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Subscribe To Nicole Kidman Flirts With Zac Efron In New Clip From The Paperboy Updates
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Nicole Kidman has had some major ups and down the past few years. Following her Academy Award-nominated performance in John Cameron Mitchell's heartbreaking indie drama Rabbit Hole, she appeared in the critically reviled Adam Sandler comedy Just Go With It then the senseless Joel Schumacher thriller Trespass, which was not only loathed by critics but made a paltry $24,000 in its domestic release. Let me say that again: Trespass made only $24,000 in US theaters.
However, there's some deeply intriguing films on the horizon for the Aussie star, from Park Chan-wook's creepy thriller Stoker, to the recently wrapped war drama Railway Man, and the Olivier Dahan-directed Grace Kelly biopic Grace of Monaco. But before any of these promising pictures make their way to theaters, Kidman will traipse across the big screen playing shameless white trash in Lee Daniel's already controversial follow-up to Precious, the sultry Southern thriller The Paperboy. Will it be a hit or miss for Kidman? Take a look at the latest clip, which features her languid femme fatale flirting with Zac Efron's titular paperboy, then sound off in comments.
Inspired by Peter Dexter's crime novel, The Paperboy centers on the quest of two brothers, one an investigative journalist (Matthew McConaughey) the other a naïve paperboy (Efron), to seek the truth in a strange murder case, urged on by the accused's wannabe bride (Kidman). David Oyelowo and John Cusack co-star as McConaughey's colleague and the jailbird object of Kidman's affections, respectively.
The Paperboy with make its US premiere October 3rd at the New York Film Festival as part of the Gala Tribute to Nicole Kidman; a limited theatrical release will follow on October 5th.


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Romney's ORCA program sank
Mitt Romney's campaign boasted for the past two weeks that it would outgun President Barack Obama's team in the Democrats' area of strength — voter-targeting. It would use a state-of-the-art system called ORCA, named for the killer whale, that cost substantial resources to build over months.
Instead, Romney campaign officials were mostly flying without instruments on Election Day.
Story Continued Below
Numerous Republicans in and around the Romney campaign called the ORCA platform a total bust, stranding thousands of volunteers without a way of reporting data back to headquarters and leaving Romney central command without a clear view of developments on the ground.
The system was not shared with officials outside a small group in Boston and was kept largely a secret until the immediate lead-up to Election Day. The system was beta-tested on its own but not within technical infrastructure of the Boston TD Garden, where the Romney campaign's massive War Room was set up. That accounted for a number of the problems, officials conceded, even as they protested to POLITICO the depth of the Election Day meltdown.
Three sources described the campaign to POLITICO as "flying blind" on Tuesday in terms of targeting, with ORCA — which had a pricetag of hundreds of thousands of dollars — failing. There was another sizable allocation of funds for emergency robocalls to goose turnout late in the day in key areas identified by ORCA, but those were never put to widespread use.
Romney political director Rich Beeson defended ORCA as a first step toward a larger-scale voter turnout system that the GOP needs to develop, telling POLITICO that the data flow was imperfect but still significant.
"Did it work perfectly? No. But did we get a hell of a lot of good data? Yes," Beeson said. "When I needed answers, I could get them."
One of the problems, Beeson said, was that ORCA was never tested in the context of the TD Garden. When so much data started flowing into the facility, it was perceived to be a hack and rejected, Beeson said.
"In all the other testing we had, we didn't have that issue," Beeson said. "When you're dealing with 34,000 people across the country, there's going to be any number of issues, whether it's user error or smart phone didn't work. The system wasn't perfect and we never claimed it would be. But as we go through cycles, this is the beginning of something that's going to be very, very effective."
In any case, Beeson said, the campaign had only a few months to assemble the turnout system: "You're sort of building an airplane as you take off from the aircraft carrier."
Romney digital director, Zac Moffatt — who did not oversee Project ORCA — conceded there were some problems but insisted the system generally worked.
"I'm not going to blow smoke," he said. "I'm not saying there weren't challenges."
But he insisted that people were taking their own individual experiences and extrapolating that the entire system failed, when in fact it worked overall.
"A lot of data came into the system that we have on file now," he said, although he couldn't answer questions about whether it was continuous throughout the day or simply at the end. He insisted that 91 percent of all counties reported into the system, 14.3 million voters were accounted for as "voted," and 5,397 reports of voting issues were reported, such as broken machines and a dearth of ballots.
"Look, we experienced challenges through it," Moffatt said, but he insisted, "It allowed us to continue our GOTV efforts and allowed us at such a massive scale to track voting irregularities."
Like where? "Florida, Ohio. We were seeing different things coming in through the alerts."

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Michel Barnier (born 9 January 1951) is a French politician of the Union for a Popular Movement (UMP) and Vice President of the European People's Party (EPP). He was named Minister for Agriculture and Fisheries on June 18, 2007 and resigned on June 7, 2009 after being elected to the European Parliament.
Michel Barnier was born in La Tronche in the Isère département of the Rhône-Alpes région, in France. He graduated from ESCP Europe in 1972. Member of the staff of some Gaullist ministers in the 1970s, he was elected to the French National Assembly at 27 as a deputy for the Savoie département in 1978, under the banner of the Neo-Gaullist Rally for the Republic (RPR) and served in this function until 1993.
Together with Jean-Claude Killy he organised the 1992 Winter Olympics in Albertville as co-president of the COJO (Comité d'Organisation des Jeux Olympiques).


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Priya Mann was born and raised near Toronto, Canada and knew from an early age that she wanted to be a reporter.
Priya graduated from Brock University with a Bachelor's Degree in Communications and completed a Post Graduate Diploma from Humber College in Advanced Journalism.
She began her career as a volunteer reporter for Rogers Television Peel, in Mississauga.
In 2008, her first job took her out West, to CFJC News in Kamloops, British Columbia. She worked as a videojournalist – shooting, writing and editing her own stories. Priya won a prestigious RTDNA award for her story 'Remembering Afghanistan'. The feature story profiled a soldier who had recently returned to Canada after serving overseas.
A year later, she moved to Windsor, Ontario, and lived on the riverfront overlooking the Detroit skyline.
As a videojournalist for CTV Windsor, she covered a number of stories including the North International American Auto Show in Detroit.
Priya was promoted to CTV London in 2010, where she also became a backup anchor and weather specialist.
In 2011 Priya was the first television reporter on the ground in Goderich, Ontario, covering the aftermath of a F-3 tornado.
In 2012, Priya moved up the 401 to work as a videojournalist for CTV Kitchener. She worked as a videojournalist, anchor and occasionally filled in for weather.
In 2013, Priya was thrilled to become a part of Detroit's number one station WDIV-Local 4.
Priya's parents and brother live in North Carolina and she has a lot of family in Chicago. She is passionate about connecting with people and sharing their stories.
Priya is a dedicated vegetarian, loves to read, do yoga and watch documentaries.
She has a cockapoo named Zen – who is not yet inspired by his name.


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A rising column of billowing steam rose from a fissure in the rocky ground. The steam spread, darkening the skies above a small volcanic island in Iceland. The island was more of a rocky outcrop in the sea, no more than 50 yards across. Blistering heat from the crater warped my view of the small fissure's depths. I was prepared for it, as an oceanic scientist should be, clad in a bulky deep sea heat suit. The digital readout on my arm unit's display told me it was 5,000 degrees in the crater's center.
Sounds good, might as well check it out.
I repelled down, through the thick steam and into the fissure. I reached the floor of what I quickly realized was a cave partially filled with water. I stopped and gaped a bit at the water's surface. It shimmered with a purple iridescence. Small fish darted about the rocks in the shallows. I noticed a dive knife on a nearby rock and put it in my belt. Might be useful.
I waded out into the water. It didn't seem to be hot at all anymore, so I sank down, submerging myself. An incredible kaleidoscope of blues and purples flowed around me. The water illuminated an underwater tunnel so I followed it. The tunnel continued for a bit before the floor came up and opened into another chamber. I surfaced to discover the water was only a couple feet deep. On each side of the chamber were rows of alcoves, each holding a sunken boat still on its trailer. The alcoves continued for 100 yards down the cavern's walls. The glittering illumination from the water let me see each alcove had a boat. There must have been dozens.
The boat chamber eventually ended with another underwater tunnel, so I continued. The water's light abruptly ended at the entrance to the tunnel and I swam on, awkwardly in my suit, into the darkness. It soon felt like the tunnel had opened up, and I turned on the lights mounted on the top of my dive helmet. A huge underwater cave appeared. Pillars of volcanic rock supported the domed ceiling.
I hardly noticed all this, however, because my lights had revealed something else. One million sleeping sharks. I could have counted them all, but I didn't have to. I knew there was one million exactly. The bright blue sharks carpeted the cave floor, gently swaying back and forth with the current. I froze, and started to back out very slowly.
Hoooolllly shit. I'm out of here.
They started to wake up, shaking themselves out of a deep sleep. It didn't take long for them to notice me and start gliding through the water in my direction. I frantically looked around for something with which to defend myself. I reached below me and grabbed a few rocks off the floor. Each rock I hurled at the approaching sharks moved through the water at an excruciatingly slow pace.
Oh right, it's underwater. This isn't going to work very well.
I retreated through the tunnel into the boat cave and stood up in the shallow water. All light coming from the water had been extinguished and not even my helmet lights could penetrate its surface now. Shark fins knifed up through the surface and began to circle me. Occasionally one would brush up against my legs and feet. They seemed more curious than hostile. I wasn't afraid. I gently tried to push them away. I took the knife off my belt, but didn't use it's razor sharp edge. There was no need to hurt them. Instead I kept the knife's tip down and just tried to nudge them away.


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Obesity is associated with low-grade chronic inflammation, and serum markers of inflammation are independent risk factors for cardiovascular disease (CVD). However, the molecular and cellular mechanisms that link obesity to chronic inflammation and CVD are poorly understood.
Methods and Findings
Acute-phase serum amyloid A (A-SAA) mRNA levels, and A-SAA adipose secretion and serum levels were measured in obese and nonobese individuals, obese participants who underwent weight-loss, and persons treated with the insulin sensitizer rosiglitazone. Inflammation-eliciting activity of A-SAA was investigated in human adipose stromal vascular cells, coronary vascular endothelial cells and a murine monocyte cell line. We demonstrate that A-SAA was highly and selectively expressed in human adipocytes. Moreover, A-SAA mRNA levels and A-SAA secretion from adipose tissue were significantly correlated with body mass index ( r = 0.47; p = 0.028 and r = 0.80; p = 0.0002, respectively). Serum A-SAA levels decreased significantly after weight loss in obese participants ( p = 0.006), as well as in those treated with rosiglitazone ( p = 0.033). The magnitude of the improvement in insulin sensitivity after weight loss was significantly correlated with decreases in serum A-SAA ( r = −0.74; p = 0.034). SAA treatment of vascular endothelial cells and monocytes markedly increased the production of inflammatory cytokines, e.g., interleukin (IL)-6, IL-8, tumor necrosis factor alpha, and monocyte chemoattractant protein-1. In addition, SAA increased basal lipolysis in adipose tissue culture by 47%.
A-SAA is a proinflammatory and lipolytic adipokine in humans. The increased expression of A-SAA by adipocytes in obesity suggests that it may play a critical role in local and systemic inflammation and free fatty acid production and could be a direct link between obesity and its comorbidities, such as insulin resistance and atherosclerosis. Accordingly, improvements in systemic inflammation and insulin resistance with weight loss and rosiglitazone therapy may in part be mediated by decreases in adipocyte A-SAA production.
Citation: Yang R-Z, Lee M-J, Hu H, Pollin TI, Ryan AS, Nicklas BJ, et al. (2006) Acute-Phase Serum Amyloid A: An Inflammatory Adipokine and Potential Link between Obesity and Its Metabolic Complications. PLoS Med 3(6): e287. https://doi.org/10.1371/journal.pmed.0030287
Academic Editor: Mitch Lazar, University of Pennsylvania School of Medicine, United States of America
Received: March 4, 2005; Accepted: February 28, 2006; Published: June 6, 2006
Copyright: © 2006 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grants DK57835 and DK62093 and an ADA Career Development Award (to DWG), R01 DK54261 and R01 DK56637 (to ARS), R01 DK52398 (to SKF), R01 AG18408 (to APG), K01 AG00747 and R01 AG19310 (to ASR), R29 A614066 (to BJN), T32 AG000219 (salary support for RBH), University of Maryland General Clinical Research Center Grant M01 RR16500, General Clinical Research Centers Program, National Center for Research Resources (NCRR), NIH, the Department of Veterans Affairs Medical Research Service, and the Baltimore Veterans Administration Medical Center Geriatric Research Education and Clinical Center (GRECC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: A-SAA, acute-phase serum amyloid protein A; BMI, body mass index; CRP, C-reactive protein; CVD, cardiovascular disease; FFA, free fatty acid; HDL, high-density lipoprotein; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; PAI-1, plasminogen activator inhibitor-1; SD, standard deviation; SEM, standard error of the mean; SVC, stromal-vascular cell; TNF-α, tumor necrosis factor-alpha
Editors' Summary
Obesity often alters an individual's overall metabolism, which in turn leads to complications like diabetes, high blood pressure, and an increased risk of cardiovascular disease (disease of the heart and blood vessels, such as stroke or heart attacks). Having established a strong link between inflammation and cardiovascular disease, scientists now think that obesity might cause persistent low-level inflammation, and that this is the reason for the cardiovascular problems seen in many obese people. By better understanding the links between obesity, inflammation, and cardiovascular disease, the hope is that scientists may be able to find medications that can be given to obese people to reduce their risk of heart attacks and strokes.
Why Was This Study Done?
Previous research had suggested that a substance in the blood called A-SAA, which is raised by inflammation, might be a "missing link" between inflammation and cardiovascular disease, since an individual's baseline level of A-SAA is associated with the risk for cardiovascular disease (in other words, the higher the A-SAA, the higher the risk of cardiovascular disease). In the new study, researchers wanted to know whether the reason that obese people have a higher risk of cardiovascular disease is because they have higher blood levels of A-SAA.
What Did the Researchers Do and Find?
They found that obese people had higher levels of A-SAA in their blood. A-SAA appears to be produced in fat cells (or adipocytes) and then released into the blood. Obese people have higher numbers of fat cells, which could by itself account for the higher blood levels of A-SAA, but the researchers also found that the average fat cell from an obese individual produces and secretes higher levels of A-SAA than fat cells from lean individuals. When the researchers studied people who underwent weight loss, they found that A-SAA levels fell in response to weight loss, and this was associated with improvements in their metabolism. They then studied obese individuals who received the diabetes drug rosiglitazone (which is known to reduce inflammation). They found that even though these individuals did not lose weight, their A-SAA levels dropped as their metabolism improved. Trying to get at the mechanisms by which A-SAA might cause inflammation and diabetes, the researchers found that exposure to A-SAA can stimulate the activation of proinflammation molecules in a number of different cells, including blood vessel cells. It can also stimulate cells to break down fat stores and release fats, which could lead to metabolic complications and ultimately contribute to diabetes.
What Do These Findings Mean?
Together with similar results from other studies, the findings here suggest that A-SAA could promote inflammation, and that elevated levels of A-SAA in obese individuals could contribute to the chronic low-level inflammatory state that puts them at higher risk for cardiovascular complications. The authors speculate that drugs that reduce the blood levels of A-SAA might be useful as treatments for obese patients (to lower their risk of heart attacks and strokes). However, as they acknowledge, additional studies are needed to establish that A-SAA is indeed a causal link between obesity and inflammation and whether it plays a major role before it could be considered a promising drug target.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030287.
• MedlinePlus pages on obesity and cardiovascular disease.
• US Centers for Disease Control and Prevention pages on obesity and cardiovascular disease.
• Wikipedia pages on obesity and cardiovascular disease (note: Wikipedia is a free Internet encyclopedia that anyone can edit).
Complications of excess fat mass, particularly central or visceral adipose tissue, include insulin resistance and resulting hyperinsulinemia, glucose intolerance and diabetes, hypertension, hyperlipidemia, and a prothrombotic state. This constellation of obesity-related complications, often referred to as the metabolic syndrome or syndrome X [1, 2], markedly increases risk of cardiovascular disease (CVD) and death [3, 4].
Although the link between excess body fat and the metabolic and cardiovascular sequelae is well documented clinically and epidemiologically, the molecular and cellular underpinnings for this link are poorly understood. Excess and/or dysfunctional adipose tissue is associated with chronic low-grade systemic inflammation, which is also associated with CVD. For example, a modest elevation in C-reactive protein (CRP), an acute-phase reactant protein produced by the liver and a long-known marker of inflammation, has been shown to be predictive of CVD risk and events [5]. Indeed, measurement of CRP is now recommended in some clinical settings to stratify individual CVD risk and to help direct therapy [3, 6]. Serum amyloid A (SAA), another acute-phase reactant protein, has also been shown to be a predictor of CVD [7, 8]. Whether these acute-phase reactant proteins are directly involved in inflammation and the atherosclerotic process or simply markers of these processes is not known.
Adipose tissue is "inflamed" in obesity, with decreased expression of the anti-inflammatory adipokine adiponectin and increased secretion of a variety of proinflammatory cytokines, e.g., tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and prothrombotic factors such as plasminogen activator inhibitor-1 (PAI-1) [9]. Infiltration of adipose tissue by macrophages is in part responsible for this inflammatory process associated with obesity [10, 11]. However, the upstream regulator(s) responsible for the inflammatory state in adipose tissue and the role adipose tissue-derived inflammatory factors play in systemic inflammation remain unclear [12]. In a systematic search of differentially expressed genes between adipocytes and stromal vascular cells, we noted that serum amyloid A1 and A2 (SAA1 and SAA2, collectively called A-SAA) were highly expressed in human adipocytes, which was unexpected but in agreement with recent publications [13, 14]. A-SAA has been regarded as merely an inflammation marker and thought to be produced primarily in liver. In this study, we aimed to test the hypothesis that adipose A-SAA may be a molecular link between obesity and its comorbidities in humans.
Human Participants
The Institutional Review Boards of the respective institutions approved all human studies, and each volunteer provided written informed consent to participate. All participants were healthy according to medical history, physical examination, and laboratory testing unless otherwise specified in the protocols. The individuals studied showed no clinical or laboratory evidence of acute inflammation such as fever or elevated white blood cell counts. Abdominal adipose tissue samples were obtained from overnight-fasted participants by aspiration with a 4-mm cannula under local anesthesia with lidocaine as previously described [19] or obtained from nondiabetic participants undergoing intra-abdominal surgeries. All blood samples were stored at −80 °C until used.
Cross-sectional study of body mass index and serum A-SAA levels.
Participants were part of the previously described Amish Family Diabetes Study [20]. Initially, A-SAA levels were measured in plasma samples from 19 sex- and age-matched (age within 5 y) sets of nondiabetic sibling pairs with a discordance in body mass index (BMI) of at least 3 kg m−2. These 38 individuals were then included in an expanded set of 134 nondiabetic individuals with BMIs ranging from 17.0 to 41.8 kg m−2. Blood samples for A-SAA measurements were obtained from an antecubital vein after an overnight fast.
Effect of weight loss on A-SAA and insulin sensitivity.
Thirty-three sedentary, overweight or obese (BMI 32.3 ± 4.0 kg m−2, mean ± standard deviation [SD]), postmenopausal (58 ± 5.7 y, mean ± SD) women were studied before and after a 6-mo weight loss program. The intervention consisted of weekly outpatient classes with a dietitian on the principles of a hypocaloric dietary program that followed the American Heart Association (AHA) Step I guidelines with restriction of caloric intake by 250–350 kcal d−1 as previously described [21]. Fat mass was determined by dual-energy X-ray absorptiometry (Model DPX-L; Lunar Radiation, Madison, Wisconsin, United States) using the 1.3z DPX-L extended analysis program. Fasting serum levels of A-SAA were measured before and after the intervention in all participants. Subcutaneous abdominal fat biopsies were obtained at baseline in a subset of 31 participants under local anesthesia for the ex vivo studies of adipose A-SAA expression and/or secretion (described below). Eight of the participants underwent 3-h hyperinsulinemic-euglycemic clamp studies at baseline and after completion of the weight loss program to assess changes in insulin sensitivity. Women were weight-stabilized (< 1 kg) for at least 2 wk prior to metabolic testing before and after weight loss. All testing was performed in the morning after a 12-h overnight fast. Arterialized blood was obtained from a dorsal heated hand vein. Basal plasma glucose and insulin levels were measured in the fasted state. A 10 min priming with insulin followed by a continuous intravenous infusion of insulin (240 pmol m−2 min−1, Humulin, Eli Lilly, Indianapolis, Indiana, United States) was performed for 180 min. Glucose was infused at a variable rate to keep the plasma glucose, measured every 5 min, at the basal (fasting) level. Blood was also drawn every 10 min for the determination of plasma insulin levels. Plasma glucose was measured with the glucose oxidase method (Beckman Instruments, Fullerton, California, United States). Insulin was determined by radioimmunoassay (Linco, St. Louis, Missouri, United States).
The mean concentration of glucose and insulin was calculated for each sample time point. The trapezoidal rule was used to calculate the integrated response over 30-min intervals from 30 to 180 min for each participant. The integrated response was divided by its time interval to compute mean concentrations. Plasma glucose and insulin levels during the clamps averaged 5.17 ± 0.10 mmol l−1 and 474 ± 14 pmol l−1, respectively. This was 97.7% ± 0.2% of the desired goal with a coefficient of variation of 5.2% ± 0.4% in all clamps. Glucose utilization (M) for the 120- to 180-min interval was calculated from the amount of glucose infused after correction for glucose equivalent space (glucose space correction).
Effect of treatment with the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone on A-SAA.
Eight healthy, nondiabetic, overweight or obese participants (age 44.7 ± 9.1 y, BMI 30.8 ± 3.1 kg m−2, mean ± SD) were recruited and treated with rosiglitazone 4 mg d−1 for 4 wk, followed by 8 mg d−1 for 8 wk. Fasting serum levels of A-SAA were measured before and at 12 wk into the rosiglitazone intervention. At the same time points, subcutaneous abdominal fat biopsies were obtained under local anesthesia for ex vivo studies of adipose A-SAA expression and secretion (described below).
Adipose Tissue Fractionation and Microarray Analysis
For microarray analysis, human omental and subcutaneous adipose tissues were obtained from four women (two obese, two nonobese) undergoing semielective intra-abdominal surgery at the University of Maryland Medical Center. Isolated adipocytes and stromal-vascular cells (SVCs) were obtained by collagenase digestion (final concentration 2 mg of collagenase per gram of fat tissue) in Kreb Ringer bicarbonate buffer containing 4% albumin and 200 nM adenosine (KRB-A). After centrifugation at ̃200 g for 1–2 min, the medium below the floating adipocytes (containing the SVCs) was removed and subjected to centrifugation at 800 g for 5 min. The pelleted SVCs were resuspended in KRB-A and washed three times using the same procedure. The floating adipocytes were washed three additional times with KRB-A. RNA was extracted from adipocyte and SVC fractions, and microarray analysis was conducted using Affymetrix (Santa Clara, California, United States) human U133A chips according to the manufacturer's instructions.
Northern Analysis
Human adipose tissue and liver specimens were purchased from the National Disease Research Interchange (Philadelphia, Pennsylvania, United States), and total RNAs were prepared with Trizol (Invitrogen, Carlsbad, California, United States) according to the manufacturer's instructions. All other RNAs were purchased from Clontech (Palo Alto, California, United States). Total RNA (15 μg) extracted from the specified mouse (C57BL) or human tissue was subjected to agarose gel electrophoresis and blotted onto Nylon membranes using standard methods. Human SAA2 cDNA corresponding to nucleotides 1–536 of BC020795, and murine SAA2 cDNA corresponding to nucleotides 1–565 of U60438, were used as probes. These probes are 97% (human) and 95% (mouse) identical to SAA1 sequence and thus would be expected to hybridize to both SAA1 and SAA2. By contrast, the mouse SAA2 probe was only 62% identical to SAA3 mRNA and thus would not be expected to hybridize to SAA3 mRNA under the stringent wash conditions used. The probes were random-labeled (Stratagene, La Jolla, California, United States) with32P-dCTP, and hybridization was carried out at 65 °C in Rapid-Hyb buffer (Amersham Biosciences, Piscataway, New Jersey, United States). Blots were washed twice with 0.5× SSC/1% SDS at 65 °C (stringent wash), and visualized by PhosphoImager (Amersham Biosciences).
RT-PCR Analysis
For semiquantitative RT-PCR analysis, reverse transcription was carried out in a reaction containing 1 μg of total RNA, poly-dT primer, and MMLV reverse transcriptase using the Advantage kit (Clontech, Palo Alto, California, United States). PCR was performed under conditions typically consisting of 28 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s. For detection of human A-SAA mRNA in fractionated adipocytes and SVCs, primers 5′-GAGAGAAGCCAATTACATCGGC -3′ and 5′-AGTATTTCTCAGGCAGGCCAGC -3′, which fully match both SAA1 and SAA2, were used. In addition, human SAA1 and SAA2 mRNAs were quantitated individually by RT-PCR using a common forward primer 5′-ATGGGGCTCGGGACATGTGGAG -3′, which was paired with reverse primer 5′-AGTCCTCCGCACCATGGCCTGT -3′ (SAA1-specific) or 5′-AGTCCTCCGCACCATGGCCAAA -3′ (SAA2-specific). Human β-actin was amplified as a control with primers 5′-TTAATGTCACGCACGATTTCC -3′ and 5′-AGACCTTCAACACCCCAGCCA -3′. RT-PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized by UV transillumination.
The level of adipose A-SAA mRNA expression was more accurately quantitated by real-time PCR. Applied Biosystems (ABI, Foster City, California, United States) TaqMan PCR kits with commercially available assay-by-design primers were used on an ABI PRISM 7900 Sequence Detection System. The primers and probe for SAA match both SAA1 and SAA2 genes and therefore measure total A-SAA (SAA1 and SAA2) mRNA. Cyclophilin A mRNA was used as an internal standard. Threshold cycle (CT) values were obtained and relative gene expression was calculated using the formula (1/2)CT SAA − CT cyclophilin A.
SAA Secretion from Adipose Tissue
To examine the relationship between adipose A-SAA secretion and BMI, and regulation by weight loss and rosiglitazone treatment, adipose tissue fragments were obtained at biopsy in premenopausal women over a range of BMI values (26.8 ± 4.2 kg m−2, mean ± SD, n = 16), in postmenopausal women subjected to weight loss ( n = 33), and in participants before and after 12 wk of rosiglitazone therapy ( n = 7). Adipose tissue fragments were incubated for 3 h in M199 medium containing 1% BSA, and the medium was collected and stored at −80 °C until analysis for A-SAA. Adipocyte size was determined by a photomicrographic method [22].
For ex vivo studies of the regulation of SAA secretion, adipose organ culture was performed as previously described [23]. In a sterile hood, fresh human subcutaneous adipose tissue was minced into 5- to 10-mg pieces, washed with warm sterile saline, and cultured with no hormones, 25 nM dexamethasone (American Pharmaceutical Partners, Schaumburg, Illinois, United States), 7 nM insulin (Novo Nordisk, Princeton, New Jersey, United States), or a combination of these hormones, with and without rosiglitazone (1 μM ) (GlaxoSmithKline, Philadelphia, Pennsylvania, United States). The culture medium was changed daily and A-SAA was assayed in the conditioned medium on day 2.
Effect of SAA on Cytokine Production
Primary human coronary artery endothelial cells were purchased from Cambrex (Walkersville, Maryland, United States) and grown in endothelial cell basal medium-2 (EBM-2) supplemented with EGM-2 BulletKit. All experiments were conducted between the third to fifth subcultures. Human primary adipose SVCs from the subcutaneous depot were isolated as described as above from a normal female participant (BMI 27.5 kg m−2) who underwent elective abdominal reconstructive surgery. The SVCs were cultured in complete EGM-2, and subcultures between the second to third subcultures were used. RAW264 monocytes ( ATCC , Manassas, Virginia, United States) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum. These cells were seeded on six-well tissue culture plates at about 75% confluence and grown to 90%–95% confluence. The growth medium was replaced with supplement-free media (EBM-2 basal medium for human coronary artery endothelial cells and RPMI1640 for RAW264 monocytes). The cells were treated 1 h after the medium change with recombinant synthetic human apo-SAA (Peprotech, Rocky Hill, New Jersey, United States), or vehicle (PBS). The endotoxin level for this commercial preparation was less than 0.1 ng μg−1 protein. The conditioned medium was collected 8 h after SAA treatment by centrifugation at 2,000 g for 5 min and frozen until use for cytokine analysis. To examine the effect of SAA on adiponectin secretion, minced adipose tissue was cultured with recombinant SAA (2.34 μg ml−1), and the conditioned medium was collected from 24 to 48 h of incubation for adiponectin assay.
Effect of SAA on Lipolysis
Minced adipose tissue samples were cultured with recombinant SAA at a final concentration of 2.34 μg ml−1. After 24 h, medium was collected and glycerol was measured using a fluorometric assay [24] to assess changes in lipolysis in response to SAA. Data are presented as micromoles of glycerol per gram of adipose tissue in 24 h.
Cytokine Analysis
Human A-SAA (BioSource, Camarillo, California, United States) and PAI-1 (American Diagnostica, Greenwich, Connecticut, United States) were measured with ELISA kits according to instructions of the manufacturers. The SAA ELISA kit detects only A-SAA (SAA1 and SAA2) and not SAA4. The intra- and interassay coefficients of variation were 5% and 8%, respectively. Human monocyte chemoattractant protein-1 (MCP-1), IL-6, and IL-8; and mouse TNF-α, MCP-1, and RANTES in tissue culture media were analyzed at the Cytokine Core Facility, University of Maryland School of Medicine with cytokine multiplex reagents (Upstate Biotechnology, Lake Placid, New York, United States) by Luminex 100 (Luminex Corporation, Austin, Texas, United States). Human serum IL-6 and TNF-α levels were measured by high sensitive Quantikine enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minnesota, United States), and adiponectin levels were measured by radioimmunoassay (Linco, St. Charles, Michigan, United States). All samples were assayed in duplicate.
Statistical Analysis
Results are expressed as mean ± standard error of the mean (SEM) unless otherwise specified. Variables that were not normally distributed were natural logarithm-transformed for analysis and back-transformed for presentation. The Student's two sample or paired t test was applied when appropriate, as specified in the figure legends. Significance of correlations between two variables was determined by the Spearman rank correlation coefficient. In order to control for relatedness among the Amish participants, variance components analysis as implemented in SOLAR [25] was used to assess the correlation between BMI and A-SAA levels in the larger set of 134 Amish individuals. Differences were considered to be significant at p < 0.05.
Acute-Phase SAA Is Highly Expressed in Human Adipocytes
Microarray analysis of mRNA preferentially expressed in fat cells compared to stromal cells of human subcutaneous and omental adipose tissue pointed our attention to A-SAA (unpublished data). Semiquantitative RT-PCR analysis with primers fully and specifically matching SAA1 and SAA2 cDNAs validated the high level of expression of A-SAA mRNA in human adipocytes but not in SVCs (Figure 1A). Northern analysis showed that A-SAA mRNA was selectively and abundantly expressed in human adipose tissue (Figure 1B, left). The higher expression of A-SAA mRNA in human adipose compared to liver tissue (at least 15-fold) was confirmed in an independent Northern analysis containing additional specimens of adipose and liver tissues (Figure 1B, right). Conversely, in mice, A-SAA mRNA was predominately expressed in liver but not in adipose tissue (Figure 1C). These studies show, unexpectedly, that in humans, A-SAA is predominately expressed in adipose tissue, more specifically in adipocytes, and that the adipose expression is species-specific.
(A) Representative semiquantitative RT-PCR analysis of A-SAA and β-actin mRNA in SVCs and adipocytes fractionated from human omental (O) and subcutaneous (S) adipose tissues.
(B and C) Northern analyses of multiple tissue blots from the human and mouse, respectively. For all Northern analyses, 15 μg of total RNA from the indicated tissues were electrophoresed, blotted onto a nylon membrane, and hybridized with a radiolabeled human (B) or murine (C) SAA2 cDNA probe, which detects both SAA1 and SAA2 (upper gels). Equality of RNA loadings was estimated by ethidium bromide staining (lower gels). Comparison of A-SAA expression was made in five independent participants (B, right). Epi, epididymal; SubQ, subcutaneous
Obesity Is Associated with Increased Circulating Levels of A-SAA due to Increased Adipose A-SAA Gene Expression and Secretion
Selective and abundant A-SAA expression in adipocytes suggests that obesity may be associated with increased circulating A-SAA levels. To test this hypothesis, we measured plasma A-SAA levels in 19 age- and sex-matched nondiabetic sibling pairs who were discordant (>3 kg m−2) for BMI. A paired t-test showed significantly higher plasma A-SAA levels in the heavier siblings ( p = 0.044) and a positive Spearman correlation coefficient was observed between the BMI and A-SAA differences ( r = 0.54, p = 0.017). In an expanded set of 134 nondiabetic men and women over a range of BMIs, BMI was a significant predictor of A-SAA level ( p = 0.025, controlling for age, sex, and family structure). When individuals were grouped (Figure 2) into lean (BMI < 25 kg m−2), overweight (25 kg m−2 ≤ BMI < 30 kg m−2), and obese (BMI ≥ 30 kg m−2), the mean plasma A-SAA level of the obese group (ln-transformed for analysis, back-transformed for presentation) was 43% higher than that of the lean group ( p = 0.013, adjusted for age, sex and family structure).
A-SAA levels were measured in plasma of normal human participants who were divided into lean (BMI < 25 kg m−2, n = 54), overweight (BMI 25–30 kg m−2, n = 49) and obese (BMI ≥ 30 kg m−2, n = 31) groups. Data are expressed as mean ± SEM (ln-transformed for analysis, back-transformed for presentation), adjusted for age, sex, and family structure. * p = 0.013 versus lean group.
Increased serum A-SAA levels in obesity could be the result of normal expression and secretion of A-SAA from an increased fat mass, and/or increased expression and secretion of A-SAA from dysfunctional adipose or other tissues. To distinguish between these possibilities, adipose tissue samples were obtained from healthy premenopausal women over a range of BMIs, and A-SAA mRNA expression and secretion were measured. Adipose tissue A-SAA mRNA levels were significantly correlated with BMI ( r = 0.47, p = 0.028, n = 22; Figure 3A). Moreover, SAA release per gram of adipose tissue was strongly correlated with BMI ( r = 0.80, p = 0.0002, n = 16; Figure 3B). Furthermore, there was a strong correlation between adipose A-SAA gene expression and secretion in seven of these individuals who had both A-SAA mRNA and A-SAA secretion measured ( r = 0.89, p = 0.007, n = 7). A-SAA gene expression was also positively correlated with average adipocyte size ( r = 0.47, p = 0.04, n = 19; Figure 3C). These data suggest that increased A-SAA secretion from adipose tissue in obesity is the result of both increased fat mass and an increased rate of secretion per unit of adipose tissue.
Adipose A-SAA mRNA levels, measured by quantitative real-time RT-PCR (top) and A-SAA release by adipose tissue (middle), were significantly correlated with BMI. Furthermore, adipose A-SAA mRNA levels were increased with the adipocyte size (bottom). Dotted lines indicate 95% confidence intervals.
Changes in A-SAA Levels Are Associated with Outcomes of Clinical Interventions: Effect of Weight Loss and Rosiglitazone Therapy
If A-SAA is a marker of or causal link between obesity and its metabolic and cardiovascular complications, we would predict that circulating A-SAA levels would decrease in response to interventions that decrease obesity or its metabolic complications, e.g., insulin resistance. We measured serum A-SAA levels before and after weight loss with a hypocaloric diet program in 33 obese (BMI 32.3 ± 4.0 kg m−2; mean ± SD) postmenopausal women. A mean (± SEM) weight loss of 6.0 ± 0.7 kg or 7.1% was associated with a 13.8% reduction in SAA levels ( p = 0.006, n = 33; paired t-test). Significantly, the relative changes in serum A-SAA concentration correlated with relative changes in BMI ( r = 0.39, p = 0.03, n = 33) and body fat mass ( r = 0.35, p = 0.04) (Figure 4), but not with changes of fat free mass ( r = 0.23, p = 0.30). Eight of these participants also underwent hyperinsulinemic-euglycemic clamps before and after the weight loss intervention. In response to weight loss, increases in insulin sensitivity were correlated with decreases in A-SAA ( r = −0.74, p = 0.034). These findings suggest that A-SAA falls in response to weight loss and that a decrease in A-SAA partially predicts the increase in insulin sensitivity seen with weight loss in obese postmenopausal women.
Correlation between changes in serum A-SAA levels and changes in body fat mass before and after weight loss. Dotted lines indicate 95% confidence intervals.
We next reasoned that if A-SAA was an inflammatory adipokine involved in the metabolic consequences of obesity, other interventions that influence inflammation and insulin sensitivity might alter A-SAA concentrations. Eight overweight or obese nondiabetic individuals (BMI 30.8 ± 3.1 kg m−2, mean ± SD) were treated for 12 wk with rosiglitazone, a drug with insulin sensitizing and anti-inflammatory actions. There were no statistically significant changes in body weight or fat mass in these individuals during the 12-wk intervention. Nevertheless, serum A-SAA levels decreased by a mean of 37% after treatment ( p = 0.033) (Figure 5A). Moreover, the secretion of A-SAA from adipose tissue explants obtained by aspiration from these same participants was significantly reduced after rosiglitazone treatment (Figure 5B). The extent of serum A-SAA decrease tended to correlate with that of adipose A-SAA secretion, although the correlation was not statistically significant, presumably due to the small sample size. Notably, one participant (green line in Figure 5) responded to rosiglitazone with a marked reduction in A-SAA. Exclusion of this individual changed the p-values for serum and adipose SAA secretion from 0.033 and 0.034 to 0.001 and 0.055, respectively. Thus, rosiglitazone reduced adipose A-SAA secretion and lowered serum A-SAA levels with no significant change in BMI or fat mass.
Serum A-SAA ( n = 8) (top) and adipose secretion of A-SAA ex vivo ( n = 7) (bottom) were measured in nondiabetic participants before and after 3 mo of rosiglitazone treatment. The data are plotted with lines connecting the A-SAA levels of each individual. Serum A-SAA and adipose secretion of A-SAA (one symbol for same person of both studies) were significantly decreased by rosiglitazone ( p = 0.033 and p = 0.034, respectively; paired t-test after log-transformation).
The mechanism by which rosiglitazone decreases adipose A-SAA secretion could be direct, through action on adipose tissue, or indirect, through its effects on circulating hormones or other factors. Thus, we further investigated whether rosiglitazone acted directly on adipose tissue to decrease A-SAA secretion. Adipose tissue obtained from nondiabetic participants was cultured ex vivo. Incubation of the fat explants for 2 d with insulin or dexamethasone resulted in an SAA accumulation in the medium, and combination of the two hormones was additive in the stimulatory effect. Addition of rosiglitazone in the presence of insulin and dexamethasone reduced A-SAA secretion into the medium by 70% ( p = 0.002) (Figure 6). These findings indicate that rosiglitazone acts directly on adipose tissue to suppress A-SAA production by adipocytes.
Human adipose tissue explants were incubated in cell culture medium 199 (basal) or medium with insulin (Ins, 7 nM) and dexamethasone (Dex, 25 nM) in the presence or absence of rosiglitazone (Rosi, 1 μM) for 48 h. A-SAA production between 24 and 48 h was measured and corrected for tissue weight. Data are expressed as mean ± SEM, n = 3 independent experiments. * p = 0.03, ** p = 0.002, two sample t-test after log-transformation.
SAA Is a Proinflammatory Cytokine
We hypothesized that A-SAA, produced by adipocytes, may be a causal link between obesity, chronic systemic inflammation, and metabolic and cardiovascular consequences through stimulation of inflammatory cytokines locally in adipose tissue as well as at distant sites. Primary human coronary vascular endothelial cells (HCVECs), adipose SVCs and mouse RAW264 monocytes were treated with vehicle (PBS), or with low (0.47 μg ml−1) or high (2.34 μg ml−1) concentrations of SAA for 8 h, and the conditioned medium was assayed for cytokine production. SAA dramatically stimulated, in a dose-dependent manner, the release of IL-6, IL-8, MCP-1, and PAI-1 in HCVECs; IL-6, IL-8, and MCP-1 in adipose SVCs; and IL-6, RANTES, TNF-α, and MCP-1 in RAW264 monocytes (Figure 7). Treatment of these cells with 1 ng ml−1 lipopolysaccharide, a concentration that is at least ten times higher than the maximum possible contamination of endotoxin in the recombinant SAA, did not stimulate inflammatory cytokine secretion in HCVECs or adipose SVCs. Thus, SAA is a potent proinflammatory adipokine.
Human coronary artery endothelial cells (HCAECs, A), adipose stromal vascular cells (SVCs, B) and mouse RAW264 monocytes (C) were treated with vehicle (PBS, white bar), low (0.47 μg ml−1, hatched bar), or high (2.34 μg ml−1, black bar) concentrations of recombinant human SAA for 8 h in serum-free medium. Cell-free supernatants were then assayed for cytokines. Data are expressed as mean ± SEM from n = 3–5 independent experiments. Statistical significance (* p < 0.05; ** p < 0.01; two sample t-test) was observed between the SAA-treated groups and vehicle.
SAA Stimulates Lipolysis
One mechanism by which obesity may be linked to insulin resistance is through increased lipolysis, which results in increased circulating levels of free fatty acids (FFAs) and decreased glucose uptake by muscle and liver [26]. Because chronic treatment with inflammatory adipokines such as TNF-α [27] and IL-6 [28] increase basal lipolysis, we next examined whether SAA might have a similar effect. Culture of human adipose tissue treated with SAA for 24 h significantly increased lipolysis, as measured by glycerol accumulation in the incubation medium by 47% ± 11% (mean ± SEM, p = 0.001) (Figure 8).
Adipose tissues (eight subcutaneous and one omental) were cultured in the presence or absence of SAA (2.34 μg ml−1) for 24 h. SAA treatment increased lipolysis by 47% ± 11% as assessed by measurement of glycerol accumulation in the culture medium. Data are expressed as mean ± SEM (log-transformed for analysis, back-transformed for presentation). * p = 0.001, n = 9.
SAA Versus Adiponectin, IL-6, and TNF-α
Another mechanism by which adipose secretion of SAA might link obesity with insulin resistance is through down-regulation of adiponectin expression and/or secretion. To test this hypothesis, we treated human adipose tissue explants with SAA (2.34 μg ml−1) and measured adiponectin secretion into the medium. We found that SAA tended to reduce adiponectin secretion, but the difference did not reach statistical significance (basal versus SAA treatment [ng g tissue−1 24 h−1], 2.80 ± 1.6 versus 2.34 ± 1.27, p = 0.07, n = 9]. We further examined whether the levels of A-SAA in plasma of human participants over a wide range of BMIs were correlated with those of adiponectin. Although adiponectin levels were negatively correlated with BMI ( r = −0.3, p < 0.0001, n = 157), and SAA levels were correlated with BMI (see above), there was no correlation between the levels of A-SAA and adiponectin ( r = 0.049, p = 0.45, n = 157). These findings do not support a role of adiponectin in SAA-mediated pathways of insulin resistance.
We further examined the relationship of serum A-SAA levels with IL-6 and TNF-α in a population of postmenopausal obese women. Serum A-SAA is positively correlated with serum IL-6 ( r = 0.54, p = 0.03, n = 16), but not with serum TNF-α ( r = −0.30, p = 0.11, n = 30), which is consistent with the observation that there is no correlation between serum IL-6 and TNF-α levels ( r = 0.03, p = 0.93, n = 16). This finding suggests that common mechanisms may regulate A-SAA and IL-6.
Increasing evidence supports the hypothesis that the low-grade chronic systemic inflammation associated with obesity may be an important mediator of the metabolic syndrome and its constituents, including insulin resistance, type 2 diabetes, dyslipidemia, and hypertension [29–31]. However, the molecular and cellular mechanisms that link obesity to inflammation are poorly understood. SAA is a multigene family consisting of four genes (SAA1–4) that are conserved in major vertebrates [32]. In humans, three of the four genes (SAA1, SAA2, and SAA4), but not SAA3 (a pseudogene), are expressed [33]. In response to acute inflammatory stimuli, SAA1 and SAA2 levels in plasma can increase as much as 1,000-fold within 5–6 h and therefore, SAA1 and SAA2 are collectively known as acute-phase SAA (A-SAA) [34]. As with other acute-phase reactants, e.g., C-reactive protein, and based on animal studies, the liver is thought to be the primary source of circulating A-SAA [35].
In this study, we demonstrate that in humans, A-SAA (both SAA1 and SAA2) is predominantly expressed in adipose tissue, specifically adipocytes. Others have recently reported similar findings [13, 14]. Adipose expression of A-SAA in humans is in sharp contrast to mice, in which A-SAA is expressed predominantly in liver. There is a report of the expression of murine SAA3 in adipose tissue [36], but this gene is not expressed in humans [33]. Similar to the results of other investigators [15, 16], we found that circulating A-SAA levels are elevated in obese compare to lean individuals and, furthermore, that A-SAA expression is correlated with BMI and fat cell size. Collectively, these results strongly support adipose SAA as a major source of circulating SAA, particularly in obese individuals.
Is A-SAA a marker of excess and/or dysfunctional adipose tissue and inflammation or is it a causal link between obesity, inflammation, and metabolic and cardiovascular sequelae? We demonstrated that interventions that are known to decrease chronic inflammation and improve the metabolic and cardiovascular consequences of obesity, such as weight loss and rosiglitazone treatment, also decrease adipose A-SAA expression and secretion as well as circulating A-SAA levels. Similar findings with regard to serum A-SAA levels were recently reported in persons who underwent weight loss [13, 14, 37], and in those treated with rosiglitazone [38]. Furthermore, previous studies indicated that A-SAA is a potent stimulus for the expression and release of TNF-α, IL-6, and IL-8 in neutrophils [17, 18, 39]. We showed also that A-SAA directly stimulates the production of inflammatory cytokines in coronary artery endothelial cells and monocytes, as well as locally by adipose tissue SVCs. Others have shown that A-SAA is induced by TNF-α and IL-6 in hepatoma cells [40], suggesting positive feedback between A-SAA and other cytokines. Collectively, these findings implicate A-SAA as a local and systemic proinflammatory adipokine, and not just a marker of inflammation.
The increased mass of dysfunctional adipose tissue in obesity is known to be a source of several inflammatory factors, including TNF-α [41], IL-6 [42], and MCP-1 [43], and also of the prothrombotic factor PAI-1 [44]. These proinflammatory cytokines are predominantly products of SVCs within adipose tissue [42, 45]. By contrast, A-SAA, like leptin and adiponectin, is a product of adipocytes and not SVCs (Figure 1A). Our finding that SAA potently stimulates the secretion of proinflammatory cytokines in adipose SVCs suggests that adipocyte A-SAA acts as a paracrine factor to enhance cytokine production by SVCs. In addition, obesity is associated with increased infiltration of adipose tissue by macrophages [10, 11], which may also be target cells of SAA action. SAA may also be a chemoattractant for macrophages, raising a possible link for the association of fat cell size with macrophage infiltration in obese adipose tissue [10]. Thus, A-SAA may act locally to alter cytokine production and fat metabolism as well as systemically on liver, muscle, cells of the immune system, and the vasculature, to impact insulin resistance and atherosclerosis (Figure 9).
A-SAA secreted from adipocytes acts locally on adipose SVCs to stimulate cytokine release and in adipocytes to stimulate lipolysis, increasing FFA release and decreasing insulin sensitivity in adipocytes, and possibly contributing to systemic dyslipidemia. In addition, A-SAA secretion by adipocytes into the circulation stimulates cytokine production at more distant sites, including in endothelial cells and monocytes, resulting in endothelial dysfunction, monocyte infiltration, accelerated atherosclerosis, and possibly insulin resistance in muscle and liver. A-SAA-stimulated lipolysis increases circulating FFA concentrations, further contributing to insulin resistance in muscle and liver. Finally, A-SAA incorporation into HDL accelerates its degradation and impairs its function, resulting in decreased HDL and accelerated atherosclerosis.
The signaling pathways of the A-SAA-mediated inflammation response are not well studied. In neutrophils, SAA induces IL-8 production through the formyl peptide receptor-like 1/lipoxin A4 receptor and activates nuclear factor kappa B [46]. The same signaling pathway recently has been shown to be an important mediator of inflammation-associated insulin resistance [47, 48]. Whether the induction of cytokine production by A-SAA in vascular endothelial cells and SVCs, as we have shown here, occurs through the same mechanism remains to be determined.
Our findings that rosiglitazone treatment significantly reduces A-SAA secretion and serum A-SAA levels suggest that SAA1 and SAA2 may be target genes of peroxisome proliferator-activated receptor-gamma. Both in vitro and in clinical studies, rosiglitazone exhibits anti-inflammatory properties [38, 49, 50], which are likely to be beneficial in slowing or reversing atherosclerosis [51]. Thus, the suppression of A-SAA may be a significant component of the anti-inflammatory and antiatherogenic action of peroxisome proliferator-activated receptor-gamma agonists, providing evidence that agents that inhibit A-SAA secretion or action may be efficacious for treatment of the metabolic syndrome and atherosclerosis.
Obesity is associated with increased basal lipolysis in adipose tissue [26] and elevated circulating FFAs that are thought to elicit systemic insulin resistance [52]. We also discovered that SAA, like IL6 [28] and TNF [27], has a long-term effect in stimulating basal lipolysis. The lipolytic activity of SAA can be an autocrine feedback mechanism by which increased SAA production (Figure 3) from enlarged adipocytes limits further triacylglycerol accumulation. The resulting increased release of FFA into the circulation may contribute to insulin resistance. The mechanism of SAA-mediated lipolysis is under investigation. A recent study indicated that SAA binds CLA-1 (CD36 and LIMPII Analogous-1), resulting in activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases [53]. Extracellular signal-regulated kinase activation is involved in TNF-α-induced lipolysis [27]. CLA-1 is highly expressed in adipose tissue (unpublished data). Thus, SAA may act through CLA-1 and the extracellular signal-regulated kinase signaling pathway to stimulate lipolysis directly. Alternatively, increased lipolysis by SAA might be indirect, through its stimulation of other lipolytic cytokines, e.g., IL-6 and TNF-α.
Recent studies suggest that in addition to its role in inflammation, A-SAA may play a direct physiological role in cholesterol metabolism. SAA is an apolipoprotein and a component of high-density lipoprotein (HDL) particles [54]. The interaction of SAA with HDL may impair the function of HDL as an antiatherogenic molecule [55] and facilitate its degradation [56]. Dyslipidemia, including low HDL-cholesterol (HDL-C), is a metabolic consequence of obesity and a component of the metabolic syndrome [29]. Thus, the increase of adipose-derived SAA in obesity that we observed may be a mechanistic link between obesity, low HDL-C, and increased CVD risk (Figure 9).
CRP is an acute-phase reactant produced in the liver and a marker of chronic low-grade inflammation. Modest elevation in CRP is associated with increased CVD risk in epidemiological studies [5, 57, 58]. In contrast to A-SAA, CRP is barely expressed in adipose tissue in humans (unpublished data). If A-SAA is a direct mediator of obesity-associated inflammation and its metabolic and cardiovascular consequences, might serum A-SAA be a better indicator of obesity-associated CVD risk than CRP? Ridker et al. [5] showed that both CRP and A-SAA levels confer similar risk for CVD events in participants of the Women's Health Study. Similarly, in the Women's Ischemia Syndrome Evaluation (WISE) Study, Johnson et al. [7] recently reported that SAA is independently associated with angiographic coronary artery disease and highly predictive of 3-y cardiovascular events. These studies did not specifically address the relationship between BMI and A-SAA (or CRP) in predicting CVD events. Our studies suggest that A-SAA may be a valuable diagnosticand prognostic marker of obesity-associated CVD risk and possibly of the effects of interventions such as weight loss and rosiglitazone therapy. Additional studies are needed to better define the biology and clinical utility of A-SAA and to further establish A-SAA as a causal link between obesity and inflammation and their cardiovascular consequences.
Supporting Information
Alternative Language Abstract S1. Chinese Translation of the Abstract by RZY and DWG
Author Contributions
RZY, MJL, ASR, BJN, APG, ARS, SKF, and DWG designed the study. RZY, MJL, HH, ASR, BJN, SS, RBH, KH, NHG, and DWG performed the experiments. RZY, MJL, TIP, ASR, BJN, and DWG analyzed the data. RZY, MJL, ASR, BJN, RBH, APG, ARS, SKF, and DWG contributed to writing the paper.
- 1. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (2002) Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation 106: 3143–3421.
- 2. Reaven GM (1993) Role of insulin resistance in human disease (syndrome X): An expanded definition. Annu Rev Med 44: 121–131.
- 3. Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO, et al. (2003) Markers of inflammation and cardiovascular disease: Application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation 107: 499–511.
- 4. Isomaa B, Almgren P, Tuomi T, Forsen B, Lahti K, et al. (2001) Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 24: 683–689.
- 5. Ridker PM, Hennekens CH, Buring JE, Rifai N (2000) C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 342: 836–843.
- 6. Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, et al. (2005) C-reactive protein levels and outcomes after statin therapy. N Engl J Med 352: 20–28.
- 7. Johnson BD, Kip KE, Marroquin OC, Ridker PM, Kelsey SF, et al. (2004) Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women: The National Heart, Lung, and Blood Institute-Sponsored Women's Ischemia Syndrome Evaluation (WISE). Circulation 109: 726–732.
- 8. Ogasawara K, Mashiba S, Wada Y, Sahara M, Uchida K, et al. (2004) A serum amyloid A and LDL complex as a new prognostic marker in stable coronary artery disease. Atherosclerosis 174: 349–356.
- 9. Lehrke M, Lazar MA (2004) Inflamed about obesity. Nat Med 10: 126–127.
- 10. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, et al. (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112: 1796–1808.
- 11. Xu H, Barnes GT, Yang Q, Tan G, Yang D, et al. (2003) Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 112: 1821–1830.
- 12. Flier JS (2004) Obesity wars: Molecular progress confronts an expanding epidemic. Cell 116: 337–350.
- 13. Poitou C, Viguerie N, Cancello R, De Matteis R, Cinti S, et al. (2005) Serum amyloid A: Production by human white adipocyte and regulation by obesity and nutrition. Diabetologia 48: 519–528.
- 14. Sjoholm K, Palming J, Olofsson LE, Gummesson A, Svensson PA, et al. (2005) A microarray search for genes predominantly expressed in human omental adipocytes: Adipose tissue as a major production site of serum amyloid A. J Clin Endocrinol Metab 90: 2233–2239.
- 15. van Dielen FM, van't Veer C, Schols AM, Soeters PB, Buurman WA, et al. (2001) Increased leptin concentrations correlate with increased concentrations of inflammatory markers in morbidly obese individuals. Int J Obes Relat Metab Disord 25: 1759–1766.
- 16. Jousilahti P, Salomaa V, Rasi V, Vahtera E, Palosuo T (2001) The association of c-reactive protein, serum amyloid A and fibrinogen with prevalent coronary heart disease—Baseline findings of the PAIS project. Atherosclerosis 156: 451–456.
- 17. Furlaneto CJ, Campa A (2000) A novel function of serum amyloid A: A potent stimulus for the release of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8 by human blood neutrophil. Biochem Biophys Res Commun 268: 405–408.
- 18. Hatanaka E, Furlaneto CJ, Ribeiro FP, Souza GM, Campa A (2004) Serum amyloid A-induced mRNA expression and release of tumor necrosis factor-alpha (TNF-alpha) in human neutrophils. Immunol Lett 91: 33–37.
- 19. Dowling HJ, Fried SK, Pi-Sunyer FX (1995) Insulin resistance in adipocytes of obese women: Effects of body fat distribution and race. Metabolism 44: 987–995.
- 20. Hsueh WC, Mitchell BD, Aburomia R, Pollin T, Sakul H, et al. (2000) Diabetes in the Old Order Amish: Characterization and heritability analysis of the Amish Family Diabetes Study. Diabetes Care 23: 595–601.
- 21. Ryan AS, Nicklas BJ (2004) Reductions in plasma cytokine levels with weight loss improve insulin sensitivity in overweight and obese postmenopausal women. Diabetes Care 27: 1699–1705.
- 22. Lavau M, Susini C, Knittle J, Blanchet-Hirst S, Greenwood MR (1977) A reliable photomicrographic method to determining fat cell size and number: Application to dietary obesity. Proc Soc Exp Biol Med 156: 251–256.
- 23. Fried SK, Zechner R (1989) Cachectin/tumor necrosis factor decreases human adipose tissue lipoprotein lipase mRNA levels, synthesis, and activity. J Lipid Res 30: 1917–1923.
- 24. Laurell S, Tibbling G (1966) An enzymatic fluorometric micromethod for the determination of glycerol. Clin Chim Acta 13: 317–322.
- 25. Almasy L, Blangero J (1998) Multipoint quantitative-trait linkage analysis in general pedigrees. Am J Hum Genet 62: 1198–1211.
- 26. Arner P (1988) Control of lipolysis and its relevance to development of obesity in man. Diabetes Metab Rev 4: 507–515.
- 27. Souza SC, Palmer HJ, Kang YH, Yamamoto MT, Muliro KV, et al. (2003) TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes. J Cell Biochem 89: 1077–1086.
- 28. Trujillo ME, Sullivan S, Harten I, Schneider SH, Greenberg AS, et al. (2004) Interleukin-6 regulates human adipose tissue lipid metabolism and leptin production in vitro. J Clin Endocrinol Metab 89: 5577–5582.
- 29. Reaven P (2004) Metabolic syndrome. J Insur Med 36: 132–142.
- 30. Yudkin JS (2003) Adipose tissue, insulin action and vascular disease: Inflammatory signals. Int J Obes Relat Metab Disord 27: Suppl 3S25–S28.
- 31. Schmidt MI, Duncan BB (2003) Diabesity: An inflammatory metabolic condition. Clin Chem Lab Med 41: 1120–1130.
- 32. Sellar GC, Jordan SA, Bickmore WA, Fantes JA, van Heyningen V, et al. (1994) The human serum amyloid A protein (SAA) superfamily gene cluster: Mapping to chromosome 11p15.1 by physical and genetic linkage analysis. Genomics 19: 221–227.
- 33. Kluve-Beckerman B, Drumm ML, Benson MD (1991) Nonexpression of the human serum amyloid A three (SAA3) gene. DNA Cell Biol 10: 651–661.
- 34. Whitehead AS, de Beer MC, Steel DM, Rits M, Lelias JM, et al. (1992) Identification of novel members of the serum amyloid A protein superfamily as constitutive apolipoproteins of high density lipoprotein. J Biol Chem 267: 3862–3867.
- 35. Gabay C, Kushner I (1999) Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340: 448–454.
- 36. Lin Y, Rajala MW, Berger JP, Moller DE, Barzilai N, et al. (2001) Hyperglycemia-induced production of acute phase reactants in adipose tissue. J Biol Chem 276: 42077–42083.
- 37. O'Brien KD, Brehm BJ, Seeley RJ, Bean J, Wener MH, et al. (2005) Diet-induced weight loss is associated with decreases in plasma serum amyloid A and C-reactive protein independent of dietary macronutrient composition in obese subjects. J Clin Endocrinol Metab 90: 2244–2249.
- 38. Mohanty P, Aljada A, Ghanim H, Hofmeyer D, Tripathy D, et al. (2004) Evidence for a potent antiinflammatory effect of rosiglitazone. J Clin Endocrinol Metab 89: 2728–2735.
- 39. Ribeiro FP, Furlaneto CJ, Hatanaka E, Ribeiro WB, Souza GM, et al. (2003) mRNA expression and release of interleukin-8 induced by serum amyloid A in neutrophils and monocytes. Mediators Inflamm 12: 173–178.
- 40. Thorn CF, Lu ZY, Whitehead AS (2004) Regulation of the human acute phase serum amyloid A genes by tumour necrosis factor-alpha, interleukin-6 and glucocorticoids in hepatic and epithelial cell lines. Scand J Immunol 59: 152–158.
- 41. Hotamisligil GS, Shargill NS, Spiegelman BM (1993) Adipose expression of tumor necrosis factor-alpha: Direct role in obesity-linked insulin resistance. Science 259: 87–91.
- 42. Fried SK, Bunkin DA, Greenberg AS (1998) Omental and subcutaneous adipose tissues of obese subjects release interleukin-6: Depot difference and regulation by glucocorticoid. J Clin Endocrinol Metab 83: 847–850.
- 43. Sartipy P, Loskutoff DJ (2003) Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A 100: 7265–7270.
- 44. Shimomura I, Funahashi T, Takahashi M, Maeda K, Kotani K, et al. (1996) Enhanced expression of PAI-1 in visceral fat: Possible contributor to vascular disease in obesity. Nat Med 2: 800–803.
- 45. Fain JN, Bahouth SW, Madan AK (2004) TNFalpha release by the nonfat cells of human adipose tissue. Int J Obes Relat Metab Disord 28: 616–622.
- 46. He R, Sang H, Ye RD (2003) Serum amyloid A induces IL-8 secretion through a G protein-coupled receptor, FPRL1/LXA4R. Blood 101: 1572–1581.
- 47. Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, et al. (2005) Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB. Nat Med 11: 183–190.
- 48. Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW, et al. (2005) IKK-beta links inflammation to obesity-induced insulin resistance. Nat Med 11: 191–198.
- 49. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK (1998) The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 391: 79–82.
- 50. Jiang C, Ting AT, Seed B (1998) PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 82–86.
- 51. Hsueh WA, Bruemmer D (2004) Peroxisome proliferator-activated receptor gamma: Implications for cardiovascular disease. Hypertension 43: 297–305.
- 52. Boden G (1997) Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46: 3–10.
- 53. Baranova IN, Vishnyakova TG, Bocharov AV, Kurlander R, Chen Z, et al. (2005) Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases. J Biol Chem 280: 8031–8040.
- 54. Benditt EP, Hoffman JS, Eriksen N, Parmelee DC, Walsh KA (1982) SAA, an apoprotein of HDL: Its structure and function. Ann N Y Acad Sci 389: 183–189.
- 55. Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, et al. (1995) Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures. J Clin Invest 96: 2758–2767.
- 56. Wu A, Hinds CJ, Thiemermann C (2004) High-density lipoproteins in sepsis and septic shock: Metabolism, actions, and therapeutic applications. Shock 21: 210–221.
- 57. Festa A, D'Agostino R, Howard G, Mykkanen L, Tracy RP, et al. (2000) Chronic subclinical inflammation as part of the insulin resistance syndrome: The Insulin Resistance Atherosclerosis Study (IRAS). Circulation 102: 42–47.
- 58. Koenig W, Sund M, Frohlich M, Fischer HG, Lowel H, et al. (1999) C-Reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men: Results from the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) Augsburg Cohort Study, 1984 to 1992. Circulation 99: 237–242.


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Latest News
After 5 days of fighting, Syrian town devastated, no one claims victory
Five days of fighting over Kafer Zaita in northern Syria came to an ignominious end Wednesday, with the town devastated and empty of people but controlled by neither the government nor the rebels.
After sometimes fierce fighting that began Saturday and was witnessed by a McClatchy reporter, the rebels had abandoned the town Tuesday.
On Wednesday, government troops moved in and, according to rebels, burned and bulldozed about 60 homes that presumably had belonged to rebel sympathizers. Then without warning the army pulled out. The rebels moved back in, planted explosives in the army command post that had been the object of most of the fighting, and demolished it. Then the rebels, too, were gone.
Left behind were many damaged and destroyed buildings, some of them still smoldering when the last rebels pulled out.
What sparked the fighting was never very clear. Rebels said the army started it by shelling the town from the command post in retaliation for a visit earlier by a United Nations monitoring team based in the city of Hama. They called for other rebels to assist, and soon more than 500 rebel fighters had arrived in the town. But in three days of fighting, they never succeeded in taking the command post.
Danish Lt. Col. Peter Dahl, the head of the Hama-based U.N. team, told McClatchy that he and other monitors had attempted to establish an outpost to observe the battle. But their plans were disrupted when fire from a Syrian tank disabled one of his two vehicles.
In an interview, he said the U.N. team had been alerted to the outbreak of fighting by a phone call Saturday night from a rebel commander. U.N. rules forbid the monitors from traveling after dark, so Dahl and his team headed for Kafer Zaita on Sunday morning, he said. They set up about a mile from the town to observe.
Later that morning, an army tank unit sought to reinforce the besieged command post. "They decided to clear a route by fire," Dahl said. "Unfortunately, my vehicle happened to be on that route."
Dahl said no U.N. personnel were injured. The team members were able to tow the downed vehicle back to their base in Hama. Dahl said he had no opinion on whether the fire had struck his vehicle "in the heat of battle" or was intentional.
"I would like people to know that we tried," Dahl said. He said he'd submitted a report of the incident to U.N. headquarters in New York.
Only one rebel died in the fighting, killed by fire from a helicopter. Army casualties are unknown, though the fighting coincided with what must surely be the bloodiest period of combat for the Syrian military since the uprising began nearly 15 months ago. In the first five days of June, the government news agency listed the names of 100 soldiers or police officers who'd died in clashes with rebels. The agency doesn't list where the soldiers died.
As for the town itself, casualties were uncertain. Most residents had fled before the final battle. Once the rebels were gone Tuesday, the army tanks took up positions in the town and additional ground troops moved in. On Wednesday, according to rebels, they detained an unknown number of suspected sympathizers and destroyed their homes.
One rebel medic said his 37-year-old cousin had been burned in his home Wednesday. Another said a 15-year-old boy had been shot and killed. There was no way to confirm or disprove their accounts.
Then the army left and the rebels returned, to finish off the command post they'd been unable to capture. The few residents who remained could be seen leaving Kafer Zaita as a pair of rockets landed outside the town at around 8 p.m.


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The little boy, wrapped like a mummy, would cry in agony every few days as his mother and a nurse changed the bandages that protect him from infection. He dreaded outings because it meant possible contamination and...


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Kids These Days
Education can never start too early. Which is why I, for one, am happy to see all the things kids are learning from their birthday cakes. You know, things like....
...friendly hand gestures:
And where Spider-Man's web comes from:
Watch it go!
Then there are the helpful anatomy lessons:
I think it's safe to say that Bailey really, REALLY enjoyed his Bar Mitzvah party.
Not to mention the legal drinking age:
Hint: that's not it.
And perhaps the best life lesson of all: one of the hazards of mixing boobs with beer:
Hey, it could be worse. You could be on 16 And Pregnant and be rewarded for your questionable life choices with money and fame.
There. Don't you feel better now?
Thanks to Sarah G., Anony M., Tanya G., M.M., and Jenae B. for the education.


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text:
New book on the demise of the railway dining car.
http://www.telegraph.co.uk/travel/artsa ... g-Car.html
I recall having great breakfasts on the early morning Manchester Pullman in the 80s, but Pullman cars no longer exist.
If you have read railway stories by Conan Doyle or Dickens or Agatha Christie or Graham Greene ( Travels with My Aunt ) or Harry Walton ( Swamp Train ) that reflect chance meetings on trains, or even seen Brief Encounter, this book would be of interest.


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Charity formed in 2005 to reduce numbers of women dying in pregnancy & childbirth due to poverty
Website: http://www.hopeforgracekodindo.org
Location: Cardiff, Wales, UK
Members: 3
Latest Activity: Dec 17, 2015
Started by Angela Gorman. Last reply by Angela Gorman Aug 30, 2012. 2 Replies 0 Likes
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Tags: Dev, Goals, Millennium, poverty, mortality


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With This Wreck, I Thee Wed
It seems I never fail to underestimate your insatiable desire for wedding wreckage. And sprinkles. You guys always want sprinkles.
Two birds? Meet your stone:
Not crumby enough for you? Try this:
Because naked wedding cakes have more Funfetti.
"Ooh, you guys, I've got it! Ok, picture this: we have the happy couple, in their wedding finery, scaling a giant turd. Eh? EH?!"
Bam. Nailed it.
"People, I know we can fit a few more pieces of plastic on here! WE JUST AREN'T TRYING HARD ENOUGH."
"Barb, you fetch the curling ribbon while Sam and I open another bag of aquarium greenery. Move, move, move!"
We all know there's a lot of crying at weddings. Fortunately, this couple decided to put all the used tissues to good use:
You might think it's icing holding all those together, but I'm here to tell you: it's not.
Thanks to Heather B., Sarah L., Brittany P., Lesley W., & Danielle N. for the tear-jerkers.
Thank you for using our Amazon links to shop! USA, UK, Canada.


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We aim to have your order to you within 2-5 working days-this can be quicker if you live in a capital city and it may take a little longer if you live in a remote area.
Damage or loss in transit is rare but if it happens we will take responsibility for it. Please contact us to organise the return of damaged goods or to report that a parcel has not arrived.
We understand that buying clothing and fashion online is very convenient but does not give you the opportunity to try things on first and sometimes items just don't fit or suit you. You may return items purchased online for a full refund or exchange if you prefer including sale items (Please Note: the refund is for the purchase price of the items purchased and does not include postage charges).
More detail is available in our "Delivery and Returns" policy found under Customer Information.


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As proudly reported in Scottish Life magazine, a British think tank called the New Economics Foundation recently determined that the Scots are the happiest people in Britain, boasting some of the most impressive "wellbeing" scores anywhere. Exactly why this is, no one seems sure. Does Scotland's rugged natural beauty make people feel better, as the magazine suggested? Is it the sound of the bagpipes? The freedom men experience wearing skirts?
Whatever the reason, if Scots are so "chirpy," as Edinburgh's Scotsman newspaper put it, why is there such grisly violence and deep psychological distress in their crime fiction? And why is crime fiction this bonnie land's favorite genre to begin with?
With the release of new books by four of the top practitioners of "Tartan Noir," the best school of detective fiction of recent years, there may be no better time to ask these questions.
Psychological thrillers don't get any darker than those of Denise Mina, a onetime criminology teacher who made her reputation a dozen years ago with the harrowing "Garnethill" trilogy and sets a new personal standard with the quietly unsettling "Gods and Beasts." Her third book featuring female Detective Sergeant Alex Morrow, it deals with the aftermath of an oddly played out post office robbery in which an elderly bystander is perforated with an AK-47.
Ian Rankin's John Rebus, the most popular of Scottish investigators, is back after a brief retirement of three novels — not as a detective, but a civilian cold case consultant — connecting the dots between missing young women in "Standing in Another Man's Grave."
Stuart MacBride, a lesser-known talent in the U.S., is seeking to raise his profile here with two new books. In "Birthdays for the Dead," his first featuring Detective Constable Ash Henderson, a psychopath kidnaps girls on the eve of their 13th birthday and marks the occasion every year by sending increasingly horrific photos of them to their fathers. The first of those girls to get snatched was Henderson's. And "Close to the Bone," MacBride's eighth Logan McRae novel (out next month), gets off to a cheery start when a man is "necklaced": chained to a stake, wedged into a tire (they spell it tyre) and set on fire.
And then there's the prolific Val McDermid, whose "The Retribution," her latest thriller featuring profilers Dr. Tony Hill and detective Carol Jordan, was published last year. It boasts a sicko serial killer from their past who makes their lives miserable again: "Vance dragged the knife across his throat and at once there was blood everywhere. He stepped back and flipped the man on his back. The blood sprayed and foamed and fountained from the carotid arteries, driven higher and faster by...."
Well, you get the idea.
The rub is, the grimmer these novels get, the edgier the gallows humor in them becomes, and the more the fiercely independent investigators thumb their noses at authority. The alcoholic, chain-smoking, chronically insubordinate Rebus is the king of incorrigibility. The bantering McRae, rising above a chattering ensemble, suffers no fools. Alex Morrow processes the violence and corruption around her with a finely honed cynicism. And the perpetually tormented Tony Hill copes by existing in his own eccentric world.
Maybe any attempt to figure out why the Scots are so chirpy should begin here: When you can deflect the worst that life has to offer with a wisecrack or a sneer — like the 60-year-old man who won a 2012 Glenfiddich Spirit of Scotland award for refusing to vacate his land so Donald Trump could build a golf course on it — how can you not be happier than ordinary folk?
"We always find a way to laugh, even if it's sick or dark," said McDermid. "That's what happens when Presbyterian guilt and shame is crossed with Gaelic song and dance, which happens in our culture."
"We have a word here, thrawn," said MacBride. "We're desperately thrawn people. It means obstinate, contrary. It's a guiding principle in the Scot psyche that if you tell us to do one thing, we'll do another. We don't react well to authority."
The Scots' piss and vinegar, manifested in brash street language, contrasts with the sobriety of the Scandinavian crime writers who have dominated the genre since the worldwide success of Stieg Larsson's "The Girl with the Dragon Tattoo" trilogy. When you think of Swedish mystery legend Henning Mankell's Wallander, acclaimed Icelandic author Arnaldur Indridason's Erlendur or any number of imitators, you think of dour, isolated individuals struggling to get through the day. (Not that this bridge is uncrossable for Mina, a onetime resident of Norway who is working on a graphic novel adaptation of the "Dragon Tattoo" novels.)
As close-knit as the Scottish crime writers are, though, it would be a mistake to stereotype them. Rebus' Old Testament morality — "Once you've sinned, you are ever a sinner in his view," said Rankin — is very different from the pragmatic code Morrow follows, having a gangster for a half-brother. And the books strongly reflect the particularities of their settings.
Rankin's Edinburgh is a smallish city with no guns, a low murder rate and a personality split between old urban sprawl and new geometrically designed downtown. Mina's Glasgow is, like Chicago, a broad-shouldered onetime industrial center with shady politics. MacBride's Aberdeen, in northeast Scotland, is defined by its granite composition and frequent changes in weather. "On sunny days, the place sparkles and the people are uplifted," he said. "When it's gray and overcast, the place just sinks."
The Oxford-educated McDermid, who grew up in the town of Kirkcaldy on Scotland's east coast, now divides her time between Manchester, England, where she worked as a reporter, and a small seaside town in Northumbria. (She crossed over into England, she said, when it became impossible for her to live as an open lesbian in Scotland.)
Strongly influenced by Chicago mystery writer Sara Paretsky, she has written three series: one featuring Glasgow reporter Lindsay Gordon, a lesbian; one with wisecracking Manchester detective Kate Brannigan, and the Tony Hill novels, which are set in the fictional West Yorkshire city of Bradfield. "A Place of Execution," her standalone 1999 masterpiece about a missing 13-year-old girl, is set in an isolated hamlet in Derbyshire.
In Scotland, as in the U.S., crime fiction has struggled for respect. In America, it got a momentous boost in 1971 when revered Southern writer Eudora Welty, in the lead review in the New York Times Book Review, praised Ross Macdonald's "The Underground Man" as "exhilaratingly well done" and "very moving." In Scotland, novelist William McIlvanney changed people's attitudes toward the genre when he moved from writing "serious" prize-winning literary fiction with a blue-collar bent to crime novels that dealt with moral and social issues, beginning with "Laidlaw" (1971).
"He made it 'all right' for me to try writing crime fiction," said Rankin. "If Willie wrote it, then it was literature and not the child of a lesser god."
While the Scots were reinventing crime fiction in their own terms, the Brits were largely confining themselves to village mysteries and police procedurals. The sheer range of Scottish mysteries is pretty overwhelming. Among the books Rankin singled out were Alexander McCall Smith's "No. 1 Ladies' Detective Agency" series, set in Botswana; Christopher Brookmyre's satirical Glaswegian thrillers (and more recent procedurals with drama student turned private eye Jasmine Sharp), and British-born Edinburghian Kate Atkinson's series with P.I. Jackson Brodie.
Three other contemporary writers leaving a deep personal imprint on Scottish crime fiction are Edinburgh noir specialist Allan Guthrie ("Kiss Her Goodbye," "Slammer"), whose deadpan intensity and rat-a-tat dialogue could have been influenced by American master Elmore Leonard; Louise Welsh, whose quietly nasty, attention-grabbing 2002 debut, "The Cutting Room," winkingly describes Edinburgh as ''a peaceful wee haven," and Peter May, whose new Isle of Lewis trilogy featuring Edinburgh detective Fin Macleod is set in the Outer Hebrides.
For Mina, political passion is the glue that bonds all the writers together. "To be taken seriously, you have to be politically engaged," she said. Paddy Meehan, the girl reporter who comes of age in "The Field of Blood" (2005), "The Dead Hour" (2006) and "Slip of the Knife" (2007), is a study in empowerment. Alex Morrow makes a political statement in "The End of the Wasp Season" (2010), the middle novel of the series, by remaining in pursuit of brutal killer while seriously pregnant with twins, neither using it as an excuse to slow down nor letting it hamper her work.
"In Scotland, we had lots of discussions about identity, about what kind of country we wanted to be as we moved forward," said McDermid. "The crime novel was a way to talk about society." Her 2003 novel, "The Distant Echo," in which a Scottish town is torn apart by an investigation into an unsolved murder, dissects the tribalism created by class distinctions.
"Crime fiction is received differently, read differently, than other kinds of fiction," said Mina. Judging by the national mood in Scotland, it's being read in the best way possible.
Lloyd Sachs, a regular contributor to Printers Row Journal, blogs at jazzespress.com.


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A little over a month ago, the news that Ben Affleck would be the next actor to take up Batman's mantle in director Zack Snyder's Man of Steel sequel - currently known as Batman Vs. Superman - came seemingly out of the blue. The expected fan backlash has since calmed down, allowing different perspectives on the ambitious project to come to light, including the fact that the film is further along in development than anyone previously thought.
Affleck waited for the dust to settle (and for the Internet to take a collective breath and relax) before addressing the casting choice on Late Night with Jimmy Fallon. The interview was short on specifics, but touched on some of the unique aspects of this new Batman, including how he would depicted as older than his previous big-screen incarnations, which presumably sets up Superman and Batman as adversaries, at least initially.
While more recent stories surrounding Batman Vs. Superman have centered on the question of whether or not Wonder Woman would have a role, we now have new insights from Ben Affleck on his take as the defender of Gotham City.
In an interview with 411mania, Affleck was asked about his beloved Red Sox, touched on his TV project The Middle Man, his favorite shows (he's a Game of Thrones fan), and how he made the decision to take the role of Batman:
"Initially I was reluctant as I felt I didn't fit the traditional mold but once Zack showed me the concept, and that it would be both different from the great movies that Chris and Christian made but still in keeping with tradition I was excited. Doing something different and new is always tricky and part of the thrill and the risk is that initially it confounds expectations. The truth is, it's the movie and the execution of it is what all the actors depend on and I believe in Zack's vision."
While this version of Batman is to be "tired, weary and seasoned," according to Warner Brothers CEO Kevin Tsujihara, Affleck was asked how this description might extend to Batman's alter-ego, Bruce Wayne. According to Affleck:
"Yes, Kevin described it aptly. I don't want to go further because I want to be able to capitalize on what is new about this iteration by having it be a surprise to the audience."
While the fan outcry over Affleck's casting echoed similar reactions to every other actor to play the role - going back to Michael Keaton in Tim Burton's 1989 Batman - one wonders if Affleck himself has a favorite previous version. His response:
"It has been done very well many times before. "
It may be a noncommittal response, but it's also very diplomatic, and possibly meant as a gracious attempt to honor all the previous Batmen, as well as deflect any premature ideas of how Affleck will interpret the role. He will be compared very closely to every other actor to play Batman, so keeping his approach under wraps is the best move at this point. Henry Cavill seemed to play the role of Superman as if it had never been played before, resulting in a fresh take on the Man of Steel - this is a tactic Affleck may be looking to echo.
"Confounding expectations" is a key phrase among Affleck's responses. Zack Snyder and David S. Goyer did just that with Man of Steel; for all its flaws, it's safe to say it was not what we were expecting, and very different from what came before.
One of the things which provoked the ire of many a fan and critic was Superman's apparent disregard for the safety of Smallville and Metropolis during his throwdowns with General Zod; now we know that Snyder and Goyer plan to address this in the sequel, setting up the potential for a story full of morally and philosophically complex issues. As more information trickles our way, we will find out just what else we should - and should not - expect from this long-awaited team-up.
Batman Vs. Superman opens in U.S. theaters on July 17th, 2015.
Source: 411mania


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Wow. I guess this guy started his celebrating early! He's even looking a little green.
And yes, sorry, dude; I'll quit screaming now.
Oh, but I can't let you go without at least one St. Patrick's day greeting, so here:
Haha, just kidding! This is actually a sobriety test. If all you can see is the word "SMEGHIT" then you should probably stay in tonight.
Speaking of which, you guys are welcome to join me, John, and my Guinness-soaked chocolate chips tonight for a few rounds of our new Supernatural drinking game. The rules are simple: just drink when any of the characters do. We should all be smashed by the first commercial break. ["SMEGHIT!!"]
Now if you'll excuse me, I have to go get ready for tonight's marathon by salting the doors and windows and finding an iron poker and also learning how to kiss in Irish.
Er, those things aren't all necessarily related, btw.
(Sam? Dean? Thoughts?)
Thanks to Constance H., Alisa B., & Laura M., who thinks someone may have actually asked that last baker to write, "Kiss me," IN IRISH.
Sadly, the world may never know.


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People sure are smelling a lot of stuff around Justin Bieber lately: The pop singer's private plane was detained and searched at a New Jersey airport Friday after authorities thought they smelled marijuana smoke onboard, according to various reports.
Customs and Border Protection agents began a search shortly after the plane arrived at Teterboro Airport from Toronto around 3 p.m. Friday, ABC News sources said. A canine team gave the whole aircraft the sniff test, but no drugs were found onboard, according to the New York Daily News.
The flight was crossing the border after Bieber on Wednesday turned himself in to Toronto police for booking on suspicion of assault on a limo driver back in December.
The smell of alcohol was mentioned prominently in the police report for Bieber's DUI arrest Jan. 23 in Miami Beach, Fla.
"I immediately smelled an odor of alcohol eminating [sic] from the driver's breath," the report read. The singer was later determined to have a negligible amount of alcohol in his system, registering a blood-alcohol level of 0.014% on a breath test (0.08% is legally intoxicated). However, he did test positive for weed and Xanax.
Bieber has pleaded not guilty to charges of DUI, resisting arrest and driving without a valid license, and will be arraigned Feb. 14 in that case.
Coincidentally, Bieber, his dad and some friends were headed into the New Jersey-New York area to attend parties around Super Bowl XLVIII, which some smarties have dubbed the "Weed Bowl," because marijuana is legal in Colorado and Washington, the states from which the competing teams hail.
"Never forget where you come from or those who supported you before anyone else," the 19-year-old tweeted Thursday night along with a link to a story about his hometown having his back.
"Still the small town kid," he wrote. "Thanks."
Kristen Bell, Dax Shepard urge boycott on celebrity kid photos
Model Christie Brinkley celebrates 60 posing in a sexy swimsuit
Scarlett Johansson-Oxfam partnership fizzles amid SodaStream tiff
Follow Christie D'Zurilla on Twitter and Google+. Follow the Ministry of Gossip on Twitter @LATcelebs and on Facebook facebook.com/ministryofgossip.


text: vulgar slang To shock or frighten one very suddenly and/or severely. (Hyperbolically alludes to frightening someone so badly as to cause them to lose control of their bowels.) Don't sneak up on me like that, you scared the shit out of me! That car accident seems to have scared the shit out of Janet—she's still shaken by it.