detectable levels of HAV antibodies when tested by the same method. Using more sensitive immunoassays and neutralization assays, low titres of HAV antibody can be detected in Ig recipients. Since Ig provides approximately 90% efficacy in preventing hepatitis A, it would appear that very low levels of HAV antibody are needed to prevent infection. Consequently, measurement of HAV antibodies elicited by HAV vaccines should provide a reasonable method to evaluate their immunogenicity and predict their efficacy.
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BAYNARD CASTLE
The remains of a medieval magnate's residence, which has been known as Baynard Castle since the 19th century. A manor house was first documented on the site in the 1170s. There are subtantial remains of a moated enclosure and rampart, also visible on aerial photograhs and lidar. Finds from the site suggest that there may have been a settlement on the site in the Mid Saxon period.
Large sections of the moat have now been obliterated by building construction. Authorised for publication on 1/1250 Resurvey as 'BAYNARD CASTLE [GT] (site of)' [TI]. 'MOAT [GT] Published as Athorised for publication on 1/1250 Resurvey as: MOAT (GT) (2)
No trace of the ramparts remain, but much of the moat is still in existence, and has been surveyed. Area consists of housing developments and small market garden. (3)
The moat is dry. Slopes as shown on 6" Qtr sheet varying in height from 2-3 metres. (4)
Cottingham. The Castle received licence for crenellation in 1327. The moat is well defined behind the house in Northgate and West End Road, and the motte can be seen from West Green. (5)
TA 041330 (centred) Baynard Castle, Cottingham, scheduled. Moated site, and such by at least 1276. Badly damaged in building work, 1951. Much of moat still, however, traceable in midst of housing and a small market garden. (6)
TA 041 331. Cottingham (Baynard Castle). A double-moated enclosure founded in the time of Stephen. William de Stuteville received a licence to crenellate it in 1201, and it had another licence in 1327. (8-10)
Baynard Castle, Cottingham, was originally a rectangular enclosure of 10.75 acres with a suggestion that the north-east corner originally as a keep. The present entrance on the south-east may represent the original position. (11)
A moat at Cottingham was mentioned in a manorial extent 1276. There were fish-ponds, but they have been destroyed. (12)
TA 041 330. Baynard Castle, Cottingham. A rectangular enclosure of c.11 acres with rounded corners and defended by a bank and ditch. The N half is further defended by an inner ditch. Building in the present century has largely obscured the outer defences, although they remain clearly fossilised in the settlement plan. Only on the E side of the site can a clear impression be gained of the former layout of the monument, through the survival of the outer rampart and ditch together with a portion of the outer bailey including some earthwork remains preserved in rough pasture.
A castle was first mentioned in c.1170-80 and in 1201 William de Stutville had licence to enclose and fortify it. The house was moated in 1272 and described as "well built with a double ditch and enclosed by a wall" in 1282. Thomas Wake had licence to crenellate in 1327, but the house was described as ruinous in 1349. Garrets, a bridge and a gatehouse are mentioned in the 15th century. Leland found no trace of Stutville's castle, but "four mean farmer's houses...within the castle garth". (13)
TA 041 330. Baynard Castle, Cottingham. Scheduled No HU/140. (14)
A trial excavation within the S outer bailey in 1991 uncovered phases of occupation dated to the 12th to 14th centuries, including chalk floors, wall footings and industrial metal-working areas, the latter still containing furnace/hearth bases. Wooden sill beams from early 12th century wall structures, and evidence for a wattle-and-daub structure were also discovered. Several sherds of mid Saxon pottery represented earlier activity in the area. A full archive report is available from the Humbs Archaeol Unit. (15)
The monument includes buried and earthwork remains of part of a medieval magnate's residence which has been known as Baynard Castle since at least the 19th century. The monument includes the inner court, the full circuit of the inner moat, part of the outer court, and the undeveloped defensive bank and surrounding outer moat bank. (16)
The earthworks of a moated enclosure and outer rampart are visible on aerial photographs and lidar. Parts of the southern and western part of the site are encroached upon by housing and gardens. The moated enclosure, centred at TA 0407 3306, has three sides visible and the southern ditch is less clearly defined. The outer rampart is visble along the eastern side.(17-18)
SOURCE TEXT
( 1) Ordnance Survey Map (Scale / Date)
OS 6" Prov 1926/38
( 2) Large Scale / Small Scale Map Revisers Comment
Large Scale Surveyor 03-MAR-1951 Km TA 0433 SW
( 3) Field Investigators Comments
Chief Surveyor Hull OS 513 27-JUN-1951 (KMS TA 0333 SE & TA 0433)
( 4) Large Scale / Small Scale Map Revisers Comment
SA Crick 7 252 SS Reviser A.06"
( 5) by Nikolaus Pevsner and David Neave 1995 Yorkshire : York and the East Riding
The Buildings of England Page(s)217
( 6) by Neil Loughlin and Keith Miller 1979 A survey of archaeological sites in Humberside carried out for the Humberside Joint Archaeological Committee
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Amorphus orientalis sp. nov., an exopolysaccharide-producing bacterium isolated from salt mine sediment.
A Gram-negative, moderately halophilic, non-motile, aerobic bacterium, designated strain YIM D10T, was isolated from a salt mine sediment sample from Yunnan, south-west China. The strain grew optimally in the presence of 3-8% NaCl and at 28 degrees C and pH 7.5. The polar lipid profile of strain YIM D10T comprised diphosphatidylglycerol, an unknown phospholipid and two unknown aminolipids. The major cellular fatty acids were C18:1omega7c (30.5%), C19:0 cyclo omega8c (29.3%) and C18:0 (13.2%). The respiratory quinone was ubiquinone 10 (Q-10). The genomic DNA G+C content was 65.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM D10T was closely related to Amorphus coralli RS.Sph.026T (96.8% gene sequence similarity). Results confirmed the placement of isolate YIM D10T within the genus Amorphus. However, DNA-DNA hybridization between strain YIM D10T and the type strain of the only recognized species of the genus Amorphus, A. coralli RS.Sph.026T, was 16.7%, showing clearly that the isolate constitutes a new genospecies. Strain YIM D10T could be clearly differentiated from A. coralli and other phylogenetic neighbours on the basis of some phenotypic, genotypic and chemotaxonomic features. Therefore, strain YIM D10T is considered to represent a novel species of the genus Amorphus, for which the name Amorphus orientalis sp. nov. is proposed; the type strain is YIM D10T (=DSM 21202T=CCTCC AA 208035T).
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Getting started with Austrian Poland research
Time period: 1782-1918.
The parish-priests were given the title of civil registrars.
Since 1784 the vital documents gained a legal status; each of the religions had a separate set of records for births, marriages, and deaths; records were written in Latin; the entries were separate for each of the villages included in a parish; copies of the records were given to the civil authorities at the end of each year. The Lutherans had their right to keep their own records, usually in German.
Since 1787 when there was a marriage of mixed religions, the entry had to be made in both of the involved parishes.
Since 1789 there were established Jewish communities, before that time the records would be maintained by the catholic priests.
In 1891 was the final division of the Jewish communities, entries were made in German and Polish, headings appeared in Hebrew or Yiddish.
In 1907 the main change to the form of the tabular records was the addition of the last column called "remarks".
Where can we find the records today: At the places where the records were originally kept; at the appropriate Civil Registrar’s Offices (usually not older records than 100 years in Poland and usually 80 years in the former Soviet Republics) then at the State Archives; at the Church Archives; at the "Beyond the Bug River Archives"–the Main Archive of Ancient Documents (Archiwum Glowne Akt Dawnych) in Warsaw, the Civil Registrar’s Office (Warszawa-Srodmiescie).
Jurisdictions
Research Tools
Helpful information for researching Germans in Galicia can be found at Galizien German Descendants website. Click Researching our Galizien Germans link on the left to learn about research. Then click Records and Archives dealing with Galizien Germans link for information about available records including Family History Library microfilm numbers.
Did you know?
The Austrian Crownland of Galizien (Galicia) is called Halychyna in Ukrainian and Halicz in Polish. The area of Galicia refers to the region that came to Poland during the first partition in 1772. Two years later, Empress Maria Theresa issued a settlement patent to encourage immigration to the sparsely settled region. Her successor Emperor Joseph II issued a second patent in 1781 and added a Toleranzpatent promising religious toleration for Protestants. Germans from the Palatinate (Pfalz), Wurttemberg, and Bohemia responded, as did Poles, Czechs, Slovaks, and others. Galicia was annexed to Poland in 1918. In 1939, it was divided between the Provisional Government of Warsaw and Ukraine, a division drawn with the modern geographical boundaries of Poland and Ukraine.
Galicia reaches north from the Carpathian Mountains across the Sarmatian Plain. It stretches from the Biala River, a tributary of the Weichsel, in the west to the Zbrucz, a tributary of the Dniester, in the east.
This area had a large Ukrainian population in the eastern section and a Polish population on the western side which was often refered to as Little Poland. Some of the localities in Austrian Poland are Tarnow, Rzezow and Nowy Sącz.
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# Base16 Material Vivid - kitty color config
# Scheme by joshyrobot
background #202124
foreground #80868b
selection_background #80868b
selection_foreground #202124
url_color #676c71
cursor #80868b
active_border_color #44464d
inactive_border_color #27292c
active_tab_background #202124
active_tab_foreground #80868b
inactive_tab_background #27292c
inactive_tab_foreground #676c71
tab_bar_background #27292c
# normal
color0 #202124
color1 #f44336
color2 #00e676
color3 #ffeb3b
color4 #2196f3
color5 #673ab7
color6 #00bcd4
color7 #80868b
# bright
color8 #44464d
color9 #f44336
color10 #00e676
color11 #ffeb3b
color12 #2196f3
color13 #673ab7
color14 #00bcd4
color15 #ffffff
# extended base16 colors
color16 #ff9800
color17 #8d6e63
color18 #27292c
color19 #323639
color20 #676c71
color21 #9e9e9e
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Filling Station for Sale in Abuja 2017 Updated
Previously I have written about fuel stations available in Abuja for sale, but in this article Filling station for sale in Abuja 2017 Updated is the most recent information about petrol stations or fuel stations or gas stations for sale in Abuja and surrounding. I also made a video about it check it out on YouTube Zondre E!
I want to reecho an idea and that which is: filling station irrespective of if you are buying as a company is very indispensible asset. Especially in a growing economy; well on this topic I have a detailed explanation on this article best time to buy a filling station check out what I mean by that.
Moving straight to the reason for this article, there are lots of filling station for sale in Abuja. Investors know that filling station is a viable asset in Nigeria. All the filling station listed on this site is available and is situated on the roads where there is an easy drive in for the customers; a viable market for the owners/operators and finally it’s in a situation where upgrades can be done.
The lists of filling Station for sale in Abuja 2017 Updated are as follows
#1 Filling station for sale in Abuja 2017 updated
Located at Kubwa Abuja; It has 10 pumps, two-one storey building, over 200,000 liters of storage tanks, with a high market viability and also a projected income of about N250Million (naira) annually (depends on the operation it can be up to or less).
The asking price is N2.5 Billion Naira. This is a very large filling station.
#2 Filling Station for sale in Abuja 2017 updated
Located at Lugbe Abuja it has about 8 pumps, over 150,000 liters storage of petroleum products, with a projected income of about N133million naira annually. Within the station there are also a functional car wash, retail shops, lubrication and service bay
For the asking price of N300 Million Naira
#3 Filling Station for sale in Abuja 2017 updated
Located at Abuja-Kaduna express way, it consists of 8 pumps, 33,000 liters storage tanks of DPK, AGO and PMS (for each). With a projected income of over N100 million naira annually(depending on the operation), it is going for the asking price of N300 million naira.
#4 Filling Station for sale in Abuja 2017 updated
Located also at Abuja-Kaduna express way, with only 6 pimps and also a same storage capacity as #3 above and goes for the asking price of N250 million naira
#5 Filling Station for sal in Abuja 2017 updated
Located near Jehre, with 8 pumps and a good market viability and over 100,000 liters of storage tanks and with an asking price of N280 illion naira
Are you interested?
Are you or your company interested in the purchase of any of these filling stations? Or do you want to go into partnership by renting and operating any of these filling stations for 5years or more? If yes, then contact us here or call the phone number right Now 08063084876.
So that we can begin the process of showing you these filling stations and leave you to make your choice but before you make your choice I want to you to go through this article the best time to buy a filling station. Watch us on our YouTube Channel.
Don’t forget to follow us on our various social media platforms @zondrealestate on facebook and instagram @zondrealestates on @twitter. Please leave your comments and questions below so that we can discuss more. Thank you for visiting my site hope you found it informing?
Comments
Zondre
My name is Uzondu Chigozie a.k.a Zondre. I am a real estate entrepreneur and Entertainer. In the world of Real estate, Entertainment, Motivation and in Life, I hope to be a positive contributor to you, so join me explore the world and make it better than we've found it.
Thank you for checking me out!!!
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ABOUT
Zondrealesate.com is a multi-content blog that features Real Estate, Motivation, Entertainment and others.
On Real estate business, investment Market and service including properties related information. Motivation is the other aspect you get from this site, because we all need to be sharpened from time to time to enable us achieve our full potentials in life so that we can be the best we can be.
Finally, Entertainment is another great content of the zondrealestate.com featuring comedy and Music. our team are dedicated to feed you with the finest content in all of our category: Real estate, Motivation and Entertainment.
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Organisms of the genus Fusobacterium are important human and animal pathogens which are isolated frequently from liver and brain abscesses. F. nucleatum is isolated from normal subgingival dental plaque but its numbers increase dramatically in cases of gingivitis and periodontitis suggesting pathogenic potential. Little is known about the physiology of the fusobacteria and most published reports refer to these organisms as asaccharolytic although sugar use is easily demonstrated. Previous work in this laboratory has shown that glucose transport by F. nucleatum is dependent on energy provided by the fermentation of amino acids. In addition, we have shown extensive sugar use by F. mortiferum strongly suggesting that this organism can be used successfully as a model for genetic studies which are lacking in the fusobacteria. In the past year we have concentrated on the proof of the cellobiose phosphoenolpyruvate phosphotransferase (PTS) use in F. mortiferum. A cellobiose PTS assumes the intermediate formation of cellobiose-6-P and its subsequent hydrolysis by a specific hydrolase. Cellobiose-6-P and a variety of other beta disaccharide phosphates were made using an ATP kinase purified from Klebsiella pnemoniae. The beta glucoside phosphates had the predicted molecular weight when analyzed by mass spectrometry and were all hydrolyzed by the 54 KD protein from cellobiose grown F. mortiferum which we suggested as the cellobiose- 6-P hydrolase. In addition, an artificial beta glucoside phosphate was made from p nitrophenyl beta D glucoside [PNP beta GP]. The hydrolysis of PNP beta GP gave a yellow color which could be used to quantitate the hydrolase activity or be used qualitatively to follow the purification of the enzyme. The cellobiose phosphate hydrolase retained activity in air in contrast to the maltose-6-P hydrolase. Washed, cellobiose grown cells of F. mortiferum used limited amounts of cellobiose from a buffered suspension when incubated aerobically. The aerobic inhibition of cellobiose use was gradual, rather than abrupt, as in the maltose system of the same organism.
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Q:
Cannot receive messages on subscriber VM (ZeroMQ on VirtualBox)
I have two OpenWrt (18.06.4) VM's (A and B) in VirtualBox and I'm trying to send messages in a publisher-subscriber scheme using ZeroMQ. A is the server, B is the client.
I'm using the following code:
Publisher code: http://zguide.zeromq.org/c:psenvpub
Subscriber code: http://zguide.zeromq.org/c:psenvsub
and it works on my computer, so I decided to try it on the VMs.
I had to compile both (using the SDK) so I can execute them in the VMs. I compiled two times, changing one minor detail:
1) client listening to the IP 10.0.1.4 of the server
2) client listening to the IP 192.168.56.10 of the server
Both versions were tested in the VMs and in both, the server sends the messages (the send function executes and prints the message sent) but the client never receives any message (message is always null).
About my network configuration. In VirtualBox, I have a Nat Network (10.0.1.0/24) and a virtualbox network (192.168.56.1/24). Both VM A and B have a host-only adapter (vboxnet0) and a NAT network adapter.
The machines can ping each other.
The network configuration of the machines is the following:
A
config interface 'loopback'
option ifname 'lo'
option proto 'static'
option ipaddr '127.0.0.1'
option netmask '255.0.0.0'
config globals 'globals'
option ula_prefix 'fd03:84ea:bc33::/48'
config interface 'lan'
option ifname 'eth0'
option proto 'static'
option ipaddr '192.168.56.10'
option netmask '255.255.255.0'
config interface 'wan'
option ifname 'eth1'
option proto 'dhcp'
Note: The NAT network IP ('wan') is currently 10.0.1.4
B
config interface 'loopback'
option ifname 'lo'
option proto 'static'
option ipaddr '127.0.0.1'
option netmask '255.0.0.0'
config globals 'globals'
option ula_prefix 'fdea:4700:64aa::/48'
config interface 'lan'
option ifname 'eth0'
option proto 'static'
option ipaddr '192.168.56.20'
option netmask '255.255.255.0'
config interface 'wan'
option ifname 'eth1'
option proto 'dhcp'
Note: The NAT network IP ('wan') is currently 10.0.1.5
Do you guys have any idea what the problem might be? Should I change the network configuration inside each VM and/or change the adapters on VirtualBox?
A:
Avoid the dependency on symbolic-address resolution:
// zmq_bind (publisher, "tcp://*:5563"); // PUB-side wildcard-address translated
zmq_bind (publisher, "tcp://10.0.1.4:5563"); // explicit address
// zmq_connect (subscriber, "tcp://localhost:5563"); // SUB-side symbolic-address
zmq_connect (subscriber, "tcp://10.0.1.4:5563"); // explicit-address
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Q:
Error: "unrecognized selector sent to instance" when clicking button
Please let me tell you what I am doing.
I have a window that has a NSView "MainContainer"
On laod of the window I am adding a custom view "Options" in the NSView of the window
There is a button on the customer view "Options" named "Customer Details"
On Customer Details click>> current view(Options) gets removed and a new view (customer details view) gets loaded. How I am doing is given below:
NSViewController* cdv = [[CustomerDetailsView alloc] init];
NSView* MainView = [[self view] superview];
[[self view] removeFromSuperview];
[MainView addSubview:[cdv view]];
Now the issue is that the last view (Customer Detail View) has some buttons and no one is working and I am getting an error which is "unrecognized selector sent to instance". Please let me know what should I do?
2015-09-17 15:45:37.872 TechHeal[5058:125394] not start
2015-09-17 15:46:05.452 TechHeal[5058:125394] -[NSSnapshotContextSignature encryptClick:]: unrecognized selector sent to instance 0x6080000e5c80
2015-09-17 15:46:05.452 TechHeal[5058:125394] -[NSSnapshotContextSignature encryptClick:]: unrecognized selector sent to instance 0x6080000e5c80
2015-09-17 15:46:05.464 TechHeal[5058:125394] (
0 CoreFoundation 0x00007fff9834a03c __exceptionPreprocess + 172
1 libobjc.A.dylib 0x00007fff8e54c76e objc_exception_throw + 43
2 CoreFoundation 0x00007fff9834d0ad -[NSObject(NSObject) doesNotRecognizeSelector:] + 205
3 CoreFoundation 0x00007fff98292e24 ___forwarding___ + 1028
4 CoreFoundation 0x00007fff98292998 _CF_forwarding_prep_0 + 120
5 libsystem_trace.dylib 0x00007fff95ef2cd7 _os_activity_initiate + 75
6 AppKit 0x00007fff9127eeb1 -[NSApplication sendAction:to:from:] + 452
7 AppKit 0x00007fff91294946 -[NSControl sendAction:to:] + 86
8 AppKit 0x00007fff91294862 __26-[NSCell _sendActionFrom:]_block_invoke + 131
9 libsystem_trace.dylib 0x00007fff95ef2cd7 _os_activity_initiate + 75
10 AppKit 0x00007fff912947bf -[NSCell _sendActionFrom:] + 144
11 libsystem_trace.dylib 0x00007fff95ef2cd7 _os_activity_initiate + 75
12 AppKit 0x00007fff91292cb3 -[NSCell trackMouse:inRect:ofView:untilMouseUp:] + 2821
13 AppKit 0x00007fff912eb34f -[NSButtonCell trackMouse:inRect:ofView:untilMouseUp:] + 770
14 AppKit 0x00007fff91291366 -[NSControl mouseDown:] + 714
15 AppKit 0x00007fff917fb2dc -[NSWindow _reallySendEvent:isDelayedEvent:] + 14125
16 AppKit 0x00007fff9118ac86 -[NSWindow sendEvent:] + 470
17 AppKit 0x00007fff91187212 -[NSApplication sendEvent:] + 2504
18 AppKit 0x00007fff910b0b68 -[NSApplication run] + 711
19 AppKit 0x00007fff9102d244 NSApplicationMain + 1832
20 TechHeal 0x00000001000048e2 main + 34
21 TechHeal 0x0000000100001224 start + 52
22 ??? 0x0000000000000003 0x0 + 3
)
(lldb)
PS: If I am loading the Customer details directly then its working fine however if I am loading it from another view then its not working.
A:
You appear to be creating a CustomerDetailsView controller, taking its view, and then letting the controller go out of scope. Assuming encryptClick is implemented in that controller, the problem is that when it's called the object has already been released. The message is being passed to whatever now occupies that chunk of memory.
Try making cdv a strong property of whatever self is in your posted code so that it stays around to handle events.
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Q:
Media Source Extensions appendBuffer of WebM stream in random order
I am trying to achieve video downloading in parallel from multiple sources. However MSE appendBuffer method always fails when not following sequence order of video file.
I would like to append parts in random order and play video "as soon as possible".
I was exploring SourceBuffer mode property as well as timestampOffset. None of those were helpful.
I am wondering if source webm file i have could be in "not supported format" for such a task (sequential approach works fine).
source video file
Thank you for any advices.
UPDATE:
I tried to analyse well known example video file and i figured out that it is possible to append parts of it out of order. Seems like it is necessary to follow Cluster byte ranges:
<Cluster type="list" offset="4357">
<Timecode type="uint" value="0"/>
<SimpleBlock type="binary" size="7723" trackNum="1" timecode="0" presentationTimecode="0" flags="80"/>
<SimpleBlock type="binary" size="5" trackNum="2" timecode="0" presentationTimecode="0" flags="80"/>
...
</Cluster>
<Cluster type="list" offset="16187">
<Timecode type="uint" value="385"/>
<SimpleBlock type="binary" size="5" trackNum="2" timecode="0" presentationTimecode="385" flags="80"/>
<SimpleBlock type="binary" size="4968" trackNum="1" timecode="13" presentationTimecode="398" flags="80"/>
...
</Cluster>
A:
After digging into webm format specification, compiling libwebm tools and studying DASH i finally figured out how to make MSE appendBuffer working in any order!
ffmpeg -i result.webm -g 10 -c:v libvpx resultClusters.webm (you can also use libvpx-vp9)
mkvmuxer_sample -i resultClusters.webm -o resultRepaired.webm
mse_json_manifest resultRepaired.webm >> manifest.json
You will get on stdout something like:
{
"type": "video/webm; codecs=\"vp8\"",
"duration": 27771.000000,
"init": { "offset": 0, "size": 258},
"media": [
{ "offset": 258, "size": 54761, "timecode": 0.000000 },
{ "offset": 55019, "size": 166431, "timecode": 2.048000 },
{ "offset": 221450, "size": 49258, "timecode": 4.130000 },
{ "offset": 270708, "size": 29677, "timecode": 6.148000 },
{ "offset": 300385, "size": 219929, "timecode": 8.232000 },
{ "offset": 520314, "size": 25132, "timecode": 10.335000 },
{ "offset": 545446, "size": 180777, "timecode": 12.440000 },
{ "offset": 726223, "size": 76107, "timecode": 14.471000 },
{ "offset": 802330, "size": 376557, "timecode": 14.794000 },
{ "offset": 1178887, "size": 247138, "timecode": 16.877000 },
{ "offset": 1426025, "size": 78468, "timecode": 18.915000 },
{ "offset": 1504493, "size": 25614, "timecode": 20.991000 },
{ "offset": 1530107, "size": 368277, "timecode": 23.093000 },
{ "offset": 1898384, "size": 382847, "timecode": 25.097000 },
{ "offset": 2281231, "size": 10808, "timecode": 27.135000 }
]
}
Now all you have to do is firstly load metadata xhr.setRequestHeader("Range", "bytes=0-257"); and then in ANY ORDER all other segments. E.g. second segment range is 55019-221449 bytes.
Explanation:
The most important thing is ffmpeg reencoding with group of frames set to the size of cluster you would like to have. In this example i choose pretty low threshold (each 10 frames) but you can choose higher causing fewer clusters are generated (less items in "media" array).
After that you have to fix cues in the classic way (using sample_muxer from libwebm) and you are ready to go.
Tested on: Chrome 51, Firefox 47.
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Q:
Table layout cutting out 4th column on android app
I am tried to creating a simple calculate app for android. I used a table layout and add 4 rows and 4 columns. In each column I add a button . My problem is the 4th column cuts out as shown in the image below,
Here is my code below
<?xml version="1.0" encoding="utf-8"?>
<RelativeLayout xmlns:android="http://schemas.android.com/apk/res/android"
xmlns:tools="http://schemas.android.com/tools"
android:layout_width="match_parent"
android:layout_height="match_parent"
android:paddingBottom="@dimen/activity_vertical_margin"
android:paddingLeft="@dimen/activity_horizontal_margin"
android:paddingRight="@dimen/activity_horizontal_margin"
android:paddingTop="@dimen/activity_vertical_margin"
tools:context="com.tashtoons.calculator.Calculator">
<LinearLayout
android:orientation="vertical"
android:layout_width="match_parent"
android:layout_height="match_parent"
android:layout_alignParentTop="true"
android:weightSum="1"
android:layout_alignParentRight="false"
android:layout_alignParentEnd="false">
<TextView
android:layout_width="343dp"
android:layout_height="77dp"
android:id="@+id/answerScreen"
android:layout_gravity="center_horizontal" />
<TableLayout
android:layout_width="371dp"
android:shrinkColumns="1"
android:layout_height="match_parent"
android:baselineAligned="true">
<TableRow
android:layout_width="match_parent"
android:layout_height="match_parent">
<Button
android:text="1"
android:id="@+id/button"
android:layout_column="0"
android:width="5px" />
<Button
android:text="2"
android:id="@+id/button2"
android:layout_column="1"
android:width="5px" />
<Button
android:text="3"
android:id="@+id/button3"
android:layout_column="2"
android:width="5px" />
<Button
android:text="+"
android:id="@+id/button4"
android:layout_column="3"
android:width="5px" />
</TableRow>
<TableRow
android:layout_width="match_parent"
android:layout_height="match_parent">
<Button
android:text="4"
android:id="@+id/button5"
android:layout_column="0"
android:width="5px" />
<Button
android:text="4"
android:id="@+id/button6"
android:layout_column="1"
android:width="5px" />
<Button
android:text="5"
android:id="@+id/button7"
android:layout_column="2"
android:width="5px" />
<Button
android:text="-"
android:id="@+id/button8"
android:layout_column="3"
android:width="5px" />
</TableRow>
<TableRow
android:layout_width="match_parent"
android:layout_height="match_parent">
<Button
android:text="6"
android:id="@+id/button9"
android:layout_column="0"
android:width="5px" />
<Button
android:text="7"
android:id="@+id/button10"
android:layout_column="1"
android:width="5px" />
<Button
android:text="8"
android:id="@+id/button11"
android:layout_column="2"
android:width="5px" />
<Button
android:text="X"
android:id="@+id/button12"
android:layout_column="3"
android:width="5px" />
</TableRow>
<TableRow
android:layout_width="match_parent"
android:layout_height="match_parent">
<Button
android:text="9"
android:id="@+id/button13"
android:layout_column="0"
android:width="5px" />
<Button
android:text="0"
android:id="@+id/button14"
android:layout_column="1"
android:width="5px" />
<Button
android:text="%"
android:id="@+id/button15"
android:layout_column="2"
android:width="5px" />
<Button
android:text="/"
android:id="@+id/button16"
android:layout_column="3"
android:width="5px" />
</TableRow>
</TableLayout>
</LinearLayout>
</RelativeLayout>
How can I stop it from cutting out?
A:
Instead of your code Use below code.
Whenever you have to give same width of all Button or TextView or etc.
There is property of LinearLayout is WeightSum . This can equal divided the part.
so change your xml code to below format.
xml code :
<RelativeLayout xmlns:android="http://schemas.android.com/apk/res/android"
xmlns:tools="http://schemas.android.com/tools"
android:layout_width="match_parent"
android:layout_height="match_parent"
android:paddingBottom="@dimen/activity_vertical_margin"
android:paddingLeft="@dimen/activity_horizontal_margin"
android:paddingRight="@dimen/activity_horizontal_margin"
android:paddingTop="@dimen/activity_vertical_margin"
tools:context="com.example.softeng.xxy.ThirdActivity">
<RelativeLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:layout_marginTop="50dp"
android:id="@+id/firstRow">
<LinearLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:weightSum="4">
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="1" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="2" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="3" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="+" />
</LinearLayout>
</RelativeLayout>
<RelativeLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:id="@+id/secondRow"
android:layout_below="@+id/firstRow">
<LinearLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:weightSum="4">
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="4" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="4" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="5" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="-" />
</LinearLayout>
</RelativeLayout>
<RelativeLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:id="@+id/thirdRow"
android:layout_below="@+id/secondRow">
<LinearLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:weightSum="4">
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="6" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="7" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="8" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="X" />
</LinearLayout>
</RelativeLayout>
<RelativeLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:id="@+id/forthRow"
android:layout_below="@+id/thirdRow">
<LinearLayout
android:layout_width="match_parent"
android:layout_height="wrap_content"
android:weightSum="4">
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="9" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="0" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="%" />
<Button
android:layout_width="0dp"
android:layout_height="wrap_content"
android:layout_weight="1"
android:text="/" />
</LinearLayout>
</RelativeLayout>
</RelativeLayout>
Output :
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Serial measurement of serum S-100B protein as a marker of cerebral damage after cardiac surgery.
We used serial measurements of serum S-100B protein to evaluate the time course of serum S-100B protein concentration after cardiovascular surgery and to determine the clinical relevance of its concentration and cerebral damage. We assessed neurologic function in 149 patients undergoing cardiovascular surgery with cardiopulmonary bypass. The patients were classified into three groups according to their early postoperative outcome: those without complications (group A), those having unconsciousness or convulsion or both but no hemiplegia (group B), and those having unconsciousness and hemiplegia either with or without convulsion (group C). Serum S-100B protein concentrations were measured with a commercially available immunoluminometric assay, Sangtec 100 LIA, at seven time-points: before cardiopulmonary bypass, at the end of cardiopulmonary bypass, and at 5, 12, 24, 48, and 72 hours after cardiopulmonary bypass. At 5 hours after cardiopulmonary bypass, the S-100B values in groups B and C were significantly higher than the value in group A. Although the S-100B level decreased in group C during the first 5 hours after cardiopulmonary bypass, it increased thereafter (12 through 24 hours) and continued at a high level until the final measurement at 72 hours. At 12 hours after cardiopulmonary bypass, S-100B was significantly higher in group C than in group B. This late increase in S-100B was associated with radiologically detected abnormalities and cerebral damage. Serial measurement of serum S-100B protein in the initial 12 hours after cardiopulmonary bypass can be used to predict early postoperative brain injury.
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Opportunistic Encryption for Firefox - cpeterso
http://bitsup.blogspot.com/2015/03/opportunistic-encryption-for-firefox.html
======
chimeracoder
I'm really glad to see this.
I really dislike that browsers seem to treat self-signed certificates as
_worse_ than plain HTTP (in that self-signed certificates cause a big scary
yellow warning that looks similar to the big scary red warning for invalid
certs).
Self-signed certs are bad insofar as you can't prove that someone else isn't
MITMing the connection and serving you an encrypted, but untrustworthy, proxy
page. But with plain HTTP, you already have no guarantees that that isn't
happening![0]
If I understand it correctly, this seems to combine the best of both:
encouraging the use of self-signed certificates over plain HTTP, while still
rewarding verified certs (ie, signed by a trusted third party) over self-
signed.
[0] If you really care, you should use a CA-signed cert. But every attack that
possible when using self-signed certs is not only possible when using plain
HTTP, but much easier to execute, and also much easier to execute _silently_.
~~~
tines
I won't defend it, but I can imagine that an argument for why self-signed
certs could be worse than plain HTTP might be that if you're using HTTP, the
communication isn't meant to be secure/trustworthy, but the presence of a
certificate means that your communication is meant to be one or both, and
since the cert is self-signed, it may not be either. This argument does
presume that site administrators know what does and doesn't need to be
confidential, which of course may not be the case.
Some argue that all communication should be encrypted, but that's another
issue.
~~~
vbezhenar
Self-signed certificate provides defense against passive attacker, and HTTP
does not. That's an important difference IMO.
~~~
copsarebastards
That's good in that it requires a slightly more sophisticated attack, but I
don't really view that as a very effective defense.
_However_ , the bigger thing a self-signed cert allows you is the ability to
verify later if your communication has been snooped by an active attacker--
which is very significant. It's problematic that the communications aren't
authenticated before they occur, but that doesn't mean they can't be
authenticated later (via CAs or other means).
~~~
Wicher
> That's good in that it requires a slightly more sophisticated attack, but I
> don't really view that as a very effective defense.
A slightly more sophisticated attack, indeed — but one that doesn't scale
well. It ups the cost of mass surveillance tremendously.
~~~
copsarebastards
I hear this frequently but I don't buy it. What about the MITM scales worse
than a passive listener?
As far as I can tell, both insertion of a passive listener and a MITM are
algorithmically O(n) on the number of connections being surveilled. All you're
increasing for the MITM is the constant factor.
When you're on the order of billions of connections being surveilled, even
linear growth is hard, but we know that _the NSA already has done that_.
Increasing the difficulty by a constant factor is not much harder, and there's
no question that the NSA has the budget to do so. And in fact, the constant
factor isn't even large: it's whatever the resource costs of two connection
handshakes per connection is, plus decryption/encryption on the data flowing
across, all of which are highly optimized algorithms at this point.
~~~
Wicher
No. Passively listening only requires dumping whatever flies over the
interface of some router. Done! Very hard to detect. You can also just scan
for keywords and only start dumping traffic when triggered. With SSL, you
_cannot_ retroactively decide that you'd want to dump traffic. You have to
MITM _from the beginning_.
That's point 1.
Point 2: Detection.
Actively MITM-ing an SSL connection requires you to (if the CA Chain-O'-Trust
works as supposed) give away the fact that you own a (valuable!) compromised
CA authoritative cert. You would want to use such a cert for a targeted
attack, not waste it on blanket surveillance and get found out (and called
out) within a couple of days.
If you don't own a root cert but rely on vulnerable implementations, or
implementations such as this one which do not rely on the CA infra, same
story. You do not want to waste that on blanket surveillance and get caught.
You'd save it for the /special occasions/.
SSL-MITMing _everyone_ , _all the time_ , as in blanket surveillance, is
unfeasable even without CA chains. You'll get called out on it by people that
_do_ check cert fingerprints once in a while.
This is a different kind of scalability than the computational order-of-
complexity one that you seem to be thinking about.
------
Navarr
This is very interesting.
I think a better approach might be to separate encryption from trust.
I'm thinking back to Chrome's announcement that they were considering making
[http://](http://) show some sort of warning.
What if:
* We make HTTP show up as "insecure" in browsers
* We make HTTPS work with self-signed certificates, and display websites encrypted that way the same way we currently show http
* We make HTTPS with Trust show up the way we currently show HTTPS
* Keep EV Certs the same
~~~
agwa
> * We make HTTP show up as "insecure" in browsers
> * We make HTTPS work with self-signed certificates, and display websites
> encrypted that way the same way we currently show http
Clear text and opportunistic encryption should have exactly the same UI.
Security UIs are already too confusing, and we shouldn't introduce more
complexity. Besides, one should never make a decision based on a clear text vs
OE distinction, since OE is so easily defeated.
~~~
belorn
I guess this is why most administrators use telnet instead of ssh, since they
don't have time to check ssh fingerprints... Well, not really. As it is, many
people do make a decision based on a clear text vs OE.
Now if just Firefox would store and check fingerprints of self-signed
certificates, we would get the exact same benefit when telnet was replaced by
ssh.
~~~
IgorPartola
You don't check ssh fingerprints?
------
peterwwillis
Opportunistic Encryption is harmful because people think it's actually useful.
Here's the problem with opportunistic encryption:
OE provides unauthenticated encryption over TLS for data that would otherwise be
carried via clear text. This creates some confidentiality in the face of passive
eavesdropping,
You should never assume that 'eavesdropping' is passive. In almost every
practical context of traffic interception, if you can read the transmission,
you can write as well. If you're going to the trouble of installing some kind
of tap, it makes more sense to make it read-write so you can actually _do_
something with that intercepted connection. Collecting data is great, but
hijacking is even better.
and also provides you much better integrity protection for your data than raw
TCP does when dealing with random network noise
This is a red herring, and if you need _real integrity_ is totally useless
since it doesn't prevent an active attacker from corrupting the data once
they've hijacked your unauthenticated connection. For the majority of
plaintext traffic, a small amount of corruption is way more efficient to allow
for than breaking down and re-creating a connection every time a single bit
gets flipped.
To use OE, you have to set up an SSL service in the first place, so just take
the extra 15 minutes and make a real signed certificate. There is no such
thing as "kind of" secure, after all. Encryption is intended to provide
security. OE is not secure.
~~~
y0ghur7_xxx
> Opportunistic Encryption is harmful because people think it's actually
> useful. [...] You should never assume that 'eavesdropping' is passive.
\- Google driving by with it's street view cars and capturing all your wifi
traffic for later analysis is passive.
\- People sitting in a coffee shop and capturing your wifi traffic is passive.
OE protects against these attack vectors. It does not protect against other
attack vectors, but that does not mean it's harmful or useless.
Moreover, with pins it could be a first step to get rid of CAs.
~~~
peterwwillis
These are both examples of how OE would _not_ help you.
With google street view you're barely even in range long enough to pick up a
couple packets, if the person was even using their computer while the car
drove by; this is not what OE is designed to protect against. If the car sat
outside their house, they'd still get owned, and OE would still not be useful.
Sitting on a cafe's wireless is literally the de-facto example of how to
actively sniff or inject traffic on an unsuspecting victim. The only more
bluntly useless case for OE is a state-sponsored mitm using coercion-induced
signed certificates.
And you can't get rid of CAs.
~~~
y0ghur7_xxx
You just continue to state the same thing: that OE does not protect against an
active attacker. We know that. Nobody says that it does. What it does is
protect against a passive attacker. And that makes it useful for some use
cases.
------
13throwaway
The problem with allowing self-signed certificates has always been
distinguishing if a site should be signed by a CA or not. Consider the follow
situation:
Alice sends Bob a link: [http://example.com](http://example.com)
Bob trusts Alice and now knows that example.com is probably ment to be
accessed over HTTP. Now for the next example:
Alice sends Bob a link: [https://example.com](https://example.com)
With the current implementation of browsers Bob knows that example.com should
present a CA signed certificate. But what if example.com wants to encrypt
their data, but for whatever reason uses a self-signed certificate? Some
people say that Bob's browser should not display a "big scary" warning, but
instead display a UI similar to when accessing a HTTP site. However, in this
situation HTTPS has lost some meaning. I think http2 should work as follows:
http2:// \- encrypted, not verified
https2:// \- encrypted and verified
This way the protocol still conveys the same level of information.
However, if it were completely up to me, I would say ditch the CAs and use
namecoin to verify certificates.
~~~
JonathonW
That's more or less what OE does. It allows the browser to use HTTP/2 (and
encryption) to connect to a site, but keeps the user experience the same as
unencrypted HTTP.
That's why self-signed certificates work in this context; the identity of the
server's not supposed to be validated (unencrypted HTTP can't validate server
identities), so the browser can accept a self-signed certificate without
warning.
There's no change to how certificates are authenticated when accessing a site
via an [https://](https://) URL.
------
pavpanchekha
This is a great step, one that I've been hoping for. Of course encryption
without authentication is much worse than true encrypted transport (as in,
with authentication), since it only prevents passive adversaries, but any sort
of encrypted transport is better than plaintext. I'm also hoping this will
ease the transition to TLS, since you can get it up and running without
worrying about mixed-resource problems, then fix those one at a time.
~~~
orthecreedence
I'd argue that the CA system is false authentication because it's fairly easy
for the right players to tamper with. In that case, unauthenticated/encrypted
transport is only a little less safe than "authenticated"/encrypted transport,
but with the latter giving a higher illusion of safety than the former.
The only real trust that would work is distributed trust. The CA system is
kind of a joke.
That said, yes, it does protect coffee shop HTTPS browsing better than a self-
signed cert.
------
y0ghur7_xxx
Does someone know how to set this up serverside? Does apache support this? I
skimmed through the mod_spdy docs, but found nothing about opportunistic
encrypion.
This, together with TACK or Certificate Transparency could be a CA killer.
------
upofadown
This is a neat idea, but we really need to define a standard for self signed
certificates. Something like certificate pinning should be mandatory. It
should be done in a way so there is no confusion with a connection with
identity protection _but_ at the same time it is essential that the user knows
they are at least getting the protection of the self signed certificate.
Perhaps we need something like a httpq:// resource identifier.
For bonus points such a standard should incorporate a web of trust system that
can not be overridden by a bogus certificate in the regular system. Ideally a
self signed certificate provided by someone you can physically visit should be
_more_ secure than what we have now.
Added: I guess my point is that we are thinking about this backwards. A
verified self signed certificate is the gold standard, not some inferior
alternative. I should be able to walk into my bank and then walk out with a
certificate on a USB key that can not be messed with. If we are going to
change things we should strive to end up with the possibility of something
better than what we have now.
------
teddyh
This is only for HTTP/2\. If _could_ have been for HTTP/1.1 also, _if_ there
had existed a registered ALPN name for “HTTP/1.1 with TLS”. As it is now,
HTTP/2 has both variants, but HTTP/1.1 stands alone¹.
① [https://www.iana.org/assignments/tls-extensiontype-
values/tl...](https://www.iana.org/assignments/tls-extensiontype-values/tls-
extensiontype-values.xhtml#alpn-protocol-ids)
~~~
patrickmcmanus
fwiw the h1 barrier was the lack of a scheme indication in an h1 transaction -
not really the alpn ids (which can always be registered if need be). But
without a scheme the server just needs to infer http vs https from the
port/address and that wouldn't work with alt-svc
~~~
teddyh
Since this is a backport of a new feature from a new protocol to its older
predecesor protocol, it is not necessary to be so wary of slightly uglier
features. Simply having “Alt-Svc: http/1.1:443” imply HTTPS would do fine to
solve this specific problem, and I doubt anyone would really have a problem
with it.
------
Dylan16807
> 443 is a good choice :)
If it's self signed, and going to throw massive warnings with a direct
connection, shouldn't you use anything other than 443?
Any subtleties I should be aware of?
~~~
opejn
The main reason I would think it's a good choice is because if you decide to
get a CA certificate later, you just drop it in and you're done; no additional
configuration required.
If you don't have a CA certificate, you're probably not advertising your
[https://](https://) URLs anyway, so unless search engines are aggressively
looking/prioritizing for https transport, it wouldn't seem to hurt anything to
run a self-signed certificate there.
~~~
Dylan16807
HTTPS everywhere. And I would not trust an entire site to go unindexed.
There's a lot more to change if I want real HTTPS support. Changing a single
port number is the least of my worries.
------
quonn
This is good, but still not a good as a service that requires at least an
unauthenticated encryption. The problem is that the attacker has to be active,
but very little effort is needed to break this without the user noticing -
it's enough to inject some packets to disrupt the TLS connection.
However, for HTTP it's the best thing possible at this point.
------
jesrui
Since the request and response sizes can reveal what public page you are
browsing over https, OE in the proposed form would not prevent user tracking:
[http://sysd.org/stas/node/220](http://sysd.org/stas/node/220)
------
zaroth
Please, please, can we have the same for WiFi?! Trade a key with the AP when
you first associate, and be done with it. The entire concept of a WiFi
password is a *%^$ waste of time.
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Q:
Missing sections and settings in Group Policy editor
I'm trying to edit an AD group policy that maps network drives. The policy works fine, I can view the settings under the Settings tab and see the existing drive mappings, the structure in Sysvol is intact, but when I go to edit it in GPME the entire Users > Preferences section does not have any sections and settings underneath it -- it just says "There are no items to show in this view." I also noticed the Computer Config Preferences section has the same problem.
I checked other GPOs and they have the same problem -- User Config > Preferences folder is there but nothing is underneath it. Missing Preferences settings
I tried re-registering all the following components and that did not fix the problem.
Administrative Templates and Scripts: gptext.dll
Folder Redirection: fde.dll
Internet Explorer Maintenance: ieaksie.dll
IP Security: ipsecsnp.dll
Public Key and Software Restriction: certmgr.dll
Remote Installation Services: rigpsnap.dll
Security: wsecedit.dll
Software Installation: appmgr.dll
I created a new GPO, same issue. Nothing under Preferences. I've asked four of my colleagues and they have no idea either. I don't know where to go from here.
A:
I was able to resolve this on my own by removing Group Policy Management from the list of installed Server Features, rebooting the server and reinstalling the GPM feature.
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L'Anse Creuse High evacuated after man in camouflage enters school
WXYZ reports that L’Anse Creuse High School in Harrison Township was evacuated after a possibly armed man wearing camouflage was seen entering and leaving the school around 6 a.m. Tuesday. Fox 2 reports that the man is a former student. The students at the school were evacuated to a nearby grocery store.
The person is described as a white man about 6 feet tall, wearing camo and a black vest.
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249 S.W.3d 922 (2008)
Earl COZART, Claimant/Appellant,
v.
BOARD OF EDUCATION, CITY OF ST. LOUIS, Employer/Respondent, and
Treasurer of Missouri as Custodian of the Second Injury Fund, Respondent.
No. ED 90439.
Missouri Court of Appeals, Eastern District, Division Two.
April 15, 2008.
*923 Stephen A. Walsh, St. Louis, MO, for appellant.
Eric S. Christensen, Jeremiah W. (Jay) Nixon, Atty. Gen., Kevin A. Nelson, Asst. Atty. Gen., St. Louis, MO, for respondents.
Before LAWRENCE E. MOONEY, P.J., BOOKER T. SHAW and KURT S. ODENWALD, JJ.
ORDER
PER CURIAM.
The claimant, Earl Cozart, appeals the final award of the Labor and Industrial Relations Commission. The Commission adopted the decision of the administrative law judge, denying workers' compensation benefits for an injury the claimant contends he suffered in August 2003 while working in a warehouse for the Board of Education of the City of St. Louis. We affirm the Commission's determination.
An opinion would have no precedential value. The parties have been provided with a memorandum, for their information only, setting forth the reasons for this decision.
The award of the Commission is affirmed. Rule 84.16(b)(4).
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