text: Annoy Your Neighbors With the Project Car Hell Song!Murilee Martin10/11/07 10:30amFiled to: Choose Your EternityProject Car HellRock-N-RollMusic38EditPromoteShare to KinjaToggle Conversation toolsGo to permalink It occurred to us that what our beloved readers really need is a special song to play while they're snapping off bolt heads and adding new scar tissue to their knuckles during bouts of Project Car Hell punishment in the garage. Something that sounds just about right blaring from a treble-centric WD-40-spattered boombox in a shed lit by yellow bug lights and full of the scent of Bondo and burning money. So we dragooned our musician friend Jack Astro, rounded up Belvedere Adrian, and a new band was born: Jack Astro & the Asscrackistanis! Play it loud! Louder! And any of you who want to download the MP3 to play when you're not at the computer, here ya go! It's a .zip, because my creaky old Pentium II webserver in the crawlspace can't cope with attempts to stream an MP3.


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Artist description:
From the music video 'Stay'. Took about 2-2,5 hours, 1 layer.
Java Draw Created May 07, 2013 1024 x 736px Unless noted Copyright 2013 hemmeligperson.
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2-2, 5 hours? huh?? tho u drew her very well! even captured her expression/features well n also like her hair n the water its just awesum--time to fav~
She would be crazy to not appreciate the time and effort someone took to draw her likeness its beautiful

subset: commoncrawl


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The Patterned Irrationality of the Left
For example, as Bomford writes, on a deep unconscious level, "one who hates has to believe that his or her hatred is returned." Note that this is a logical operation, only based upon a different sort of logic. This logic is no doubt the source of the psychotic fear of Israel in the Islamic world. Their unconscious hatred is so profound that it simultaneously reverses the relation, so that they can't help perceiving that Israel hates them. But Israelis just want to shop, raise their families, or read the Torah. They couldn't care less about Muslims, except to the extent that bloodthirsty Muslim barbarians harbor murderous rage toward them.
It's fine to hate evil, but in the Islamic world, what is hated is transformed into evil. Something is not hated because it is evil, but evil because it is hated. One could say the same of the left, which habitually fears what it eternally hates. The left cannot be comprehended unless one appreciates the extent of their unbound hatred. Once this is grasped, what seems illogical is suddenly seen to obey the dictates of symmetrical logic. For example, the unconscious feeling that I hate America and want us to lose in Iraq is transformed to General Petraeus is a traitor, or I am a racist becomes America is racist, or I am unbearably envious becomes the wealthy are engaged in class warfare against me!
Another characteristic of the unconscious is that it is timeless, in the sense that it can reverse temporal relations. For example, in the unconscious mind, if A is the cause of B, B can also be the cause of A. Thus, "before" and "after" become meaningless. Therefore, although we were inexcusably attacked by Islamists on 9-11, within minutes, leftists were saying that the real reason for the attack was that we had done something to offend Muslims.
Likewise, throughout the Cold War, leftist scholars wrote "revisionist" histories, in which the United States was the cause of the Cold War, or at least equally responsible for it. You will notice that there are no conservative revisionists who write, for example, that blacks were the cause of their own lynching, or that Japanese Americans were the cause of their own internment. You can only think in this manner if you are pathologically under the sway of unconscious symmetrical logic.
Also in the unconscious mind, there is no distinction between the memory of something that actually occurred vs. the memory of a fantasy. Here we can understand how and why the left is so prone to mythologizing the past, as their fantasies are mingled with reality.
Thus, no amount of reality and asymmetrical logic will ever convince them that FDR made the Great Depression worse, not better, or that the black family only began to disintegrate after the imposition of all the "Great Society" programs of the mid to late '60s. No amount of logic could convince a leftist that his policies harm the "little guy," since his ruling myth, in the face of all evidence to the contrary, is that he is here to rescue the hapless little guy (for whom the leftist always feels rich contempt in the unconscious mind, contempt which only seeps out everywhere).
One thing you will notice about the left is that they are passionate. Because the left is guided by feelings and intentions, they are blind to the results of their actions. If their feelings are infinitely good, then in the unconscious mind, the results must also be infinitely good.
As I have written before, this is a religious passion in the absence of religion, so it has no traditional means to structure and channel it. Just as religion partakes of symmetrical logic in an adaptive way (i.e., the meek shall inherit the earth, the Golden Rule, humans are made in the image of the Creator, etc.), leftists do so in a terribly unhealthy way. That is, because of the intensity of their feelings, these feelings reach way down into the symmetrical realm, with no way to structure or make sense of them. This is why you always see so much highly charged, "unfiltered" unconscious material coming out of the left. To borrow a metaphor from someone, reading dailykos or huffington post is like taking a ride through a sewer in a glass bottom boat.
As Bomford writes, the dictates of symmetrical logic mean that deductions "do not follow the path of fact, but of feeling or emotion." And although this inevitably leads to "crazy" deductions based upon a chain of feelings, in a sense, it is much more "free" than asymmetrical, Aristotelian logic. For example, the latter "has a deterministic feel. That is to say, it never delivers a new truth, though it may deliver truths that had not been clear before. Everything is already 'there' in the premises."
Not so symmetrical logic, which has considerably more freedom to "deduce." It can easily arrive at patent falsehoods while still obeying its own logic. For example, the knuckleheads at Columbia University believe that having a genocidal sociopath speak on their campus is an instance of defending "freedom of speech." I would agree, but only in a psychotically cluelessidal way, rooted in symmetrical logic. By the standards of normal logic, it makes no sense whatsoever. It's crazy.
One of the most fascinating aspects of unconscious logic is the way it can shift attributes from agent to agent. For example, as mentioned above, it is the work of a moment for a leftist to turn a perpetrator into a victim and a victim into a perpetrator, based upon the emotional needs of the day. For example, the standard leftist logic would be Larry Craig --> Homosexual --> Ultimate Victim. But place an "R" after the name, and the overriding logic becomes Republican --> Homophobic Victimizer --> Burn him!
Likewise, the normal train of leftist logic would be ROTC --> Don't ask, Don't tell policy --> Homophobia --> Get off our campus, fascists! But Ahmadinejad -- whose government's policy toward homosexuals is "don't tell, because we'll bury you alive" -- is given a pass because he shares the left's passionate hatred of America and of President Bush. Their interests converge in the deep, symmetrical unconscious. Ironically, it is obvious that Ahmadinejad is much more conscious of this than the left to which his manipulative talking points are tailored. You might say that he is consciously speaking to the left's unconscious, pushing every one of their happy buttons he can think of.
Don't believe me?
Daily Kos: 45% Want Ahmadinejad As US President
I Am a Jewish Lesbian, and I Have a Crush on Ahmadinejad:
"... the guy speaks some blunt truths about the Bush Administration that make me swoon... Okay, I admit it. Part of it is that he just looks cuddly. Possibly cuddly enough to turn me straight. I think he kind of looks like Kermit the Frog. Sort of. With smaller eyes.... I can't help but be turned on by his frank rhetoric calling out the horrors of the Bush Administration and, for that matter, generations of US foreign policy preceding...."
Homosexuality? In Iran? We don't allow it. Makes the goats jealous.


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I just wanted to add what else will go into the overall decorations.
The plan is to add what my wife calls my "Redneck Tiki Torch" which is simply a propane regulator attached to a valve and a long brass pipe with a tin can poked full of holes on top. It looks like a big ball of flames on the end of the pipe. in the grass alongside the tank will be an EMPTY, repeat EMPTY oxygen tank with the safety cap on it and another EMPTY CO2 tank. Inside the yellow barrel will be a green light with dry ice making green fog coming out.
If I can find one I plan on having a yellow rotating safety light, like those on top of a tow truck, flashing and that sound of an alarm blaring. (Same as the siren ring tone on the iPhone)
I will have video when it is all done from Halloween night.


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Heroin, cocaine and drug paraphernalia landed three people in the Brunswick County jail last week.
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text: The Synthesis CH2 Series of uninterruptible power supplies from Chloride Systems is available in four capacity ratings (600W, 1,000W, 1,500W, and 2,000W). The series accommodates advanced lighting systems by using double conversion continuous UPS technology. Standard features include an automatic battery system and remote communication capabilities. The standard battery run time listing is UL 924, 90 minutes; but a UL 924 auxiliary lighting and power listing increases battery run times by 10 or 20 minutes. The Synthesis CH2 Series allows for up to 12 single-pole output circuit breakers (without trip alarm) and up to six single-pole output circuit breakers (with trip alarm) for load distribution. chloridesys.com


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Traditional folding power chair which easily maneuvers and negotiates obstacles both inside and outside the home. Featuring an attractive silver vein and black finished frame with charcoal gray nylon upholstery, it disassembles and unfolds in seconds to a narrow 13" for easy transport. Maximum speed of 4mph with a turning radius of 36". Adjustable height back 17-19". Overall length 47", overall width 24.5". Features MK5 NX electronics. Batteries not included; uses two U1 Batteries, Item # ISGU1BAT sold separately. Off board charger. Overall weight 91lbs, weight capacity 250lbs. 3 year warranty on frame, 1 year on motor and electronics.


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All eyes are opened, or opening, to the rights of man.
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Thousands of child sex slaves uncovered in DC/NC ring...MEDIA BLACKOUT!
October 07, 2009, 03:18:58 PM »
Teens become prey in Charlotte sex trade
Human trafficking case shows the desperate plight of girls forced into prostitution.
By Franco Ordoñez
Posted: Sunday, Oct. 04, 2009
In his east Charlotte apartment less than a mile from Windsor Park Elementary, Jorge Flores Rojas created a religious shrine to a mystical figure known as the patron saint of death, who is said to protect pimps and other criminals.
Each day, Flores prayed to Santa Muerte, or "Saint Death," joined by the teenage girls whom he forced to have sex with as many as 20 men a day.
Flores, 45, was a notorious operator in a city that has become a center for sex trafficking along the East Coast.
Local and federal authorities are not sure how extensive the Charlotte sex rings have become. They say Flores' ring brought in hundreds of young women each year to work as prostitutes.
Flores was convicted of trafficking in April. But authorities say other pimps in Charlotte continue to prey on young girls from poor countries.
"I don't think we really realized how big this was," says Delbert Richburg, assistant special agent in charge of U.S. Immigration and Customs Enforcement's Office of Investigations in Charlotte. "We're probably just scratching the surface."
The growth is so extensive that this month ICE stationed a team of agents in Charlotte to focus on human trafficking, smuggling and exploitation. Across the Carolinas, immigrant sex rings have been broken up in Monroe, Durham and Columbia.
Jennifer Stuart, a staff attorney for Legal Aid of North Carolina, says her office has seen a "sharp increase" in trafficking case referrals the last few months.
Federal agents say Flores, an illegal immigrant from Mexico, picked up vans full of eight to 10 young women each week outside the McDonald's on West Sugar Creek Road near Interstate 85, where other traffickers had brought them. Others, he smuggled in directly from Latin America.
Two pairs of children's sneakers, pink and green, now sit outside Flores' old apartment off Sharon Amity Road near Eastland Mall. It was one of two apartments where, just a year ago, he hid his teenage victims.
Authorities say he brought customers there, but mostly took the girls to hotels and brothels set up around the city.
He favored teenagers because he could charge more. Clients paid $25 to $30 for 15 minutes with one of the girls. One teenage victim testified in court that, on many occasions, Flores would drive her to a house or an apartment where men would be waiting.
An undercover agent says the teenagers would be made to have sex with up to 100 men a week.
"I have daughters," he says. "... Every time I think of that number, it's something I can't fathom."
Trading in New York, D.C.
To keep a fresh cycle of women in Charlotte, Flores traded with traffickers, including relatives, in Washington, D.C., and New York.
In November 2007, court documents say, he "sold" at least two teenagers from Mexico to Yaneth Martinez, a D.C. madam, who advertised her services with cards offering "Hair Cuts for Men Only."
Martinez worked the girls in the capital city and gave Flores a cut of the profits. A month later, she returned them to him.
Their business relationship worked like this for more than a year, federal authorities said. Then, Flores took a liking to Martinez's teenage daughter.
He asked her if she'd work with him. She refused. Flores didn't give up.
He later called the girl's cell phone and asked her to meet him. He threatened to hurt her mother if she didn't.
She agreed to meet him. She hoped he only wanted to talk, but Flores threw her in his car, authorities said.
"'Sit there, don't say anything. Don't even try to look where we're going,'" agents said he told her.
Martinez tipped off a women's center in Washington that her daughter had been kidnapped. The center contacted authorities.
On Feb. 7, 2008, ICE agents stormed Flores' apartment in Charlotte. He wasn't there, but authorities arrested him a day later in Myrtle Beach. He had brought some of his victims to South Carolina because they had become "overused" in Charlotte, according to court records.
Martinez's daughter spent about three weeks as Flores' captive. Authorities say he raped her repeatedly. He forced her to have sex with dozens of men.
He stuffed her underwear in a small glass vase on his shrine. They prayed together to Santa Muerte.
If you run away, the saint will punish you, he told her.
Charlotte is particularly vulnerable to human trafficking. It's the largest city between Atlanta and Washington, D.C., at the junction of two interstate highways.
In addition, the size of the city's illegal immigrant community allows pimps like Flores to conceal their activities.
As with past waves of immigrants, many of the Latinos are men who left their families to find work. Many victims were lured here with promises of other jobs.
The women are often in the country illegally and dependent on their captors for food and shelter. They're easy to coerce.
"You don't have to have chains or bars to keep somebody under control," said John Price, a special agent with the FBI in Charlotte. "You can do it psychologically and emotionally, and that's typically what traffickers will use. It's a lot cheaper. It's a lot easier to threaten somebody. To beat them up."
Thousands of victims
The FBI estimates that some 18,000 people are trafficked into the United States for sex or forced labor. About a fourth end up in the Southeast; thousands come to the Carolinas.
Most victims of the sex rings are from Latin America, others from Asia and Eastern Europe.
One girl forced into prostitution thought she was coming to North Carolina to be a nanny, says Stuart of Legal Aid, which gives free legal services to low-income people.
Another 14-year-old from Mexico, who thought she was to work at a restaurant, was forced to have sex with men in Greenville, S.C., Columbia and Charlotte.
Martinez's daughter is like any other teenager, said her attorney Christopher Nugent of Washington. She enjoys her iPod and loves to shop. She often draws the dresses she'd like to wear.
In court, she asked if she could answer questions without looking at Flores.
His Charlotte attorney, Lucky Osho, said his client admits arranging women to have sex with men. But he said no one was kidnapped or forced to have sex against her will. Osho said Martinez's daughter was working for Flores in return for some of his women working for Martinez.
"It was part of the business," Osho told the Observer. "It was an exchange."
Flores told authorities that Martinez ran the sex ring.
Martinez's attorney, Lane Williamson, said his client did help Flores' girls find work, but that she did not coerce them. Williamson said Martinez was herself a victim, forced into prostitution earlier in life.
"This is something she was in, from her standpoint, as a matter of necessity," Williamson said. "The (women) were free to go and they did go on their own volition. That was not the case with Flores."
ICE is bringing in more agents and another supervisor to work on its new trafficking team. Victims will not be targeted for arrest or deportation, Special Agent Richburg said. Instead they will be offered special visas in exchange for their help prosecuting traffickers.
In April, Flores pleaded guilty to sex trafficking involving a minor and was sentenced to 24 years, after which he will be deported. He is currently in a federal prison in South Carolina. In July, Martinez pleaded guilty to transporting individuals for prostitution and was sentenced to time served. She will be deported to Honduras.
Martinez's daughter is doing much better, Nugent said. She's living with a foster family. She is getting a special green card for abused or abandoned children.
She wants to go to college and be a lawyer.
Two other girls found with Flores at the time of his arrest were also placed with foster families through a Charlotte women's center, authorities said.
The center arranged medical care and new clothes. ICE agents arranged work permits.
Before the permits arrived, the girls disappeared.
All eyes are opened, or opening, to the rights of man. The general spread of the light of science has already laid open to every view the palpable truth, that the mass of mankind has not been born with saddles on their backs, nor a favored few booted and spurred, ready to ride them legitimately
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Clippers silence Thunder to end playoff drought
(Reuters) - The streaking Los Angeles Clippers knocked Oklahoma City out of first place in the West with a forceful 92-77 victory on Monday that earned the hosts a first playoff berth in six years.
Nick Young led a balanced attack with 19 points off the bench, Blake Griffin added 17 and 11 rebounds and the Clippers overwhelmed the visiting Thunder in the second half to reach the post-season for the first time since the 2005-06 campaign.
"It feels great, especially to do it against one of the top teams in the league," Young told reporters. "I was too excited (after the game). I ran to the back and was slapping hands with everyone."
Winners in 12 of their last 14 games, the Clippers (38-23) also have a shot at their first ever division title, trailing the Lakers by a game in the Pacific Division, and for third in the West.
The Thunder (44-17), meanwhile, slipped percentage points behind San Antonio in their see-saw tussle for the top seed in the conference.
Kevin Durant had a team-high 24 points for Oklahoma City, who built a nine-point halftime lead before being outscored 49-25 in the second half.
It was their second defeat at the hands of the Clippers in less than a week following a two-point loss last Wednesday.
In the latest showdown, Los Angeles scored the game's first basket and did not lead again until the fourth quarter, and as the Clippers warmed up to pull away, the Thunder suddenly went cold and shot just 37 percent on the night.
All-Star Russell Westbrook had a particularly off game and finished with just nine points. Continued...


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DIV Expand/Collapse Script
This project was awarded to simplyrahul for $30 USD.
Get free quotes for a project like thisProject Budget
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10Project Description
Modify scripts (referenced below), or create new script as follows:
1. open a single div for a specific period of time.
2. close div automatically without end-user intervention after 30 seconds.
3. keep div open if end-user hovers over div with mouse; div closes automatically on mouseout.
4. if end-user leaves site, the aforementioned behavior occurs again.
Special Instructions:
1. If the scripts referenced below are not feasible solutions, Service Buyer has the option of writing a script to achieve the functionality/behavior as described herein.
2. The script must NOT rely upon client-side dependencies such as having cookies enabled. Server-side preferred. Service Buyer uses an Apache/php operating environment.
3. Must be able to place css-controlled border around div when div is in an "open" state.
4. Must have the ability to control div background color and disable color fade-in.
Script References:
dynamicdrive . c o m / dynamicindex17 / animatedcollapse . h t m
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Spry Collapsable Panel
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The ESD series was redesigned for increased efficiency and improved specific performance, as the one-to-one direct drive design includes a premium efficiency drive motor with three PT 100 temperature sensors. All models come standard with the new Sigma Control 2 intelligent compressor control for enhanced communications capabilities and equipment protection. Also standard is a variable speed fan for the fluid cooler that helps save energy by adjusting to cooling demands. Maintenance points are more easily accessible than ever before with five access doors and a removable panel.
Kaeser Compressors Inc., Fredericksburg, VA 877-586-2691 www.kaeser.com


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News: Encore Closes [1]
As has become an all-too familiar story [5], Encore [6] owners and siblings Paul and Aileen Reilly announced on Friday that failed lease negotiations forced the restaurant's closure. But the Reillys didn't go silently: They served huge crowds Friday, Saturday, and Sunday, with patrons flooding through the doors to show their support. I went for their last service—Sunday brunch—and the Colfax eatery was on a 90-minute wait. The first thing to sell out: The legendary bacon-wrapped jalapeño poppers.
The five-year-old Encore (which the Reillys purchased in 2010) has become more than a restaurant, it has become a community gathering spot and a platform for chef Paul's creative, sometimes tongue-in-cheek seasonal American cuisine.
The good news: The Reillys have been shopping locations (including the former JR's [7] space on 17th Avenue) for as long as a year and will be back in business. I just hope it's soon. In the meantime, I had my fill of the fantastic thin-cut fries with hot mustard drizzle (pictured) yesterday.
Follow Encore on Facebook [8] and Twitter [9] for information about the new location.


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Dysregulation of the complement cascade in the hSOD1G93Atransgenic mouse model of amyotrophic lateral sclerosis
© Lee et al.; licensee BioMed Central Ltd. 2013
Received: 1 August 2013
Accepted: 6 September 2013
Published: 26 September 2013
Components of the innate immune complement system have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS); however, a comprehensive examination of complement expression in this disease has not been performed. This study therefore aimed to determine the expression of complement components (C1qB, C4, factor B, C3/C3b, C5 and CD88) and regulators (CD55 and CD59a) in the lumbar spinal cord of hSOD1G93A mice during defined disease stages.
hSOD1G93A and wild-type mice were examined at four different ages of disease progression. mRNA and protein expression of complement components and regulators were examined using quantitative PCR, western blotting and ELISA. Localisation of complement components within lumbar spinal cord was investigated using immunohistochemistry. Statistical differences between hSOD1G93A and wild-type mice were analysed using a two-tailed t-test at each stage of disease progression.
We found several early complement factors increased as disease progressed, whilst complement regulators decreased; suggesting overall increased complement activation through the classical or alternative pathways in hSOD1G93A mice. CD88 was also increased during disease progression, with immunolocalisation demonstrating expression on motor neurons and increasing expression on microglia surrounding the regions of motor neuron death.
These results indicate that local complement activation and increased expression of CD88 may contribute to motor neuron death and ALS pathology in the hSOD1G93A mouse. Hence, reducing complement-induced inflammation could be an important therapeutic strategy to treat ALS.
C1q C4 Factor B C3 C5 CD55 CD88 Motor neuron disease NeuroinflammationBackground
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder characterised by selective loss of upper motor neurons within the motor cortex, and of α-motor neurons of the spinal cord and brainstem [1]. This results in symptoms of muscle weakness and atrophy of skeletal muscles, leading to paralysis and eventual death due to failure of respiratory muscles [2]. The mechanisms leading to ALS are still unclear but there are compelling data that suggest neuroinflammation may contribute to the disease progression of ALS [1, 3, 4]. These data include the presence of reactive microglia and astrocytes, infiltration of T lymphocytes and upregulation of cyclooxygenase 2 and prostaglandin E2 in the spinal cord of ALS patients and animal models [5–9]. The classical complement system is also implicated in ALS pathology, as studies have shown activation fragments of complement components C1q, C3 and C4 are increased in the serum, cerebrospinal fluid and neurological tissue (including spinal cord and motor cortex) of ALS patients [4].
In addition to evidence suggesting complement involvement in human ALS pathology, several studies have demonstrated the involvement of complement factors in animal models of ALS. Upregulation of the classical pathway complement components C1q and C4, as well as of the central factor C3, has been shown in human SOD1 transgenic rodent models of ALS [4]. Other studies have also shown upregulation of the major proinflammatory C5a receptor, CD88, during disease progression [10, 11]. In addition, our group has shown that chronic administration of a specific CD88 antagonist in hSOD1G93A transgenic rats delayed the onset of motor symptoms and increased survival compared with untreated animals [11]. Overall, these studies indicate that overactivation of the complement system and increased C5a-CD88 signalling contribute to the progression of disease in these animal models of ALS.
In the present study, we examined the expression and cellular location of major complement factors and regulators during defined disease stages in hSOD1G93A mice in order to provide a comprehensive overview of complement's involvement in ALS. Additionally, given the importance of C5a in disease pathology in ALS models [10, 11], we also examined mRNA, protein levels, and the cellular localisation of C5, C5a and its cognate receptor, CD88, during disease progression. Our findings demonstrate a global dysregulation of complement, as disease progressed in these murine models of human ALS.
Ethical statement
All experimental procedures were approved by the University of Queensland Animal Ethics Committee (Permit Number: 227–09), and complied with the policies and regulations regarding animal experimentation and other ethical matters [12]. They were conducted in accordance with the Queensland Government Animal Research Act 2001, associated Animal Care and Protection Regulations (2002 and 2008), and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th Edition (National Health and Medical Research Council, 2004). ARRIVE guidelines have been followed in the preparation of the manuscript.
Different stages defined in amyotrophic lateral sclerosis
Stage | Age | Phenotype |
Pre-symptomatic | 30 days postnatal | No signs of motor deficit |
Onset | 70 days postnatal | Initial signs of motor deficit (grip strength) |
Mid-symptomatic | 130 days postnatal | Weakness in hind-limb and tremor when suspended by the tail |
End | 150 to 175 days postnatal | Full paralysis of lower limbs and loss of righting reflex |
Weight measurements and behavioural tests
hSOD1G93A and WT mice were weighed weekly at the same time of day (4:00 p.m.), from 42 days of age until end-stage when they lose their righting reflex. Two neuromotor tests, the Rota-rod and hind-limb grip strength test, were conducted on hSOD1G93A and age-matched WT mice (detailed below). These tests were performed blinded to genotype.
Rota-rod test
Mice were tested for their motor coordination from 42 days of age using Rota-rod apparatus (Ugo Basile, Comerio, VA,Italy) at a constant speed of 20 rpm. Each mouse was given three attempts and the longest latency to fall was recorded; 180 seconds was chosen as the arbitrary cutoff time. One week prior to the test, mice were trained twice to remain on the Rota-rod apparatus to exclude differences in motivation and motor learning. In the training phase, mice were placed on the Rota-rod at a constant speed of 20 rpm for a maximum duration of 240 seconds [13].
Hind-limb grip strength test
A digital force gauge (Ugo Basile) was used to measure maximal muscle grip strength. The mice were held by their tail and lowered until the mice grasped the T-bar connected to the digital force gauge with their hind limbs. The tail is lowered until the body is horizontal and the mice are pulled away from the T-bar with a smooth steady pull until both of their hind limbs released the bar. The strength of the grip was measured in gram force. Each mouse was given 10 attempts and the maximum grip strength was recorded.
hSOD1G93A and WT mice were fixed by transcardiac perfusion with 2% sodium nitrite buffer (Ajax Finechem Pty Ltd, Cheltenham, VIC, Australia) followed by 4% paraformaldehyde (Sigma, St. Louis, MO, USA) in 0.1 M phosphate buffer pH 7.4. The lumbar spinal cords were collected and then placed in 4% paraformaldehyde for another 2 hours at 4°C. The lumbar spinal cords were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA,) and snap frozen in liquid nitrogen. Serial transverse cryosections (16 μm) were collected on Superfrostplus slides (Menzel-Glaser, Braunschweig, Germany) for estimation of motor neuron numbers and fluorescence immunohistochemistry.
For the motor neuron numbers in the spinal cord, the sections are stained using 0.1% thionin (v/v) in acetic acid buffer solution (Sigma) for 3 minutes and processed as per our previous studies [14]. The lumbar lateral motor column extending from the second lumbar dorsal root ganglia to the sixth lumbar dorsal root ganglia was selected from our serial spinal sections, with the aid of the mouse spinal cord atlas [15]. Alpha-motor neurons within the lumbar lateral motor column were identified and counted on both sides of the spinal cord in every 10th section following previous guidelines [16–18]. The mouse genotypes were not made available to the researcher (JDL) conducting the counts until it was completed.
Summary of antibodies used for immunohistochemistry
Antibody | Manufacturer | Dilution | In combination with |
Rat anti-mouse C1q | Hycult Biotechnology1 | 1:1,000 | GFAP, Iba-1 and ChAT |
Rat anti-mouse C3b | Hycult Biotechnology1 | 1:50 | GFAP, Iba-1 and ChAT |
Mouse anti-mouse C5 | Hycult Biotechnology1 | 1:1,000 | GFAP, CD11b, ChAT |
Rat anti-mouse CD88 | AbD Serotec2 | 1:250 | GFAP, Iba-1, ChAT |
Mouse anti-mouse GFAP | BD Biosciences3 | 1:1,000 | C1q, C3b, C5, CD88 |
Rabbit anti-mouse Iba-1 | Wako4 | 1:400 | C1q, C3b and CD88 |
Rat anti-mouse CD11b | Abcam5 | 1:300 | C5 |
Goat anti-mouse ChAT | Chemicon6 | 1:100 | C1q, C3b, C5, CD88 |
Quantification of immunofluorescence for C1q and C3b was performed on ~25 to 35 lumbar spinal cord sections (per animal; n = 3) spaced 160 μm apart and expressed as the percentage immunoreactive area per section. Staining procedures and image exposures were all standardised between genotype and between sections.
Real-time quantitative PCR
Taqman probes used for quantitative PCR
Gene of interest | Catalogue number |
C1qB | Mm01179619_m1 |
C4 | Mm00437896_g1 |
Factor B | Mm00433909_m1 |
C3 | Mm01232779_m1 |
CD55 | Mm00438377_m1 |
CD59a | Mm00483149_m1 |
C5 | Mm00439275_m1 |
CD88 | Mm00500292_s1 |
Glyceraldehyde-3-phosphate dehydrogenase | Mm99999915_g1 |
Mannose binding lectin 1 | Mm00495413_m1 |
Mannose binding lectin 2 | Mm00487623_m1 |
Western blot analysis
Lumbar spinal homogenates from hSOD1G93A and WT mice were resolved on a 10% SDS-PAGE gel and electrotransferred onto nitrocellulose membranes. Membranes were blocked in 2.5% skim-milk–Tris-buffered solution–0.1% Tween 20 for CD88 and 5% BSA–Tris buffered solution–0.1% Tween 20 for CD55 and were incubated overnight with one of the following antibodies; anti-CD88 (1:1,000; BMA Biomedical, Augst, Switzerland), or anti-CD55 (1:1,000; Hycult Biotechnology). All primary antibodies were diluted in 5% BSA–Tris buffered solution–0.1% Tween 20. Anti-CD88 was detected with goat anti-chicken horseradish peroxidase (HRP) (1:15,000; GE Healthcare, Pittsburgh, PA, USA) and anti-CD55 was detected with goat anti-rat HRP (1:10,000; GE Healthcare). These secondary antibodies were detected by enhanced chemiluminescence (ECL; Amersham, Pittsburgh, PA, USA). Blots were stripped and re-probed with anti-GAPDH, (1:15,000; Millipore, Billerica, MA, USA) and detected with sheep anti-mouse HRP (1:4,000; GE Healthcare) to ensure equal protein loading. Densitometric analyses of immunoreactive bands were performed by deducting background pixels from the grey-scale pixel density of the band multiplied by the area value using Image J software [20]. The integrated pixel value for each band was normalised to its corresponding anti-GAPDH band. The normalised integrated pixel values of hSOD1G93A bands were compared with WT bands.
In situhybridisation
Synthesis of digoxigenin-labelled probes was performed using digoxigenin RNA labelling mix according to the manufacturer's instructions (Roche, Brisbane, QLD, Australia) using PCR-amplified cDNA templates generated with primers specific for CD88: forward, TAATACGACTCACTATAGGGATCATCTACTCGGTGGTGTTCC and reverse, AATTAACCCTCACTAAAGGGGAGAGACCTTAGGAGTCGTCCA. Lumbar spinal cords from hSOD1G93A and WT mice were collected and fixed overnight in 4% paraformaldehyde at 4°C. Samples were processed and embedded in optimal cutting temperature compound (Sakura Finetek), sectioned at 16 μm and probed with CD88 riboprobes and sense control as previously described [21].
Enzyme-linked immunosorbent assay
Ninety-six-well plates (Greiner Bio-One, Frickenhausen, Germany) were precoated with monoclonal rat anti-mouse C5a capture antibody (Clone I52 – 1486; BD Pharmingen, San Diego, CA, USA) diluted in coating buffer (100 μM, NaHCO3, 34 μM Na2CO3, pH 9.5) overnight at 4°C in a sealed humidified container. This capture antibody is specific for a neo-epitope exposed only in mouse C5a/C5a desArg and does not cross-react with C5 [22, 23]. Following the plate being blocked for 1 hour at room temperature with assay diluent (10% FCS/PBS), C5a standard and lumbar spinal cord homogenates was incubated for 2 hours at room temperature. The plates were subsequently incubated with biotinylated rat anti-mouse C5a detection antibody (clone I52-278; BD Pharmingen) for 1 hour at room temperature, and then incubated with streptavidin–HRP conjugate for 30 minutes at room temperature. Tetramethylbenzidine (Sigma) substrate was used as the chromogen and the plate was read at 450 nm. Levels of C5a in lumbar spinal cord samples were adjusted to micrograms per protein and expressed as nanograms of C5a per microgram of protein.
Statistical analysis
All measures were performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The statistical differences between hSOD1G93A and WT mice for body weight, Rota-rod test and hind-limb grip strength test were analysed using a two-tailed Student's t test at each time point. For the results from motor neuron counts, quantitative real-time PCR, western blotting, ELISA, statistical differences between hSOD1G93A and WT mice were analysed using a two-tailed t test at each stage of disease progression. All data are presented as mean ± standard error of the mean and differences were considered significant when P ≤0.05.
Motor deficits in hSOD1G93Amice correlate with lumbar motor neuron loss during disease progression
To monitor the decline in neuromotor performance and loss of motor neurons during ALS progression in hSOD1G93A mice, body weights, motor behavioural tests and motor neuron counts were performed. The onset of disease was defined as a stage in which a neuromotor deficit was measurable. In this study, hSOD1G93A mice showed a decrease in their body weight, hind-limb grip strength and Rota-rod performance when compared with WT mice. The weight of the hSOD1G93A mice reached the maximum at 133 days of age and was significantly decreased when compared with WT mice at 140 days (mean body weight, hSOD1G93A = 20.4 ± 0.23 g and WT = 22.3 ± 0.25 g, n = 9, * P < 0.05, #P < 0.001; arrow in Additional file 1: Figure S1A).
The Rota-rod is a widely used measure of neuromotor performance in hSOD1G93A mice [24]. In our study, both hSOD1G93A and WT mice remained on the Rota-rod for the full duration of the test until 119 days when hSOD1G93A mice showed ~30% reduction in the time remained on the Rota-rod (n = 12, *P < 0.05, #P < 0.001; arrow in Additional file 1: Figure S1B). Next, we measured maximal hind-limb grip strength as an alternate measure of neuromotor function [25, 26]. At 70 days, hSOD1G93A mice showed a significant reduction in grip strength (~35% reduction; n = 12, *P <0.05, +P <0.01, #P <0.001) when compared with WT mice (arrow in Additional file 1: Figure S1C). Our results suggest that the hind-limb grip strength test is a more sensitive measure of detecting motor deficit symptoms in hSOD1G93A mice compared with weight loss and Rota-rod performance [26]. The Rota-rod test mainly evaluates balance and coordination, and does not necessarily reflect muscle denervation (that is, loss of muscle function [27]). By contrast, the decline in grip strength in the hSOD1G93A mice closely correlated with the onset of lumbar motor neuron loss at 70 days (n = 6, ***P <0.001; in Additional file 1: Figure S1D). Post 70 days, we observed further declines in hind-limb grip strength presumably resulting from a progressive drop in lumbar motor neuron numbers up to end stage (in Additional file 1: Figure S1C, D).
Components of the classical and alternate pathways of complement are upregulated along with decreased expression levels of complement regulators in hSOD1G93Amice
Previous studies have identified various members of the complement system that are upregulated in ALS and in ALS animal models, but it is unclear which of the major complement pathways are being activated. To investigate this further, we examined the mRNA and protein levels for some of the key initiators of the complement pathways, the classical, alternate and lectin pathways, as well as the major complement regulators CD55 and CD59a [28].
The regulators of complement system CD55 and CD59a were also investigated, because they are important in maintaining homeostasis and keeping the complement system in its proper physiological state. Specifically, CD55 and CD59a negatively regulate complement activation by accelerating C3 convertase decay and inhibiting the assembly of membrane attack complex respectively [29, 30]. CD55 mRNA expression was initially increased by 1.4-fold at pre-symptomatic, and decreased at mid-symptomatic and end stage of disease by 0.4-fold and 0.5-fold respectively when compared with WT (n=6, *P <0.05 and ***P <0.001; Figure 2A). This was confirmed at protein level using western blotting, where a 41 kDa CD55 immunoreactive band was observed in all stages of hSOD1G93A mice and their respective WT mice (Figure 2B, upper panel). Semi-quantitative densitometry analyses of these bands with respect to GAPDH loading controls (Figure 2B, lower panel), revealed increased CD55 protein in the lumbar spinal cord of hSOD1G93A mice by 3.2-fold at pre-symptomatic and decreased by 0.7-fold at mid-symptomatic and 0.6-fold at end stage respectively when compared with WT mice (n = 4, *P <0.05 and ***P <0.001; Figure 2C). CD59a mRNA was also increased initially at onset by 1.3-fold and decreased at end stage of disease by 0.2-fold when compared with WT mice (n = 6, *P <0.05 and **P <0.01; Figure 2F).
Collectively, the above results suggest that regulation of complement system is perturbed, which leads to activation of classical and alternate pathways of complement system in the lumbar spinal cord of hSOD1G93A mice, which may contribute to the disease progression of ALS.
C5 is expressed by motor neurons but is not altered in hSOD1G93Amice
Next, we immunostained lumbar spinal cords from hSOD1G93A and WT mice for C5 with specific cellular markers for motor neurons (anti-ChAT), astrocytes (anti-GFAP), and microglia (anti-CD11b). C5 was clearly present in ChAT-positive lumbar motor neurons from end-stage hSOD1G93A and WT mice (white arrows in Figure 4C, D), but not in GFAP-positive astrocytes (Figure 4E, F). For microglia, we did not see any C5 in CD11b-positive microglia in WT spinal cords (Figure 4G), but we did see some activated microglia (enlarged cell shape with thickening of proximal processes and decrease in ramification of distal branches [32]) expressing low amounts of C5 in the spinal cords from end-stage hSOD1G93A mice (white arrows; Figure 4H).
CD88 is upregulated during disease progression in hSOD1G93Amice
Previous studies have shown an increase in CD88 expression in multiple rodent models of ALS [10, 11]; hence this study aimed to investigate whether there were similar differences in expression of CD88 between hSOD1G93A and WT mice during disease progression of ALS.
CD88 is localised to motor neurons and activated microglia with minimal localisation to astrocytes in hSOD1G93Amice
Next, we aimed to determine the cellular localisation of CD88 that has contributed to the increased expression seen in hSOD1G93A mice. To achieve this, we performed immunolabelling for CD88 on lumbar spinal cord sections from hSOD1G93A mice and WT mice. These sections were immunostained for CD88 and with specific cellular markers to identify motor neurons (anti-ChAT), microglia (anti-Iba-1) and astrocytes (anti-GFAP).
In WT mice, CD88 staining was observed on lumbar motor neurons (Figure 5D). CD88-stained cells were readily identified as motor neurons due to their large size, location and distinctive morphology [17]. This was confirmed by double-labelling with the motor neuron marker ChAT (white arrows in Figure 5F). CD88 immunostaining was localised predominantly to the motor neurons' soma (Figure 5D, E, F). To further support these immunohistochemical findings, we confirmed that motor neurons expressed CD88 mRNA transcripts by in-situ hybridisation (Figure 5D′, inset). Next, we examined whether CD88 immunostaining was present on surrounding microglia and astrocytes in sections of WT lumbar spinal cords. We observed minimal co-localisation of CD88 to Iba-1-positive microglia (white arrows in Figure 5G,I), but none to GFAP-positive astrocytes (Figure 5J, K, L).
Following this demonstration of CD88 immunolabelling in WT lumbar spinal cord, we then investigated CD88 cellular localisation in hSOD1G93A mice. CD88 was also expressed on the few remaining motor neurons seen at the end-stage of disease (white arrow in Figure 5O). By contrast to WT lumbar spinal cords, we observed prominent CD88 immunostaining on other cellular structures surrounding motor neurons in the lumbar spinal cords of hSOD1G93A mice by the end stage of disease (for example, yellow arrows in Figure 5M, O).
This additional CD88 immunoreactivity was investigated in hSOD1G93A end-stage mice, using microglia marker Iba-1 and astrocyte marker GFAP. By contrast to Iba-1-positive microglia in WT mice, where these cells expressed little observable CD88 and appeared to be nonactivated (that is, small size with slender processes; [32]; Figure 5H, I), Iba-1-positive microglia in hSOD1G93A mice demonstrated an activated morphology with increased expression of CD88 (white arrows in Figure 5P, R).
As expected, astrocytes were seen to increase in numbers as disease progressed in hSOD1G93A mice [33]. This was noted by increased GFAP immunolabelling in the lumbar spinal cord of hSOD1G93A mice at end stage compared with WT mice (Figure 5K, L for WT, and Figure 5T, U for hSOD1G93A mice). Minimal CD88 co-localisation to GFAP-positive astrocytes was observed in WT mice (Figure 5J, K, L) and at the end stage of disease in hSOD1G93A mice (Figure 5S, T, U).
While the pathogenesis of ALS is still unclear, there is persuasive evidence that complement factors are involved in promoting disease progression. Previous studies have demonstrated that C1q and C3 mRNA expression are significantly increased during ALS progression in hSOD1G93A mice [4]. In addition, upregulation of CD88 has also been observed in numerous neurodegenerative diseases in rodents [34–37], including ALS [10, 11], so it is plausible to propose that the complement system could be involved in the pathophysiology of ALS. The present study demonstrates that components of the classical and alternative complement pathways are upregulated during the course of disease progression in hSOD1G93A mice, and that C5a receptor CD88 expression level is also increased. In addition, we found a reduction in two major regulatory inhibitors of complement activation as the disease worsened, which is suggestive of a progressive dysregulation of complement in this model. Furthermore, we show that C5, the precursor of C5a, is expressed predominantly by motor neurons, suggesting that diseased motor neurons could be a major source of C5a generation during disease progression. This local complement self-signalling in the central nervous system might therefore contribute to motor neuron death in hSOD1G93A mice as shown previously for cortical neurons [22]. Taken together, our results indicate that motor neurons may generate C5a under stress, and that this may promote self-damage under disease conditions that exist in ALS.
Classical and alternate complement pathways are activated in ALS progression in hSOD1G93Amice
The present study provided evidence for the activation of classical (C1qB and C4) and alternate (factor B) pathways of the complement system in the lumbar spinal cord of hSOD1G93A mice during ALS disease progression. This is consistent with numerous studies in mouse models of ALS and human patients where increased levels of C1q, C3 and C4 have been found [4, 38]. Furthermore this study also extended upregulation of C1qB and C3 mRNA expression in previous studies to protein levels and localisation where C1q and C3b immunolabelling was increased in hSOD1G93A mice and expressed on motor neurons and microglia compared with WT. This may suggest that upregulation of these components could assist in the removal of dying motor neurons via opsonisation, during disease progression in hSOD1G93A mice [39].
It is also possible that cell fragments or protein aggregates from dying motor neurons could lead to complement activation in the degenerating spinal cord [28]. In our study, complement activation was seen at disease onset (P70) and was restricted to the areas of motor neuron death in the spinal cord of hSOD1G93A mice. Other studies have demonstrated that complement components C1q and C3b are located at the neuromuscular junction during the early stages of disease (P47) in hSOD1G93A mice [40]. These findings are consistent with the idea that C1q and C3b may contribute to the cellular destruction of motor nerve terminals in these mice [41–43]. Taken together, these findings and our own are consistent with the hypothesis that the early loss of motor neuron terminals is followed by the subsequent death of motor neurons within the spinal cord. However, there is current debate about the initiating site of degeneration in the cortical-motor system (upper motor neurons [44]) versus peripheral (neuromuscular junction [41]). Future studies could contrast complement activation temporally at these different sites to determine the initiating site of complement-mediated neurodegeneration.
We also showed decreased mRNA expression levels of complement regulators CD55 and CD59a at later stages of the disease, which suggests that the homeostasis of the complement system is perturbed, which may lead to dysregulation and overactivation of the complement system. This supports other studies, which have shown that deficiency in CD55 and CD59a exacerbates neuronal degeneration [45–47]. Our findings are also consistent with those of Heurich and colleagues [40], where decreased (but nonsignificant) levels of CD55 mRNA were observed during the later stages of disease in hSOD1G93A mice. In the present study, we also confirmed the mRNA changes in CD55 at the protein level, which similarly showed decreased CD55 levels at later stages of disease. Interestingly, we also observed an initial increase in the mRNA expression levels of CD55 and CD59a during early stages of disease. This may indicate an early negative feedback mechanism to delay the activation of complement in host cells, but this needs further investigation.
Collectively, our data add further support to the notion that complement activation may play an important role in accelerating motor neuron loss and ultimately in progression of ALS.
C5 is expressed by wild-type and hSOD1G93Amotor neurons during disease progression
C5, the precursor of C5a, is expressed by motor neurons in both WT and hSOD1G93A mice. This suggests that motor neurons are a major source of C5 generation in this tissue. We recently showed murine cortical neurons also expressed endogenous C5, and generated C5a in response to ischaemia, which contributed to neuronal cell death [22]. It is plausible the same phenomenon is occurring in the hSOD1G93A mouse, where stressed and dying motor neurons generate their own C5a, to act in an autocrine fashion by binding to CD88, present on these neurons, to promote their death. Indeed, C5a has been suggested to directly cause neuronal cell death in a separate model of ALS [10]. C5a protein level in the lumbar spinal cord only appeared to be significantly decreased by end stage in hSOD1G93A mice when compared with WT mice. This could be due to fewer motor neurons (a source of C5/C5a), and the increased CD88 receptor levels by surrounding activated microglia as disease progressed. Increased CD88 on these cells would act to internalise and degrade C5a post activation of its receptor [48], which could account for the decline in C5a levels over time. The consequence of C5a–CD88 signalling in WT motor neurons is not yet fully understood and will need further investigation.
C5a receptor CD88 is upregulated during disease progression in hSOD1G93Amice
The present study provided evidence for a pathophysiological role of CD88 in hSOD1G93A mice. Specifically, a significant increase in CD88 protein was observed at mid-symptomatic and end stage in hSOD1G93A mice. This is in agreement with our previous studies conducted in hSOD1G93A rats [11]. The increase in CD88 protein in hSOD1G93A mice also parallels observations in other models of neurodegenerative diseases, such as Huntington's disease and Alzheimer's disease [35, 36]. Taken together, this suggests that complement activation is a generalised response to neuronal injury in neurodegenerative diseases. Furthermore, the ability of CD88 antagonists to attenuate both neurodegeneration and disease progression in rat models of ALS and Huntington's disease, and in mouse models of Alzheimer's disease, further suggests that increased CD88 activation actively contributes to neurodegeneration [11, 37, 49].
In addition to an increase in CD88 in hSOD1G93A mice, the present study observed CD88 on motor neurons in WT mice, as well as in hSOD1G93A mice. The fact that CD88 was found on WT motor neurons suggests that it may play a non-inflammatory role in motor neuron function. Indeed, studies including our own, have shown CD88 is also present on other neurons within the brains of WT adult mice, including pyramidal neurons in the CA subfields of the hippocampus and neocortex, and Purkinje cells in the cerebellum [50, 51]; and CD88 has also been documented to be expressed on human motor neurons [10]. Hence the physiological significance of C5a receptor presence in motor neurons awaits further study.
Previous studies have shown that motor neuron death in animal models of ALS is exacerbated by toxic signals emanating from non-neuronal neighbouring cells (astrocytes and microglia), via an inflammatory response that accelerates disease progression [52, 53]. The present study also showed upregulation of CD88 on activated microglia, but minimal expression on astrocytes in hSOD1G93A mice. The latter is in contrast to our previous study conducted in hSOD1G93A rats, where CD88 was expressed primarily on proliferating astrocytes [11], but is in support of other studies that show CD88 on microglia in other neurodegenerative diseases [36, 54] The differential expression of CD88 on these proliferating glial cells between hSOD1G93A rat and mouse models may suggest a differential role for CD88 in these two species.
The strong co-localisation of CD88 with activated microglia in hSOD1G93A mice suggests that CD88-activated microglia contribute to the propagation of disease as opposed to the aetiology of the disease. This is supported by previous studies where transplanted WT microglia produced no delay of disease onset but survival was greatly extended through slowing of disease progression in hSOD1G93A mice [55]. The exact mechanism by which C5a–CD88 signalling in microglia plays a role in neurodegeneration is still unknown, but may involve the release of reactive oxygen species through NADPH oxidase, or proinflammatory cytokines, which have been shown to be upregulated in ALS [56].
In summary, the present study has demonstrated the upregulation of classical and alternative pathway complement components, together with decreased levels of complement regulators, suggesting that complement activation and/or its dysregulation may play an important role in motor neuron loss and ultimately in progression of ALS. Expression of C5a receptor CD88 was upregulated in hSOD1G93A mice, and the increased expression of CD88 in end-stage hSOD1G93A mice appears to be due to increased microglial CD88 expression. Taken together, these results indicate that CD88 may play an important role in the pathophysiology of ALS. These results pave the way for preliminary pharmacological experiments using specific downstream complement inhibitors in hSOD1G93A mice, such as C5 inhibitors or antagonists to C5a receptor that could conceivably be extended to humans with positive therapeutic outcomes.
Amyotrophic lateral sclerosis
Bovine serum albumin
Enzyme-linked immunosorbent assay
Foetal calf serum
Glyceraldehyde-3-phosphate dehydrogenase
Glial fibrillary acidic protein
Horseradish peroxidase
Phosphate-buffered saline
Polymerase chain reaction
Wild type.
The authors would like to thank Maryam Shayegh and Mary White for their technical support and David Simmons for assistance in in situ hybridisation. JDL holds an APA scholarship from the Australian government. The work was funded by project grant APP1004455 from NHMRC to PGN and TMW, and by a MNDRIA project grant to PGN, SMT and TMW. BJT is supported by grants APP1008910 from NHMRC and MNDRIA, and JDA by grants APP1006141 and APP1030513from the NHMRC and grants from MNDRIA.
Authors' Affiliations
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A unified quality health care system which provides equal access to HIV prevention and treatment services for all people
To ensure that every person living with HIV has access to quality comprehensive prevention and treatment services to live a healthy life
Strategic Approach
TAC will achieve its mission by –
Overview of TAC
Founded on 10 December 1998 in Cape Town, South Africa, The Treatment Action Campaign (TAC) advocates for increased access to treatment, care and support services for people living with HIV and campaigns to reduce new HIV infections. With more than 16,000 members, 267 branches and 72 full time staff members, TAC has become the leading civil society force behind comprehensive health care services for people living with HIV&AIDS in South Africa. Since 1998, TAC has held government accountable for health care service delivery; campaigned against official AIDS denialism; challenged the world's leading pharmaceutical companies to make treatment more affordable and cultivated community leadership on HIV and AIDS. Our efforts have resulted in many life-saving interventions, including the implementation of country-wide mother-to-child transmission prevention and antiretroviral treatment programmes. For our efforts TAC has received world-wide acclaim and numerous international accolades, including a nomination for a Nobel Peace Prize in 2004. On 30 August 2006 the New York Times named TAC, "the world's most effective AIDS group".
Together TAC's programmes facilitate access to comprehensive HIV and TB prevention, treatment, care and support services; provide scientific literacy training on prevention and treatment on an increasingly mass scale; coordinate, distil and disseminate scientific research and policy developments on HIV&AIDS and TB at the community grass-roots level ; and organize and advocate with member communities to mobilise and take up issues of health policy and service delivery.
TAC's Strategic Objectives
The TAC's objectives are described in the organisation's Constitution and are as follows:


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Featured Blu-ray / DVD Review: Tinker Bell: Secret of the Wings
October 21st, 2012
Tinker Bell: Secret of the Wings - Buy from Amazon: DVD or Blu-ray Combo Pack, or 3D Combo Pack
The Character of Tinker Bell is one of the most famous is all of animation history. First appearing in Peter Pan almost 60 years ago, the character became the defacto mascot for Walt Dinsey since then. In 2008, Tinker Bell was released direct-to-DVD and despite some trepidation, the film earn amazing reviews and was a big hit on the home market. This year, the four direct-to-DVD release comes out, but will Tinker Bell: Secret of the Wings live up to the previous releases? Or is the franchise getting tired.
The film begins with Tinker Bell and two of her fellow Tinker fairies, Clank and Bobble , weaving baskets. They are to be taken to the Winter Woods where they will collect the snow needed to change the season from fall to winter. Tinker Bell would love to go into the Winter Woods, but the others warn her against doing that. Bobble says its too cold, while Clank warns her about the stealthy glaciers. The dire warnings are nothing compared to seeing the snowy owls fly to the Winter Woods.
The basket weaving is interrupted when a runaway bunny leaps through the Tinker Fairies' workshop. Fawn (Angela Bartys) is trying to get all the hibernating animals to the Winter Woods and this one bunny is a bit of a handful. Tinker Bell immediately offers to help, but is disappointed to learn they won't get a chance to go to the Winter Woods. No Warm Fairies can go into the Winter Woods, just like to Winter Fairies are not allowed to leave. It's the rules of Lord Milori, the lord of Winter. However, as anyone who watches these movies knows, Tink isn't one to let rules get in her way and while Fawn is distracted, she leaps into the Winter Woods. When she crosses, she doesn't freeze, in fact, she kind of likes the cold and her wings begin to sparkle. Before she can get any deeper into Winter, Fawn "rescues" her and brings her back.
Once back home, Tinker Bell is sent to the doctor to make sure she didn't damage her wings, but no one believes her about the sparkling. She tries to look up what sparkling wings mean at the library, but a bookworm ate the pages out of the book. When she asks the librarian if he knows, he tells her only the Keeper knows that. The Keeper wrote all of the books and keeps all of the knowledge about fairies. However, The Keeper is a Winter Fairy. With that, Tinker Bell is even more determined to go to the Winter Woods. This time, she sneak into one of the baskets that is to be delivered to the Winter Woods. She makes it all the way to to the Keeper, but before she can talk to him, another fairy comes to see him. Periwinkle is worried because earlier that day, her wings started to sparkle and they are starting to sparkle again. This is when she learns...
At this point we have a bit of a spoiler. It turns out both Tinker Bell and Periwinkle were created from the same baby's laugh. When a baby laughs for the first time, a new fairy is created, first in the form of a little light, then as a dandelion seed, then as a full fairy. But this time, the light split into two, but one traveled to Pixie Hollow and became Tinker Bell and the other blew into the Winter Woods and became Periwinkle. This means the two are sisters.
The pair spend the day with Periwinkle's friends, Gliss and Spike (Debby Ryan). That night Tinker Bell has to go back home, because it really is too dangerous for a Warm Fairy in the Winter Woods. However, when they say goodbye, Tinker Bell reveals she has a plan to help Periwinkle cross into Pixie Hollow to see her world. Tinker Bell will create, with the help of her friends, a snow machine to help keep Periwinkle cold long enough to get to visit all of Pixie Hollow. It works... for a little bit. However, it becomes clear that the warmth of Pixie Hollow isn't just uncomfortable for Periwinkle, it could melt her wings. She barely gets back to Winter Woods in time. And when she gets there, she greeted by Lord Milori, who is not happy the fairies broke the rules. He again says that that the two worlds must remain separate and when Tinker Bell objects to his rule, Queen Clarion arrives to say it's her rule. But why would Queen Clarion make such a rule?
As Lord Milori leaves the border between the two worlds, he flies upon his snowy owl and knocks the snow machine into the river hoping to destroy it. However, instead it gets stuck with the water moving the pedals and ice building up on the front, it begins to make snow. It begins to make a tremendous amount of snow. This can't be good.
When I first heard of the plans to create a direct-to-DVD franchise based around Tinker Bell, I was cautiously optimistic. Walt Dinsey's direct-to-DVD films have had a bad reputation, and for good reason, as most of them were terrible. However, the first film wasn't just good compared to the average direct-to-DVD Disney film, it was good enough that I thought it could have had a theatrical release. Since then, each of the films have maintained this high quality level, including Tinker Bell: Secret of the Wings.
The movie has all of the strengths of the previous films, including a terrific story that is filled with heart and adventure. Maybe the movie has a little too much plot. There's a lot crammed into the film's 75-minute running time. We have the initial plot about preparing for winter and Tinker Bell wanting to go into the Winter Woods. We have her meeting Periwinkle and all that entails. We have her learning about the dangers of Warm Fairies and Winter Fairies crossing over. Then we have the final adventure portion of the film. That's a lot to deal with. Fortunately, the characters and the world have been firmly established, so we can get right into the plot and don't need to worry about setting up characters or motivations. We know why Tinker Bell would be so willing to break the rules for an adventure, because that's just who she is. We get a few more characters added into the mix, but only Periwinkle gets much screen time. The animation is also excellent and is much closer to theatrical quality than the quality of the average direct-to-DVD release. Granted, it's not up to the level of Brave, for instance, but it never looks cheap.
Overall, Tinker Bell: Secret of the Wings is absolutely worth checking out and is a worthy addition to one of the best Direct-to-DVD franchise around.
Extras start with Pixie Hollow Games, which is a 22-minute TV special based on the franchise. In the short film, we are introduced to a new garden fairy, Chloe, who is training to compete in the Pixie Hollow Games, the fairy equivalent to the Olympics. Rosetta (Megan Hilty) dismisses their chances and the Garden Fairies are on a major losing streak, so much so that none of the other fairies are even interested in competing and Chloe's teammate will be chosen at random from a basket. It's Rosetta. After trying to get out of it, she relents. She's not too worried; after all, it's not like the Garden Fairies are going to last more than one competition. No Garden Fairy team has lasted past the first day. They are certainly no match for Rumble (Jason Dolley) and Glimmer (Tiffany Thornton), who are going for a record-breaking fifth win in a row. However, when they make it past the first competition by accident (literally) the rest of the Garden Fairies start to cheer for Chloe. And when Chloe makes Rosetta feel like part of the team, the two decide to try and win it all. It's a fun short film that is entertaining and does have a nice message as well. It is also nice to see a different fairy given the spotlight. Perhaps other fairies will get their own specials later on.
The other extras are two music videos, the first is for "Great Divide" by McClain Sisters and the second is for "Dig Down Deeper" by Zendaya. There is also a 1-minute preview for Fright Light.
As we've become accustomed, the Blu-ray looks incredible. The animation isn't quite as detailed as a tentpole animated release would be, but there's absolutely nothing wrong with this transfer. The colors are incredible, the blacks are deep, and there's never any compression issues. The audio is strong, but not as strong as the video. There's no problems to deal with and the dialogue is always clear and there's no issue with high or low frequency sounds, but the audio isn't as complicated. There are enough ambient sounds and directional effects to be effective, but it isn't showy. 3D effects are well done and help add a lot of depth to the film's visuals. This is more important that throwing things at the screen, although there is some of that as well. Snow looks awesome in 3D. The video quality isn't quite as strong in 3D as it is in 2D and there's some aliasing and banding, but it is very minor.
As for the prices, the DVD costs just $17, which would be good for this type of release without the TV special. With the TV special, it is a bargain. The Blu-ray combo pack costs $6 or about 30% more and given the quality of the video, it is worth it. Finally, the 3D combo pack is $30 or about 30% more than the Blu-ray combo pack. This is on par with similar releases. $7 or $8 is the usual premium for 3D.
If you liked the previous Tinker Bell releases, then Tinker Bell: Secret of the Wings is absolutely worth adding to your collection. Thanks to the bonus TV special, the DVD is a great deal; however, the Blu-ray Combo Pack is worth the upgrade. And if you've made the leap to 3D, the 3D Combo Pack is worth paying $7 more for.
The Movie
The Extras
The Verdict
- Submitted by: C.S.Strowbridge
Filed under: Video Review, Secret of the Wings, Jeff Bennett, Timothy Dalton, Anjelica Huston, Rob Paulsen, Mae Whitman, Grey Delisle, Lucy Hale, Brenda Song


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From | "Hadoop QA (JIRA)" <j...@apache.org> |
Subject | [jira] Commented: (HADOOP-3135) if the 'mapred.system.dir' in the client jobconf is different from the JobTracker's value job submission fails |
Date | Thu, 05 Jun 2008 10:41:45 GMT |
[ https://issues.apache.org/jira/browse/HADOOP-3135?page=com.atlassian.jira.plugin.system.issuetabpanels:comment-tabpanel&focusedCommentId=12602609#action_12602609 ] Hadoop QA commented on HADOOP-3135: ----------------------------------- -1 overall. Here are the results of testing the latest attachment http://issues.apache.org/jira/secure/attachment/12383436/patch-3135-v7_1.txt against trunk revision 663487. +1 @author. The patch does not contain any @author tags. +1 tests included. The patch appears to include 6 new or modified tests. +1 javadoc. The javadoc tool did not generate any warning messages. -1 javac. The applied patch generated 453 javac compiler warnings (more than the trunk's current 452 warnings). +1 findbugs. The patch does not introduce any new Findbugs warnings. +1 release audit. The applied patch does not increase the total number of release audit warnings. -1 core tests. The patch failed core unit tests. -1 contrib tests. The patch failed contrib unit tests. Test results: http://hudson.zones.apache.org/hudson/job/Hadoop-Patch/2586/testReport/ Findbugs warnings: http://hudson.zones.apache.org/hudson/job/Hadoop-Patch/2586/artifact/trunk/build/test/findbugs/newPatchFindbugsWarnings.html Checkstyle results: http://hudson.zones.apache.org/hudson/job/Hadoop-Patch/2586/artifact/trunk/build/test/checkstyle-errors.html Console output: http://hudson.zones.apache.org/hudson/job/Hadoop-Patch/2586/console This message is automatically generated. > if the 'mapred.system.dir' in the client jobconf is different from the JobTracker's value job submission fails > -------------------------------------------------------------------------------------------------------------- > > Key: HADOOP-3135 > URL: https://issues.apache.org/jira/browse/HADOOP-3135 > Project: Hadoop Core > Issue Type: Bug > Components: mapred > Affects Versions: 0.16.1 > Environment: all > Reporter: Alejandro Abdelnur > Assignee: Subramaniam Krishnan > Priority: Critical > Fix For: 0.18.0 > > Attachments: patch-3135-v3.txt, patch-3135-v4.txt, patch-3135-v5.txt, patch-3135-v6.txt, patch-3135-v6_2.txt, patch-3135-v6_3.txt, patch-3135-v7.txt, patch-3135-v7_1.txt > > > Until Hadoop 0.13 or so, at submission time the full path of the job.xml and all supporting files in DFS was given by the client to the jobtracker. > Since 0.15 onwards (we did not test 0.14) the jobclient is obtaining the job ID from the jobtracker and creating the directory for all the supporting files using the a system-dir computed from the local jobconf. > Line 696-7 in the JobClient: > String jobId = jobSubmitClient.getNewJobId(); > Path submitJobDir = new Path(job.getSystemDir(), jobId); > This makes submissions to fail when the value of the 'mapred.system.dir' on the client is different from the one in the JobTracker. > A simple way o fixing this would be to introduce a new method in the JobSubmissionProtocol 'getSystemDir()' that would return the jobtracker system dir and use that dir for uploading all the files on submission. > ---- > For the future: A more comprehensive way of this doing would to obtain a base jobConf from the jobtracker, carrying final information for each element and the construct the job.xml on the client using the final semantics. And, in this case the 'mapred.system.dir' property should be set as final in the jobtracker. As there may be some configuration properties that are sensitive and for security reasons should not be exposed to the clients a new flag 'private' could be introduced and only properties that don't have the 'private' flag would be send over from the jobtracker to the jobclient for job.xml resolution. -- This message is automatically generated by JIRA. - You can reply to this email to add a comment to the issue online. | |
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Wrap It Up Pretty Little Black Book!
I know, I know. We are in the thick of the holiday season and all that is dancing through our heads revolves around hot cocoa, snuggling under blankets, and shopping, shopping, shopping. Not to mention winter white weddings galore. ButView Credits
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