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Live Bands Return To The Intersection in Grand Rapids
Wally Londo Published: July 9, 2021
kyle-wong--unsplash
The Intersection in Grand Rapids is a well known music venue that's been around since 1972, and moved to its current larger location in 2003. With a capacity of 1800 they regularly feature national acts of all different genres. Grand Rapids local music scene has always been very active. Unfortunately due to the pandemic, the last performance at the Intersection had been 483 days, but all that changed yesterday with their first live performance.
Their first show was presented at Elevation inside the venue, with fans lined up at the door ready to for some live music. That's the first of the three day run of entertainment. They are doing an outdoor show today and an inside show on Saturday. They are taking precautions to keep everyone safe by offering hand sanitizer and requiring staff to wash hands often.
"We just ask that everybody follows the CDC guideline applicable to them. Please don't enter the venue if you are sick or have any kind of symptoms," said Scott Hammontree, the operating partner and talent buyer with The Intersection. "If you do become sick while here at the venue, please ask the manager on duty and we'll refund your ticket and you can come to a different show."
They have been booking talent and have a few shows scheduled already. Some of the shows coming up:
Tonight it's Country performer Chase Rice, with Brendon Monroe opening
Saturday Night it's Blunts and Blondes, with opening acts Antiserum, Bawldy, and Attak of the Chads.
You can view all of their upcoming shows at the Intersection here
Read the entire story at WOODTV
Check Out the Best-Selling Album From the Year You Graduated High School
Do you remember the top album from the year you graduated high school? Stacker analyzed Billboard data to determine just that, looking at the best-selling album from every year going all the way back to 1956. Sales data is included only from 1992 onward when Nielsen's SoundScan began gathering computerized figures.
Going in chronological order from 1956 to 2020, we present the best-selling album from the year you graduated high school.
Concerts Are Coming Back: Opening Night Plans for Rock's Big Acts
Who Had the Best 10 Year Run in Rock History?: Roundtable
Filed Under: live music venues


WASHINGTON—A top Chinese official offered an olive branch to America, saying that Beijing wants to resume trade talks with Washington.
"The Chinese side is ready to have a discussion with the United States on issues of mutual concern and work for a solution on trade acceptable to both sides," Chinese Vice Chairman Wang Qishan said at the Bloomberg New Economy Forum in Singapore on Nov. 6.
Wang's speech came amid an effort by top Communist Party leaders to calm global investors disturbed by the U.S.–China trade war and a worsening economic slowdown in the world's second-largest economy.
Escalation of the trade war and a collapsing economy caused panic selling in the Chinese stock market this year. Overall, the Shanghai Composite has lost almost 25 percent of its value since hitting a peak in January. And the smaller-cap Shenzhen Composite Index declined more than 30 percent. Both benchmarks are now deep in bear-market territory.
Asia stocks had a mixed response to Wang's comments. Hong Kong's Hang Seng Index gained 0.8 percent while mainland China's markets ended the trading day lower on Nov. 6.
Wang's speech had a more conciliatory tone than Xi's confrontational remarks at the opening of an international business fair in Shanghai on Nov. 5.
In his speech, the Chinese leader criticized Trump's trade approach, denouncing "law of the jungle" and "beggar-thy-neighbor" trade practices. Xi's comments dented hopes that both sides could end the tension at the G20 summit in Argentina this month.
Xi also tried to ease concerns about China's economic situation.
Both Wang and Xi portrayed themselves as free traders, promising to lower import tariffs and improve access to the Chinese market.
Chinese officials, however, have given no indication that they're ready to meet Washington's long list of trade demands, such as ending cyber theft, forced joint ventures, and intellectual theft.
Wang's promises for more liberalization and compromise weren't convincing enough, according to Scott Kennedy, head of China studies at the Center for Strategic and International Studies in Washington.
Following Wang's speech, he wrote on Twitter that Eurasia Group President and founder Ian Bremmer, who was a moderator at the forum in Singapore, asked audience members to raise their hands if they thought Wang's speech showed a new willingness by Beijing for more liberalization and compromise.
"Almost no one raised their hand. Wang didn't win the room over," Kennedy wrote.
Europe is also aligned with Washington in its efforts to end the unfair economic and trade policies of Beijing.
European companies are concerned about China's rules requiring forced joint ventures, intellectual property theft, and unequal treatment of foreign companies, said Carlo D'Andrea, vice-president of the European Union Chamber of Commerce in China.
For decades, Beijing hasn't taken any concrete actions to fulfill its promises. And that's the reason why the Trump administration has chosen to take a tougher stance on China's decadeslong protectionist and trade-distorting policies.
To end China's "economic aggression," Washington has levied duties on roughly $250 billion worth of Chinese goods and imposed restrictions on Chinese investments.
The last round of trade talks with China ended in August with no concrete steps toward a deal.
Trump said in a tweet on Nov. 1 that he had "a long and very good conversation" with Xi, offering some hope that trade tensions may cool after the G20 meeting.


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Dark Mofo cuts close to bone with confronting works about violence that involve the audience
ABC Arts
By arts editor Dee Jefferson
Photo: Audiences queued down the street to see Slaughterhouse-15 by Japanese 'latex artist' Saeborg. (Supplied: Dark Mofo/Jesse Hunniford)
Related Story: 'I'm not sure we can get much bigger': Dark Mofo wraps up with record-breaking crowds
Related Story: MONA's latest attraction uses a giant board game to teach visitors about data mining
Related Story: Dark Mofo using art to lure tourists and help with bushfire recovery
Map: TAS
More than 180 Tasmanian children have been killed or gone missing within a period of roughly 50 years — and nobody knows exactly how each of their stories unfolded.
We know that many of these children were abducted; some were shot, some drowned, and some were thrown into fires to burn alive; we know that some were dismembered and their body parts "pickled" and traded to scientists and collectors.
These are extraordinary, shocking statements of fact — perhaps more so in a country where missing children have such a powerful resonance (in real life and literature). They are unthinkable in an age where just one missing child makes headlines across decades.
But the Tasmanian children in question lived (and in many cases died) in Tasmania between 1803-1850 and they were Aboriginal children.
Most of them did not make the papers, let alone headlines — and most didn't receive burials. Most of their families never knew what happened to them.
The story of the missing and dead children has been unearthed by Tasmanian artist Julie Gough, who created a temporary "memorial" for 185 of them in Queens Domain: a winding bushland walk "fly-postered" with A4 sheets inscribed with the headline "MISSING" or "DEAD", a name (or if unknown, "Girl" or "Boy"), and a silhouette in profile, followed by biographical essentials.
Photo: Missing or Dead is an offshoot of Julie Gough's major survey exhibition Tense Past. (Supplied: Dark Mofo/Rémi Chauvin)
For the last two weeks, members of the public could be found day and night poring over these posters as they trawled the scrub — on their way along Dark Path (the outdoor iteration of Dark Mofo's popular annual Dark Park precinct).
Dark Mofo's darkest moments
Dark Mofo has a justifiable reputation as a locus for confronting work — but you'd be hard-pressed to find a darker story in the festival's seven-year history than the missing and dead children memorialised in Gough's installation.
Missing or Dead is one of several works that haunted this year's Dark Mofo, seeping into the greater narrative of the festival and beyond.
In the performance Take This, For It Is My Body, audiences were invited to tea — and then served scones containing an "admixture of Aboriginal blood"; in the VR work Real Violence, we became bystanders to a shocking assault; in Pigpen, we were witness to a "birthing" — a supersized inflatable pig giving birth to human-sized latex-piglets — followed by a ritual slaughter of mother and babies.
These works cut through the hype by being not just confronting but timely; relevant to the time and place we live now.
Crucially, they also gave audience members "skin in the game", involving them in a situation and making them more than passive observers or recipients of a message or image.
Photo: Each performance of SJ Norman's Take This, for It is My Body, accommodated eight people. (Supplied: Elliot Hughes @_slate_grey_)
Gough's Missing or Dead involved the audience emotionally through the "Missing" poster format — something ripped from the everyday life, rather than a history book.
It inevitably provoked in viewers the question: How would we react if this was happening right now in Australia? And does the fact that these events happened 200 years ago make them any more acceptable?
Gough's installation also overwhelmed walkers with its sheer scale and layout; following the winding paths and side-shoots into the scrub to "find" each child became almost a compulsive activity. No poster — no child — should be neglected.
I found myself in this position, and across two visits I saw many others making their way slowly, meticulously through the bush.
Gough told me that several people told her they felt they had to find each child; read every poster and biographical detail.
Photo: "For me, each tree could have grown out of the essence of that child. The child could be under that tree," says Gough. (Supplied: Dark Mofo/Rémi Chauvin)
After all, what more powerful duty of care do we have than to a child?
Imagine if the child on the poster was your child: son or daughter, niece or nephew, grandchild.
And then imagine that the crime wasn't reported; that the people who did this to your child were not punished; that in fact many of them prospered.
Finally, imagine if your child had survived, had their own children and made their mark, across generations, on Tasmania.
Gough has been imagining all of this since the mid-1990s, when she moved to Tasmania and began to map her own family history: a maternal line from the Trawlwoolway people (north-east Tasmania) and a paternal line tracing to Scotland and Ireland.
Photo: Julie Gough has studied anthropology, English literature, drawing and sculpture; these days her practice is focused largely on video work. (Supplied: TMAG/Alastair Bett)
Her ancestor Dalrymple Briggs is one of the children who survived; survived separation from her parents (who like the majority of surviving Tasmanian Aboriginal people, were exiled to Wybalenna on Flinders Island from 1830-1946), servitude, and even being shot by her "master".
The Art Show live from Dark Mofo
Radio National's The Art Show broadcasting live from this year's festival.
Later in life, Dalrymple successfully petitioned Queen Victoria for the release and return of her mother from Wybalenna.
But as Gough combed newspapers, journals, official correspondence and government ordinances from the era, she discovered many stories of Aboriginal children who were taken from their families as part of the Tasmanian colonial project, and who didn't survive.
Consider the story of John Shinall (aka Shiney), separated from his family by the time he was five years old and brought up amongst white people while working as a labourer. After he died, he was decapitated and his head embalmed then sent to the UK.
"Imagine bringing up a child from another culture and then going, 'They're dead and now I'll use their body parts, put their head in a jar'," Gough says incredulously.
"If this happened to my nephew or grandchild now … if I was face to face with any of the people who did these things …". She trails off, shaking her head.
This is the legacy of trauma and unresolved tension that Tasmanians live with today.
Bloody scones
Visitors to this year's Dark Path art walk could book tickets to a Devonshire-style supper of scones, jam, cream and tea in Government House.
This being Dark Mofo, there was a twist; this was not a polite or pleasurable experience. In fact, it's fair to say that it subverted most expectations.
The show's premise was hinted at in its title: Take This, for It is My Body.
Audience members were served scones containing an "admixture of Aboriginal blood", drawn from the individuals who made and served the meal: artist SJ Norman (a diasporic Koori of Wiradjuri descent, born on Gadigal country) and performers Carly Sheppard (a diasporic descendent of the Kurtjar people, born and raised on Kulin country), Naretha Williams (a Wiradjuri woman) and Sinsa Jo Mansell (Trawoolway nation; a rrala luwana from Larapuna, the northern region of Lutruwita).
Photo: The Aboriginal Protection Act (1909) defines as Aboriginal as "any full-blooded aboriginal native of Australia, and any person apparently having an admixture of aboriginal blood". (Supplied: Elliot Hughes @_slate_grey_)
A "menu card" beside each place setting contained a brief history of race relations in Australia from first contact to the present, taking in the 146-year Frontier Wars, policies of protectionism and assimilation, the Stolen Generations, slavery, and the 1967 referendum.
The menu card continued with a chapter of personal history: the show's creator, SJ Norman, writing about their great grandmother Sally, "an itinerant cook and domestic".
"Sally was known for her scones and her temper. My mother recalls the sight of Sally's knuckles split open to the bone, her blood streaking the dough that she kneaded for the men's afternoon tea.
My mother was raised reluctantly Catholic. She recalls the residue of the communion wafer in her mouth and the brutality of nuns. She recalls the incantations of the Latin mass and she recalls the voice of her Granny, who sang the now lost songs of her Country."
The card concluded: "You are invited to consume, keep or refuse this offering as you see fit."
Photo: Performer Naretha Williams watches silently during a performance of Take This, for It is My Body at Government House. (Supplied: Elliot Hughes @_slate_grey_ )
However quickly you may have made a choice in your mind just now, I can guarantee the decision takes a far more complex flavour when you are seated at a table with strangers (likely to be majority white) and being served in silence by women whose blood is in the supper, while contemplating the contents of that menu card — and the fact that, if you're a non-Indigenous Australian, you're inevitably, inherently, a benefactor of this history of violence.
From entering the grounds of Government House to exiting, the whole experience takes 20 minutes — but manages to up-end power structures and reflect the audience back to themselves in a way that is likely to stay with each person for some time afterwards.
The venue was a key ingredient in this iteration of Norman's work: a site of power, occupied from 1858 to the present by Her Majesty's representative; and a place where colonial architecture facilitates the acts of immersion and imagination from which the performance derives its potency.
Norman has performed the work in varying types of venues since its 2010 premiere in Bristol, but never before at an official site of colonial power.
"None of us can believe we got this site," Norman admits, adding that Tasmanian performer Sinsa Jo Mansell had told them: "This is history-making shit to be [performing this work] here."
Photo: SJ Norman is a Berlin-based Australian cross-disciplinary artist. (ABC Arts: Teresa Tan)
The work has also never received such a wide audience: Dark Mofo draws on a broad spectrum of the public, not just arts types. The season sold out and the performers extended their nightly hours to add more shows.
Speaking on Sunday night after their final performance, Norman, Williams and Sheppard said that the reactions over the course of five nights and more than 50 performances during Dark Mofo had ranged from awkward silence to tears; from nervous laughter to lively discussion — and some blatant racism.
"It's been a pretty gruelling run, I won't lie," Norman told the ABC.
Some people ignored the performers serving them; others demanded their assistance or verbal interaction. One audience member said their tea was too strong, and asked Williams to top it up with hot water.
"There were a few really good sessions; tables who were really in it and having a yarn about history and complicity and privilege and guilt and grief — really talking through the complexity of what the work brings up," Norman says.
"The dynamic of each table is really different, and they [the participants] kind of set each other off — which is part of the work."
Norman describes the mechanism of the show as "a series of power flips".
"There's a volatile question around who has the agency in that space and who is actually performing? There's an assumption that we're the ones performing for them — but sometimes it's really the audience performing for us," Norman says.
"Sometimes what they're performing for us is pretty ugly."
Meat as murder
Two years ago, Austrian artist Hermann Nitsch slaughtered a bull as part of Dark Mofo. The furore started when his artwork was announced — and continued in its aftermath.
It's arguably the most confronting and controversial event staged by Dark Mofo — a festival that is not shy on either count.
Animal rights activists may have been happier with this year's slaughter event: Pigpen, by Japanese artist Saeborg.
Photo: In Pigpen, Japanese artist Saeborg wanted to explore factory farming but also the way women's bodies are seen as sites of production. (Supplied: Dark Mofo/Jesse Hunniford)
No animals were hurt in this production, which featured a giant inflatable pig and humans wearing latex piglet costumes.
Even so: this entirely un-sexy, un-kawaii performance haunts you.
The supersized sow, doe-eyed, lies along a wall of the Avalon Theatre — confined by her litter and by inflatable floor-to-ceiling bars.
As we watch, she impassively "births" eight wriggling human-sized piglets, one by one, and they quickly attach themselves to a teat apiece.
Enter the farm girl: a buxom latex blow-up doll in pinny and boots, brandishing a meat cleaver.
First comes the slaughter of the piglets (in mime, followed by unzipping of costume compartments to reveal guts and organs). Then the mother is de-robed of her most delectable flesh.
And then the striptease: inexplicably, our murderous meat maiden de-robes and does a bump 'n grind, presumably dedicated to meat-lovers everywhere.
Finally, the piglets — reborn, zombie-like — whip the audience into a booty-shaking dance frenzy.
Later that night I contemplated the IRL carcass of my latex dance partner, artfully displayed as part of Dark Mofo's Winter Feast food hall.
It still wasn't as confronting as Saeborg's bizarre, disorienting pantomime.
Part of the work's effect is having piglet-performers and audience members in the same space, unmediated by a stage or cordon; individual piglets scurried amongst us, hiding from the meat cleaver, shaking with terror, squealing.
I saw several people reach out with a soothing hand — instinctively protective; involved.
Photo: Winter Feast is one of Dark Mofo's most popular components. (Supplied: Dark Mofo/Jesse Hunniford)
'Real violence'
Not even Dark Mofo's avid detractors appear to have sniffed out this year's most notorious work: Jordan Wolfson's VR work Real Violence, in which audience members step into a short, realistic scene to watch the artist assault a man kneeling in front of him on the sidewalk — beating him with a baseball bat, stomping on his face and skull with his boots.
When it premiered at the Whitney Biennial in 2017, reactions to the work merited their own round-up article, with breathless tweets including "the most disturbing, horrifying artwork I have ever seen" and, "Can you get PTSD from an artwork? Because I think Jordan Wolfson gave it to me".
Frieze critic Dan Fox labelled it "pantomime snuff movie".
For at least some audience members at Dark Mofo, the notoriety, shock value and trigger warning would have been reasons enough to queue for the work (which you invariably had to).
Photo: Audiences queued to see confronting VR works by Jordan Wolfson and Paul McCarthy. (Supplied: Dark Mofo/Jesse Hunniford)
It's possible to find merit in the work, however: New York Times critic Roberta Smith said Wolfson's work showed "real violence that many Americans rarely see. Horrible to watch, it should at least shock almost anyone into a better understanding of how scarring it is to witness physical savagery".
Interestingly — pointedly — Wolfson has said: "I'm no moralist trying to shock people into behaving better."
For me, the work left a lingering sense of dread — it was genuinely a locus of dark thoughts.
It seems broadly effective in exposing the viewer's reaction to real versus staged violence; violence IRL versus violence on screen.
On a subtler level, it subverts expectations of on-screen violence by distributing power (somewhat) and disrupting the abuser/victim dynamic: the men are the same age, race, height and build. The assault is sound-tracked by a voiceover track of Hebrew prayers, read by the artist.
Some viewers have questioned whether the act of violence is also an act of retribution.
These are interesting factors to consider while analysing your response to the scene.
As with Gough's installation and Norman's "high tea", the venue was key: this was not a "safe" gallery space, or a polite white cube.
There was no doubt that this experience felt more fraught at night, in the cold, in a cavernous space in which Marco Fusinato's noise art blasted intermittently — an assault on the visitor's ears.
Jarrod Rawlins, part of Dark Mofo's visual arts curatorium (alongside Hannah Fox and Theia Connell), told the ABC he was drawn to Wolfson's Real Violence because of the way it "undermines" typical VR experiences.
"It's quite a cinematic experience; it's not like a first-person player game with a narrative where you're running around exploring the environment — you're actually standing still watching a scene that could be from a film," Rawlins says.
"It's a very simple way of putting you actually inside a scene that is confronting … you've become part of the work."
Dark Mofo ran from Jun 5-23. The writer attended the festival with the assistance of Dark Lab.
Topics: arts-and-entertainment, contemporary-art, visual-art, performance-art, theatre, indigenous-culture, indigenous-aboriginal-and-torres-strait-islander, aboriginal, history, tas, japan, australia, hobart-7000, united-states
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redpajama_set_name: RedPajamaCommonCrawl


In a tribute to Lou Reed (and Hulk Hogan, apparently).
How cool were your musical tastes when you were 15? Because mine were a humiliating mix of listening to My Chemical Romance, playing that one Just Jack song on repeat and telling anyone who would listen that there would never be a band as good as The Libertines. Not as cool then, as Finn Wolfhard, who at 15, already fronts a band playing to screaming fans in NYC and covering The Velvet Underground.
Not content with just being a certified Hollywood It boy, the Stranger Things star also has his own band, Calpurnia, who played this Friday at Brooklyn's Rough Trade. Their set included a pretty impressive cover of The Velvet Underground classic Here She Comes Now, dedicating the performance to "Lou Reed and Hulk Hogan". Fair enough.


Black/Gray screen when booting Final Fantasy XV? Try this!
Trying to play Final Fantasy XV but you can't get into the game due to a gray or black screen when trying to launch it? Don't fret! The fix is super easy and will only take a few minutes at most, hopefully.
Fixing this bug requires editing the GraphicsConfig.ini file found under "C:/Users/[your username]/Documents/my games/FINAL FANTASY XV/[lots of numbers]/savestorage". You can use any basic text editor, like Notepad, to open the file and make the required changes.
All you have to do is scroll down to [DisplaySettings] and change the "HardwareFullScreenMode=1" line to "HardwareFullScreenMode=0". Once you've done that all you have to do is save the file and try to launch FFXV again! Chances are, it'll work without a problem this time!
That's it! After that you should be able to get into the game without a problem. I mean, as far as black/gray screen problems go, you might run into something else but who knows!
Another, less preferable option, is simply hitting alt+enter and putting FFXV into windowed mode. Although this isn't ideal, it'll work in a pinch or if you're worried about editing game files.
If nothing else, I hope this helped you fix Final Fantasy XV's black/gray screen problem! Have another solution or do you feel I overlooked something? Let me know in the comment section!
Full disclaimer: I didn't discover this fix, instead I found it on Steam's discussion board for FFXV. I wish I knew who the first person to write about it was as I would definitely like to give them credit! If you know who they are, leave a comment and let me know!
What if i do not have those folder in "my documents".
Ah, that likely means you bought FFXV on the Windows Store instead of Steam.


Both total and cell surface expression of Fas on luteal cells was greater for early versus late stage bovine CL (89% vs. 44% of cells for total Fas; 65% vs.18% of cells for cell surface Fas; respectively, P<0.05, n=6-9 CL/stage). A similar increase in the steady-state concentration of mRNA for Fas, as detected by quantitative real-time polymerase chain reaction, however, was not observed. Transient disruption of K8/K18 filaments in the luteal cells with acrylamide (5 mM), however, had no effect on the surface expression of Fas (P>0.05, n=4 CL/stage), despite evidence these conditions increased Fas expression on HepG2 cells (P<0.05, n= 3 expts). Exposure of the luteal cells to cytokines induced cell death (P<0.05) as expected, but there was no effect of K8/K18 filament disruption by acrylamide (P>0.05) or stage of CL (P>0.05, n= 4 CL/stage) on this outcome.
The receptor molecule CD95 (Apo-1) or Fas, is considered an integral component of immune-response mechanisms within the corpus luteum (CL) which potentially influence luteal function. It is a member of the TNF receptor superfamily  and is thought of as the prototypical death receptor because when bound by Fas ligand (FasL), cells undergo apoptosis . The binding of FasL to Fas triggers trimerization of Fas receptor on the cell surface. This complex then leads to the activation of Fas associated death domain and pro-caspase-8 proteins. The cleavage of pro-caspase-8 signals the caspase cascade, which then leads to the activation of pro-caspase-3 and apoptosis [3, 4]. Indeed, in the cow, expression of Fas mRNA within the CL occurs throughout the luteal phase , and exposure of luteal cells to FasL, induces apoptosis [5, 6]. Recently, Kliem and coworkers determined Fas and FasL mRNA increase in bovine CL within 30 min to 2 h of injecting cows with a luteolytic dose of prostaglandin F2-alpha , further supporting the death-inducing role of Fas and FasL in the CL. These observations collectively suggest Fas-induced mechanisms within the bovine CL constitute a plausible pathway for the cell-specific death observed during luteal regression.
The attractiveness of the Fas-induced death pathway in luteal regression is that it is relatively conserved among species and it provides for the selective elimination of cells (i.e., via apoptosis) without invoking an inflammatory response. Indeed, regression of the CL is characterized by cells undergoing apoptosis while neighboring cells remain unaffected . The relative amount of expression of Fas on the surface of luteal cells might account for at least some of this selectivity and specificity, but this has not been directly evaulated in the CL. Instead, most studies to date have examined only gross expression of Fas mRNA or FasL in luteal tissue to propose a role for the Fas-FasL system in luteal function. In addition, potential mechanisms influencing Fas expression on the luteal cell surface have yet to be explored. Here we speculated cytoskeletal components, specifically intermediate filaments, regulate expression of Fas on the surface of luteal cells, and hence lend specificity to the process of Fas-induced apoptosis of luteal cells in the CL.
The cytoskeleton of cells consists of microtubules, microfilaments, and intermediate filaments. Intermediate filaments have a diameter ranging between 7–11 nm and consist of a family of five different subtypes . One of the subtypes is the keratin-like proteins, which are found in epithelial tissues, including the steroidogenic cells of ovarian follicles and CL [10–16] . Keratin filaments are obligate heterodimers, forming filaments of an acidic keratin (type I, K9-K20), and a basic keratin (type II, K1-K8) [9, 17]. The more prominent types of keratin filaments found in epithelial cells include filaments containing K7, K8, K18, and K19 . In the bovine CL, K8/K18 filaments are observed in luteal cells throughout the estrous cycle, yet their relative expression diminishes with advancing age of the tissue . Functionally, K8/K18 filaments provide structural integrity to cells, but they also influence intracellular transport mechanisms and signaling [18, 19]. In particular, the expression of these filaments in certain types of epithelial cells provides a mechanism of resistance to apoptosis. For instance, K8/K18 filaments in hepatocytes impair cytokine receptor trafficking and cell surface expression [20–22]. Whether or not K8/K18 filaments similarly impair Fas expression on luteal cells has not been tested.
In the present study, the objective was to quantify Fas expression on bovine luteal cells during the early developmental (Day 5, postovulation) and late functional stages (Days 16–18, postovulation) of the CL, examine the relationship between luteal Fas and K8/18 filament expression, and assess the susceptibility of the luteal cells to cytokine-induced death. Our null hypothesis was that the surface expression of Fas on luteal cells does not differ between the two stages of corpora lutea (i.e., early vs. late stage CL). In addition, we tested whether the disruption of K8/K18 filaments in the luteal cells increases the cell surface expression of Fas, and thus their susceptibility to cytokine-induced apoptosis. Experimentally, cultures of bovine luteal cells from early and late stage CL were exposed acutely to acrylamide to disrupt the K8/K18 filaments. The effects of filament disruption on Fas expression and cytokine-induced apoptosis were then measured.
All animal studies described herein were approved by the UNH Institutional Animal Care and Use Committee (IACUC# 090205). Estrous cycles of Holstein dairy cows were monitored using transrectal ultrasonography, and corpora lutea (CL) were removed by colpotomy at days 5 (early stage; n=6 cows) and 16–18 (late stage; n=9 cows) postovulation (ovulation = day 0). Luteal cells obtained from CL at these two stages of luteal function express relatively high and low amounts of keratin intermediate filaments, respectively, based upon previous findings [16, 23]. Prior to CL removal, blood samples were obtained by coccygeal venipuncture using heparinized tubes to measure plasma progesterone concentration and verify the relative stage of the estrous cycle. Corpora lutea and blood samples were transported to the laboratory on ice where the CL were extracted for total RNA (described below) and enzymatically dissociated using collagenase type I (Worthington, Lakewood, NJ) as described previously by others . Following enzymatic dissociation, the viability of the luteal cells was estimated to be 88-93% as determined by trypan blue exclusion. The dissociated luteal cells were then either freshly-fixed in paraformaldehyde for flow cytometric analysis, or placed in serum-free culture for further experimentation (described below). The heparinized blood samples from the cows were centrifuged at 2056xg for 20 min at 4°C to obtain plasma, which was then frozen at −20°C until assayed for progesterone by radioimmunoassay (RIA) as described previously .
Forward and reverse primers, respectively for bovine Fas were: 5′-ATGGGCTAGAAGTGGAACAAAAC-3′ and 5′- TTCTTCCCATGACTTTGATACC-3′. Forward and reverse primers, respectively, for bovine β-actin were: 5′- GAGGATCTTCATGAGGTAGTCTGTCAGGTC-3′ 5′-CAACTGGGACGACATGGAGAAGATCTGGCA-3′.
A thermal cycler was used to conduct the Q-RTPCR with the cyclic conditions as follows: an initial Taq activation at 95°C for 2 min, followed by 40 cycles of 95°C for 1 second, 55°C for 30 seconds and 72°C for 30 seconds. All reactions were carried out on a 7500 Fast Real-Time PCR System. The data were collected during the last 30 seconds of cycling and the amplification signals of Fas transcripts were quantified using a standard curve based upon an absolute quantitation method. The results were expressed as a ratio of Fas relative to β-actin transcripts as the reference (i.e., internal control gene). Melting curve analysis was performed with conditions as follows: 95°C for 15 seconds, 60°C for 1 min, and 95°C for 15 seconds.
Freshly dissociated luteal cells were seeded in T25 flasks at a density of 2×106 viable cells/flask and in 8-well microchamber slides at 2×104 viable cells/well. The cells were cultured in serum-free Ham's F12 culture medium (Invitrogen, Carlsbad, CA) supplemented with insulin, transferrin, selenium (ITS; 5μg/5μg/5ng/mL; Sigma Aldrich, St. Louis, MO) and gentamicin (20μg/mL; Invitrogen, Carlsbad, CA) and incubated at 37°C, 5% CO2 in air and 95% humidity overnight. The purity of the cultures under these serum-free conditions is estimated to be 70-75% steroidogenic cells because other types of cells (e.g., endothelial cells, fibroblasts, etc.) are unable to persist. The day after seeding, the flasks and chamber slides were rinsed and the conditioned medium replaced with fresh culture medium prior to treatments. Initial treatments consisted of flasks and chamber slides treated with either culture medium (control) or 5mM acrylamide (Fisher Scientific, Pittsburgh, PA) for 4 h to disrupt K8/K18 filaments and potentially increase the cell surface expression of Fas. Acrylamide is a selective, reversible, disrupter of K8/K18 filaments in mammalian cells  that under short-term culture conditions does not adversely affect microtubules [28, 29], organelles (e.g., mitochondria, ), steroid synthesis , or cell viability . After the initial 4 h treatment period, all flasks and chamber slides were rinsed twice and the medium replaced. Cells from several flasks were immediately prepared for flow cytometric analysis of Fas and K8/K18 expression as described below. The remaining flasks were treated with a cytokine cocktail containing bovine interferon-γ (IFN, 200 IU/mL; R&D Systems, Minneapolis, MN), murine tumor necrosis factor-α (TNF, 10ng/mL; US Biological, Swampscott, MA), and human recombinant soluble Fas ligand (FasL, 50ng/mL; R&D Systems, Minneapolis, MN) with a murine monoclonal anti-6x histidine cross-linking antibody (1mg/mL; R&D Systems, Minneapolis, MN) for 24 h to induce cell death. Others have previously shown this mixture of cytokines is appropriate, and necessary, to induce Fas-mediated death of bovine ovarian steroidogenic cells in vitro[5, 6, 26, 32, 33]. After 24 h incubation, the flasks were re-treated with the cytokine cocktail for an additional 24 h, prior to assessment of cytokine-induced cell death.
Murine hepatocytes were among the first cells used to demonstrate that disruption of K8/K18 filaments enhances Fas trafficking to the cell surface . Here we utilized human hepatocyte carcinoma cells (HepG2 cells) to corroborate this finding under the experimental conditions used to disrupt K8/K18 filaments in bovine luteal cells with acrylamide. Briefly, HepG2 cells were seeded into T150 flasks at 2×106 cells/flask. The cells were cultured in Eagle's Minimal Essential Medium (Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS) and incubated at 37°C, 5% CO2 and 95% humidity. At approximately 70% confluency, the HepG2 cells were subcultured in T25 flasks using approximately 1x106 cells/flask. The following day, the medium was changed and the cultures were exposed to vehicle (control) or 5mM acrylamide for 4 h. Following treatment, the cultures were prepared for flow cytometry to assess cell surface expression of Fas.
Luteal cells from freshly dissociated CL and from serum-free culture were used to analyze Fas and K8/K18 filament expression by flow cytometry. For cells obtained through dissociation of CL, approximately 1.5x106 cells/tube in 0.4mL of Ham's F12 culture medium were centrifuged using screen-capped tubes (Ref # 352235, BD Falcon, San Jose, CA) for 5 min at 276xg, 4°C. The screened cells were then fixed for 2 h on ice by adding 0.4mL 2% paraformaldehyde to the cell suspension for a final concentration of 1% paraformaldehyde. After fixation, the cells either remained in fixative (for detection of cell surface Fas) or were rinsed twice with PBS and then permeabilized using 70% ethanol (for detection of total Fas and K8/K18 filament expression). Both the fixed and permeabilized cells were stored at 4°C and −20°C, respectively, until further processed for flow cytometry.
Luteal cells in serum-free culture and the HepG2 cells cultured in serum-containing conditions were fixed in a similar manner to the freshly isolated luteal cells described above. Briefly, the flasks of cells were rinsed twice (5 min each) with Hank's Balanced Salt Solution (Sigma Aldrich, St. Louis, MO), followed by two quick washes with trypsin-EDTA (Cell Gro Mediatech, Manassas, VA). After the second trypsin-EDTA rinse, the remaining trypsin was removed and the flasks were left for 10 min. The trypsinized cells were then collected in Ham's F12 culture medium containing 10% fetal bovine serum (JRH Biosciences, Lenexa, KS), centrifuged for 5 min at 276xg, 4°C and resuspended in Ham's F12 culture medium without serum. As above, approximately 1.5x106 cells/tube were centrifuged using screen-capped tubes for 5 min at 276xg, 4°C. The filtered cells were fixed for 2 h on ice in 1% paraformaldehyde and either remained in fixative (detection of cell surface Fas; luteal and HepG2 cells) or were permeabilized with 70% ethanol (detection of total Fas and K8/K18 filaments; luteal cells only). Both the fixed and the permeabilized cells were stored at 4°C and −20°C, respectively, until analyzed by flow cytometry.
Bovine luteal cells cultured in microchamber slides were used to evaluate microscopically the efficacy and specificity of acrylamide as a disrupter of K8/K18 filaments. The cells were rinsed twice with PBS, fixed using 4% paraformaldehyde in PBS for 20 min on ice, and then stored in PBS at 4°C until permeabilized with methanol and analyzed for K8/K18 expression and microtubule expression (negative control) by fluorescent microscopy. Briefly, the previously-fixed luteal cells were rinsed twice with PBS-BSA followed by a 1 h block/permeabilization step with 0.3% triton x-100 in PBS containing 10% normal goat serum (Vector Labs, Burlingame, CA) and 3% BSA. The slides were rinsed 3 × 5 min with PBS-BSA and incubated overnight at 4°C with either a mouse anti-human K18 monoclonal antibody (clone CY-90; Sigma Aldrich, St. Louis, MO; diluted 1:800 in PBS-BSA with 10% normal goat serum), or a mouse anti-bovine alpha-tubulin monoclonal antibody (clone 236–10501; Invitrogen, Carlsbad, CA; diluted 1:200 in PBS-BSA with 10% normal goat serum). The following day, after 3 × 5 min washes with PBS-BSA, fluorescent detection of the K18-containing filaments or tubulin-containing microtubules was achieved by incubating the slides with a goat anti-mouse Alexa 488-conjugated IgG antibody (K18; Invitrogen, Carlsbad, CA) or a goat anti-mouse Texas Red-conjugated antibody (microtubules; Santa Cruz, Santa Cruz, CA). Both secondary antibodies were diluted 1:200 in PBS-BSA with 10% normal goat serum (Vector Labs, Burlingame, CA). The slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) mounting medium (Vector, Burlingame, CA) and then coverslipped.
The data were analyzed by 1-way or 2-way ANOVA followed by Tukey's multiple comparison test using the general linear model of Systat 12.0 (Point Richmond, CA). Results are expressed as mean ± SEM, with each experiment repeated three to nine times (i.e., n= 3–9). For experiments requiring cultured cells, the cells were cultured in triplicate for a given experiment and were derived from individual CL or from a frozen stock of cells (HepG2 cells). Thus, the total number of experiments (n=) is equivalent to the total number of CL or frozen aliquots (HepG2) used to establish the cultures; shown in figure legends). Differences among means at a value of P<0.05 were considered statistically significant.
Freshly dissociated luteal cells from early and late stage CL were characterized for total Fas expression (Figure 1) and expression on the cell surface (Figure 2) relative to a non-specific IgG control. Measurement of plasma progesterone revealed the cows used to obtain early stage CL had lower systemic progesterone than cows used for late stage CL (1.8 ± 0.2 versus 5.9 ± 0.7 ng/ml, respectively; P<0.05, n=6-9 CL/stage). However, a higher percentage of luteal cells expressed total Fas in early stage CL compared to late stage CL (Figure 1A, P<0.05). Mean fluorescence intensity (MFI), a measure of staining intensity for each cell, was also higher among cells from early stage CL compared to late stage CL (Figure 1C,P<0.05). Similarly, the expression of Fas on the cell surface was greater for cells of early stage CL compared to late stage CL (Figure 2B, P<0.05), as was MFI (Figure 2C, P<0.05). Overall, quantification of the percentage of cells expressing Fas on the cell surface relative to total Fas expression revealed cells from early stage CL express the majority of Fas on the cell surface (76%), whereas less than half these cells from late stage CL do so (47%). In terms of relative steady-state concentrations of Fas mRNA in the luteal tissue, Q-RTPCR indicated there was no difference between early versus late stage CL (Figure 3; P>0.05, n=5-7 CL/stage).
Flow cytometric analysis of total Fas in cells of early and late stage bovine CL. A representative histogram depicting the total amount of Fas detected in bovine luteal cells of early and late stage CL is shown (Figure 1A). Relative number of cells expressing total Fas is depicted for early versus late stage CL (Figure 1B). Relative mean fluorescence intensity (MFI) is also depicted for the two stages of CL (Figure 1C). Values shown are mean ± SEM; different letters indicate significant differences (P<0.05; n=6-9 CL/stage).
Flow cytometric analysis of Fas expression on the surface of cells of early and late stage bovine CL. A representative histogram depicting the expression of Fas on the surface of bovine luteal cells of early stage and late stage CL is shown (Figure 2A). Relative number of cells expressing Fas on the cell surface is depicted for early versus late stage CL (Figure 2B). Relative mean fluorescence intensity (MFI) for the two stages of CL is also shown (Figure 2C). Values shown are mean ± SEM; different letters indicate significant differences (P<0.05; n=6-9 CL/stage).
Relative expression of Fas mRNA in bovine CL during the early and late stages of the estrous cycle. Values shown are mean ± SEM fold-change of Fas expression (normalized using β-actin). Different letters indicate significant differences (P<0.05; n=5-7 CL/stage).
Interestingly, a comparison of Fas expression for freshly dissociated luteal cells versus luteal cells placed in culture for 24 h revealed that culture alone substantially increased the relative cell surface expression of Fas for cells of both early and late stage CL. Cell surface expression of Fas increased from ~65% to ~97% as a result of culture for cells of early stage CL (P<0.05, n=4 expts.), and from ~18% to ~66% for cells of late stage CL (P<0.05, n=4 expts.).
A higher percentage of freshly dissociated luteal cells from early stage CL expressed K8/K18 filaments than late stage CL (Figure 4, P<0.05). Average number of cells expressing K8/K18 filaments in early stage CL was 46% compared to 26% for late stage CL (Figure 4B). In contrast to what was observed for cell surface expression of Fas, culture of luteal cells for 24 h did not enhance K8/K18 expression in cells of early or late stage CL. Relative percentage of K8/K18-positive cells was 46% vs. 49% for freshly dissociated vs. cultured cells, respectively, in early stage CL, and was 26% vs. 23% for freshly dissociated vs. cultured cells, respectively, in late stage CL (P>0.05, n=4 expts., data not shown).
Flow cytometric analysis of K8/K18 expression in cells of early and late stage bovine CL. A representative histogram depicting the expression of K8/K18 filaments in bovine luteal cells of early stage and late stage CL is shown (Figure 4A). The relative number of cells expressing K8/K18 filaments is depicted for early stage CL versus late stage CL (Figure 4B). Values shown are mean ± SEM; different letters indicate significant differences (P<0.05; n=6-9 CL/stage).
Exposure of cultured bovine luteal cells to acrylamide disrupted K8/K18 filaments without adversely affecting microtubule organization (Figure 5). Cells in control cultures exhibited extensive, filamentous networks of K8/K18 staining (Figure 5A) that became aggregated around the perinuclear region of the cells following acrylamide exposure (Figure 5C). Conversely, microtubule organization when compared between control and acrylamide-treated cultures remained unaffected (Figure 5B and D, respectively). In addition, there was no observable effect of stage of CL on these outcomes, and the acrylamide treatment overall had no effect on the number of cells expressing K8/K18 filaments, luteal cell viability or progesterone secretion (P>0.05; n=2-4 CL/stage, data not shown).
Fluorescent detection of K8/K18 filaments and microtubules in control and acrylamide-treated cultures of bovine luteal cells. K8/K18 filaments (green fluorescence) and microtubules (red fluorescence) were immunostained in cultured bovine luteal cells following 4 h exposure to vehicle (Control, Figure 5A and B) or 5mM acrylamide (Figure 5C and D). Cells in control cultures exhibited a filamentous, K8/K18 intermediate filament network which spanned the cytoplasm (Figure 5A). Microtubles of these cells was similarly filamentous (red fluorescence; Figure 5B). Conversely, cells of acrylamide-treated cultures exhibited peri-nuclear aggregation of K8/K18 filaments (Figure 5C), yet the microtubules were unaffected (Figure 5D). Magnification: 40x.
Although acrylamide disrupted K8/K18 filaments, no increase in the cell surface expression of Fas was observed for luteal cells of either stage of CL (Figure 6A-C; P>0.05). Moreover, K8/K18 filament disruption failed to enhance Fas cell surface expression on specific cells, as reflected by the lack of change in relative MFI (Figure 6D; P>0.05). Consistent with the observations of freshly isolated luteal cells, cultured luteal cells of early stage CL expressed higher amounts of Fas on the surface than cultured cells of late stage CL (Figure 6C and D; P<0.05). In contrast, disruption of K8/K18 filaments in HepG2 cells, using identical experimental conditions to those for bovine luteal cells, increased the number of cells expressing Fas on the cell surface (Figure 7; P<0.05).
Flow cytometric analysis of Fas expression on the surface of cells of early and late stage bovine CL following K8/K18 filament disruption with acrylamide. Representative histograms depicting the expression of Fas on the surface of bovine luteal cells of early stage and late stage CL are shown (Figure 6A and B, respectively). The relative percentage of cells expressing Fas on the cell surface is depicted for early versus late stage CL, and for control versus acrylamide-treated cells (Figure 6C). Relative mean fluorescence intensity (MFI) is also depicted for the two stages of CL and the treatment conditions (Figure 6D). Values shown are mean ± SEM; different letters indicate significant differences (P<0.05; n=4 CL/stage).
Flow cytometric analysis of Fas expression on the surface of HepG2 cells following K8/K18 filament disruption with acrylamide. A representative histogram depicting the expression of Fas on the surface of HepG2 cells is shown (Figure 7A). The relative percentage of cells expressing Fas on the cell surface is depicted for control versus acrylamide-treated cells (Figure 7B). Values shown are mean ± SEM; different letters indicate significant differences (P<0.05; n=3 expts.).
Exposure of the cultured bovine luteal cells for 48 h to a cytokine cocktail consisting of IFN, TNF, and FasL induced cell death, as expected, but there was no effect of K8/K18 disruption by acrylamide (P>0.05) or stage of CL (P>0.05, n= 4 CL/stage) on this outcome (Figure 8). Similar results were observed when the luteal cells were exposed to cytokines and acrylamide for only 24 h (data not shown).
Cell death in cultured luteal cells from early and late stage bovine CL following exposure to acrylamide and cytokines. The relative percentage of cells undergoing death after 48 h exposure to cytokines (Cyto) is depicted for early and late stage CL (Figure 8A and B, respectively). The cultures were also exposed to a 4 h pretreatment with 5mM acrylamide (Acryl) to disrupt K8/K18 filaments before cytokine treatment. Values shown are mean ± SEM; different letters indicate significant differences (P<0.05; n=3 expts.).
The current study is the first to directly measure relative changes in the expression of Fas on the surface of bovine luteal cells across the estrous cycle. The observation of quantifiably higher Fas receptor expression on luteal cells from early stage compared to late stage CL was unexpected and somewhat contrary to what was anticipated based upon earlier published studies. In these studies, the investigators examined the gross expression of Fas mRNA [5, 7] and protein [34, 35] in ovarian tissues, without reference to cell-specificity and they found that Fas increased only in CL undergoing regression. In the current study, Fas protein was quantified for individual cells obtained from CL following tissue dissociation and cell culture, and then analyzed using flow cytometry. Similarly, Fas mRNA expression for the two stages of CL was measured by Q-RTPCR. The current methods are arguably more quantitative than the mRNA detection, immunoblot analysis, and immunohistochemistry methods described in the cited studies, but fall short of identifying specific cell type(s) known to exist within the CL. Nevertheless, dissociation of the luteal tissue and establishing serum-free culture conditions, as described, removes many of the various cell types, while enriching the population of luteal steroidogenic cells. Thus, we suggest the pattern of Fas expression observed in the current study is essentially representative of the luteal steroidogenic cell population within the bovine CL at the two extremes of the estrous cycle. Moreover, our observation of no measureable difference in relative steady-state amounts of mRNA for Fas in early versus late stage CL, as evaluated by Q-RTPCR, is consistent with a previously published study .
Overall, a 72% decline in the number of bovine luteal cells expressing Fas at their cell surface, and a 59% decline in the density of Fas expressed at the cell surface across the estrous cycle was observed. Total Fas expression (surface and intracellular) for freshly isolated cells was higher for early stage CL than late stage CL. A similar difference in Fas surface expression was observed for cultured luteal cells, but was further enhanced by culture alone. Exposure of the cultured cells to the cytokine cocktail of IFN + TNF + FasL, however, resulted in similar estimates of cell death for both stages of CL. This indicates cultured luteal cells from both stages of CL are equally vulnerable to cytokine-mediated cell death despite clear differences in the cell surface expression of Fas.
The observation that Fas expression is elevated on luteal cells of early stage CL without further enhancing their susceptibility to cytokine-induced death indicates mechanisms exist to protect the cells against Fas-induced apoptosis. For instance, a soluble secreted isoform of Fas has been identified in other tissues that sequesters FasL prior to binding at the target cell surface, thus preventing cell death [36–38]. This isoform of Fas lacks the transmembrane domain of wild-type Fas, causing it to be secreted rather than expressed on the surface of cells . The murine ovary expresses a soluble form of Fas, which has protective effects . Thus, it is possible a soluble form of Fas exists within the bovine CL to modulate the effect of elevated Fas expression in early stage CL as seen in the current study. Certainly this possibility merits additional exploration.
Another intrinsic "protective" mechanism of cells of early stage bovine CL might include the expression of membrane-bound splice variants of the Fas receptor. The cytokine TRAIL (TNF-related apoptosis-inducing ligand) for example, which is structurally similar to FasL, binds to receptors, DR4 and DR5, yet membrane-bound decoy receptors also exist for TRAIL. These receptors, named DcR1 and DcR2, have a cytoplasmic domain structurally similar to DR4 and DR5, respectively, but lack the intracellular death domain necessary for transmitting an apoptotic signal [39–41]. Recently, Sugimoto and coworkers identified a putative Fas decoy receptor, DcR3, in granulosa cells of porcine ovaries . Similar to DcR1 and DcR2, DcR3 contains an extracellular and cytoplasmic domain similar to wild-type Fas, but lacks the intracellular death domain. Unlike soluble Fas, the decoy receptor is expressed on the plasma membrane and retains its ability to bind FasL, but does not induce cell death . It is tempting to speculate that a decoy receptor of Fas may exist on bovine luteal cells, explaining the high prevalence of Fas expression observed for cells of early stage CL, but not late stage CL, in the current study. Further research is needed to determine whether or not a Fas decoy receptor exists within the bovine ovary, and to explore its possible role in ovarian function.
Alternatively, enhanced expression of Fas on cells of early stage CL can be explained by a non-apoptotic or even proliferative role of Fas in the early stage CL. In recent years, diverse non-apoptotic functions of Fas have been documented , such as the acceleration of liver regeneration after partial hepatectomy , the induction of cell migration and invasiveness of apoptotic-resistant tumor cells , and the stimulation of cardiomyocyte hypertrophy . The ability of Fas to control the fate of the cell likely hinges on the regulation of Fas-induced downstream signaling events, such as activation/inhibition of the ERK, JNK, p38, and NF-κB pathways. These same pathways have suggested roles in luteal cell function and fate [5, 48–50], but their influence on the developing early CL, especially in the context of elevated Fas expression, is unknown. Overall, the concept that Fas might facilitate development of the CL is consistent with the premise suggested by Pate and Keyes , in which immune-response mechanisms exist within the ovary to abate damaging inflammatory responses caused by dead or dying cells. In the current scenario, these cells would arise from postovulation trauma during the initial development of the CL.
In the present study, acrylamide selectively disrupted the K8/K18 filaments in the luteal cells, but did not enhance Fas expression or otherwise influence Fas-mediated cell death. In effect, this result did not support the concept that K8/K18 filaments influence Fas trafficking at the cell surface. However, acrylamide causes intermediate filaments to only partially disassemble and undergo acute dephosphorylation . Dephosphorylation provokes a 50% loss of phosphate from the keratin protein which corresponds with the morphological changes observed for intermediate filament expression . At best, the dephosphorylation event is transient, and the striking changes in intermediate filament organization are reversible. In fact, the filaments re-establish their 'net-like' organization generally within 12 h after acrylamide removal , and complete rephosphorylation occurs within 18 h . In the current investigation, the K8/K18 filaments of bovine luteal cells were exposed to acrylamide for only 4 h. This was sufficient time to noticeably disrupt the filaments, but perhaps insufficient to sustain a change in Fas trafficking or in downstream signaling that would otherwise enhance cell death. It is noteworthy, however, that these same conditions increased Fas expression on HepG2 cells in the current study. For the time-being, we cannot reject the possibility that K8/K18 filaments influence events downstream from Fas binding; however, it seems unlikely that the filaments directly impair Fas expression on the cell surface as has been suggested in other studies [20–22].
In conclusion, the elevated expression of Fas on cells of early stage bovine CL compared to late stage bovine CL raises a provocative question concerning the physiological role(s) of Fas in the corpus luteum. Although there is little doubt about the apoptotic function of this receptor during luteal regression, its purpose during early luteal development has yet to be defined. We suggest, as others do, that a broader view of Fas-mediated activities merits consideration, including the need to identify the signaling components linking Fas to non-apoptotic pathways. These insights may provide new targets to influence fertility, and treat diseases such as inflammation and cancer.
Alice Duncan, Jennifer Forcina contributed equally to this work.
This work was supported by National Research Initiative Competitive Grant no. 2007-35203-18074 from the USDA National Institute of Food and Agriculture. Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is Scientific Contribution Number 2491. The authors wish to thank Sarah Kinsman, Amanda Rivers, and Mark Townley for their contributions to this manuscript.
AD and JF equally carried out the cell culture studies, performed the flow cytometric analyses, conducted the microscopy work and assisted in the preparation of the manuscript. AB conducted the Q-RTPCR analysis. DT conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


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at the Adler Planetarium*
Resources for classes, field trips, and outreach programs
at the Discovery Center Museum*
Inner-city K-12 enrichment program in science
at the Kavli Institute for Cosmological Physics (KICP) at the University of Chicago*
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Kavli Institute for Cosmological Physics (KICP) at The University of Chicago connects local, middle and high school, under-represented minority students with the university research community*
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The Website from Fermi National Accelerator Laboratory contains K-12 program information and instructional materials and resources for educators and students
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Explore your options in this week long summer camp held at the University of Illinois Urbana-Champaign campus*
Scientific Learning Programs
Whether you are a K-12 student, undergraduate, graduate student, or faculty member, the Division of Educational Programs (DEP) at Argonne National Laboratory has a program that will engage you in a scientific learning experience


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Lucie Bertoldo
Sports Journalist
Lucie Bertoldo is a sports reporter for the Canberra Times.
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Finals dream of Commerford's Chill reignited by Tigers upset
3 Nov 2019, 7:58 p.m.
History threatens to repeat itself at PM's XI
23 Oct 2019, 2 a.m.
Breen to boost Olympics bid in Berlin with career first
14 Oct 2019, 11:54 a.m.
Australia thrash Nepal to continue impressive start to qualifying bid
10 Oct 2019, 7:26 p.m.
British Beckie's back to beat bronze
28 Sep 2019, 6:19 p.m.
Caroline given all clear to crash
21 Sep 2019, 3 p.m.
Staunton strikes for finals after 400th match
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Q: how can I filter on the second element in a tuple of tuples? In my model I have a field:
   country = models.CharField(_('Country'), max_length=2, choices=COUNTRIES)

Where COUNTRIES is a tuple of tuples like this:
COUNTRIES = (
    ('AF', _('Afghanistan')),

... and so on
Now I want to filter an instance of that model, by the country name.
This:
   i = MyModel.objects.filter(country__iexact=query)

only lets me filter by the country code.
How can I filter by country name?

A: You cannot filter directly by the country name (the choices are only used in the UI, not in the database).
If you get the full name as an input, lookup the code in the COUNTRIES tuple-of-tuples. For example:
# ... initialize a lookup dictionary
country_to_id_dict = dict((t[1], t[0]) for t in COUNTRIES)

# ... use the dictionary in the query
i = MyModel.objects.filter(country__exact=country_to_id_dict[query])

redpajama_set_name: RedPajamaStackExchange


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Apart from the unnecessary electricity consumption, there are other disadvantages attached to it too, for instance if there is a power failure, you' ll have to begin the entire downloading procedure again ( though certain sites or systems give you the option of resuming it from where it was left; but most of them don' t).


Kilrush village lies at the mouth of the River Shannon in West Clare.
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Of all the books I read in college one of the most valuable and one of the few that I still find myself returning to regularly is Dorothy Sayers' The Man Born to Be King, a cycle of twelve plays about the life Christ that was written and produced for BBC radio during World War II. If you want to get a better grasp on "the historical Jesus" this is a far better place to start than some dense tome of New Testament scholarship. Sayers weaves together all of the main details of the gospel accounts into a single tale that succeeds at being both a believable harmonization and being genuinely entertaining. And her detailed introduction and extensive character notes make it easy to imagine oneself inside the stories, to see them as living and breathing things.
[Shepherd's Wife]: Come in, my lords, come in. Please mind your heads. I fear 'tis but a poor, lowly place.
Caspar: No place is too lowly to kneel in. There is more holiness here than in King Herod's Temple.
Melchior: More beauty here than in King Herod's palace.
Balthazar: More charity here than in King Herod's heart.
Caspar: O lady clear as the sun, fair as the moon, the nations of the earth salute your son, the Man born to be King. Hail, Jesus, King of the Jews!
Melchior: Hail, Jesus, King of the World!
Balthazar: Hail, Jesus, King of Heaven!
Caspar, Melchior, & Balthazar: All hail!
Mary: God bless you, wise old man; and you, tall warrior; and you, dark traveller from desert lands. You come in a strange way, and with a strange message. But that God sent you I am sure, for you and His angels speak with one voice. "King of the Jews"––why, yes; they told me my son should be the Messiah of Israel. "King of the World"––that is a very great title, yet when he was born, they proclaimed tidings of joy to all nations. "King of Heaven"––I don't quite understand that; and yet indeed they said that he should be called the Son of God. You are great and learned men, and I am a very simple woman. What can I say to you, till the time comes when my son can answer for himself?
Caspar: Alas! the more we know, the less we understand life. Doubts make us afraid to act, and much learning dries the heart. And the riddle that torments the world is this: Shall Wisdom and Love live together at last, when the promised Kingdom comes?
Melchior: We are rulers, and we see that what men need most is good government, with freedom and order. But order puts fetters on freedom, and freedom rebels against order, so that love and power are always at war together. And the riddle that torments the world is this: Shall Power and Love dwell together at last, when the promised Kingdom comes?
Balthazar: I speak for a sorrowful people––for the ignorant and the poor. We rise up to labour and lie down to sleep, and night is only a pause between one burden and another. Fear is our daily companion––the fear of want, the fear of war, the fear of cruel death, and of still more cruel life. But all this we could bear if we knew that we did not suffer in vain; that God was beside us in the struggle, sharing the miseries of His own world. For the riddle that torments the world is this: Shall Sorrow and Love be reconciled at last, when the promised Kingdom comes? . . .
. . . Mary: These are very difficult questions––but with me, you see, it is like this. When the Angel's message came to me, the Lord put a song into my heart. I suddenly saw that wealth and cleverness were nothing to God––no one is too unimportant to be is friend. That was the thought that came to me. I am quite humbly born, yet the Power of God came upon me; very foolish and unlearned, yet the Word of God was spoken to me; and I was in deep distress, when my Baby was born and filled my life with love. So I know very well that Wisdom and Power and Sorrow can live together with Love; and for me, the Child in my arms is the answer to all the riddles.
Caspar: You have spoken a wise word, Mary. Blessed are you among women, and blessed is Jesus your son. Caspar, King of Chaldaea, salutes the King of the Jews with a gift of frankincense.
Melchior: O Mary, you have spoken a word of power. Blessed are you among women, and blessed is Jesus your son. Melchior, King of Pamphylia, salutes the King of the World with a gift of gold.
Balthazar: You have spoken a loving word, Mary, Mother of God. Blessed are you among women, and blessed is Jesus your son. Balthazar, King of Ethiopia, salutes the King of Heaven with a gift of myrrh and spices.
Zillah: Oh, look at the great gold crown! Look at the censer all shining with rubies and diamonds, and the blue smoke curling up. How sweet it smell––and the myrrh and aloes, the sweet cloves and the cinnamon. Isn't it lovely? And all for our little Jesus! Let's see which of his presents he likes best. Come, Baby, smile at the pretty crown.
Wife: Oh, what a solemn, old-fashioned look he gives it.
Wife: He likes the tinkling of the silver chains.
Joseph: He has stretched out his little hand and grasped the bundle of myrrh.
Wife: Well, there now! You never can tell what they'll take a fancy to.
Mary: Do they not embalm the dead with myrrh? See, now, you sorrowful king, my son has taken your sorrows for his own.
I have read this passage dozens of times, and I still cannot reach the end with dry eyes. The image Sayers' wordless infant Jesus, reaching past the other gifts to grasp hold of the symbol of sorrow touches something in me deeper than I know how to fully express. All I know is that this is the King that my true, damaged self needs––the one who has come for our sorrows and who is one day coming again.


I see the strike culture seems to be coming back, although I expected this in the days of Gordon Brown. British Airways ( the world's favourite luggage losers) have already started. followed by British Telecom, this announced in today's news. (are either of these Chinese owned yet).
You have to support the right to strike,wewill have kids up chimneys again if we do not have rights re employment.
I always find that those people who are against strikes are in comfortable secure jobs with good pay. Does that describe your situation SB.
What you describe as "The Strike Culture" is the only way that a working man can get a fair deal from employers. OK at one time the unions were calling the workforce out for silly reasons. But the explanation was simple most of the higher union posts were taken up by communists and other agitators and they had another agenda.
This situation was allowed to develop because the average working man could not be bothered to attend union meetings or stand for office.
I was never in a union for any great length of time and there was never one in my own firm, but I would have never stopped the lads having one if they wanted it.
I have always been and always will be a strong supporter of the unions, because I just cannot think what this country would be today without one. It would be full of people like a certain ex female prime minister of this country and a certain CEO of BA or the High Court Judge who gave judgement against UNITE on a ridiculous technicality.
No not the sort of union bashing world I would want to live in, but I suppose with the Conservatives holding the reins to the country it's all we have to look forward to. Roll on the election next year.
There is nothing in my post that says I am against striking (for the right reasons).
I was a union rep for thirteen years. In fact if you read any of my previous posts I have critcised our government for years.
The unions should have been more forceful in recent years.
The unions should have been more forceful in the MT years. But most of the unions and their membership were content to sit back in their nice comfortable and well paid jobs and forget that those high wages and rights were fought for and gotten by the unions.
And when others fought to save their jobs they were content to sit back with the attitude "I'm alright Jack"
They ably assisted MT to dismantle our industry and some are to be held more responsible than her.
I could weep when I think what has happened to us all,remember the workers of old that put everything they had on the line for a better life.


Forget all that you once knew about scavenger hunts and prepare for the most packed weekend of your Middlebury experience. The Hunt is a three day competition during J-term for students to try their hands at taking risks, pushing their comfort zones, and completing creative, wild tasks compiled by their all-powerful Hunt Masters. The challenges call on historical, theoretical, mathematical, and practical knowledge; creative thinking; strong organizational skills; boundless energy and enthusiasm; and an appreciation of the absurd.
Red Bull gives you wings. What else does it give you?
How long does it take to leap frog from Proctor to Ross?
Perform a bird's courtship dance.
Thread the needle in BiHall.
Participants can form teams of up to ten students and should come up with a fun name under which they will submit any videos, materials or simple answers to fulfill clues. The more tasks or clues completed, the more points earned for that team -- with particularly imaginative solutions often earning extra points. At the end of the three days, the Hunt Masters, along with some qualified judges, will review the materials submitted by each team and determine the winner.
This year's "Hunt Master" is Ben Freedman. If you have questions before September contact Alex Brockelman (abrockelman@middlebury.edu).


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There is no private parking but you can pull in briefly outside the property to unload. (Motor bike parking possible in the garage below the terrace).
Large enclosed sun terrace, with table and chairs, sun umberella and bench, acessed down two steps via the French Windows from the living room, or via a gate near the entrance door.