text:
If "waifu" is "wife"
should "kung-fu" be "knife" ?
thelastpsychiatrist.com - 8 Characteristics of Family Annihilators. Adnotated.
Thursday, 18 July, Year 11 d.Tr. | Author: Mircea Popescu
Mcfall murders - guess why
[He] beat his wife and daughteri to death with a rubber mallet before hanging himself...
Hugh McFall, 48, of Oswestry, Shropshire, was found hanged on 5 February, hours after wife Susan, 55, and Francesca, 18, were found dead.
An inquest heard how he left a note by his body saying "I hope I rot in hell"ii.
Why did he do it? More specifically: why does the news report say he did it?iii
No one is surprised when the news reports murders; they're interesting in a pornographic wayiv. ("Now I have these feelings, whatever shall I do with them?")
But the murder is secondary to the report; the murder is an excuse to release an already worked out narrative. Murder suspects and victims become unpaid actors for a reality show CBS calls "The News."v
An example from this week: Hundreds Attend Vigil for Slain Kansas Cheerleader.vi
When you click on the link, what do you want to see?
Here is a picture of a tiger and nitrogen while you consider your prejudices:
Be honest: you want to see a picture of a cheerleader.vii You sure as hell don't want to see a picture of a vigil -- but "vigil" = "pics of her while alive." Also: "Kansas"= white, which means she's hot. Otherwise they wouldn't have used the word "Kansas," they would have used the word "Trenton."
And what kind of a guy would kill her? How about "Rocco?"
Three burly cops="violent predator."viii Do I need to tell you what his criminal history is? If I told you that Rocco has a history of burglary but no prior rapes or sex offenses, would that make you suspect him less?ix No, because we all understand how a cheerleader might end up dead. Well, how did you come to understand that?x
GREAT BEND, Kan. -- A 14-year-old central Kansas girl whose charred remains were found at an asphalt plant last week was a vivacious teen who loved bright colors and preferred wearing flip flops over any other type of shoesxi, according to those who knew her.
Hmm. Doesn't really match the cheerleader type. In fact, this girl wasn't really a cheerleader -- she was going to be a cheerleaderxii when she started 9th grade in the fall. She was also going to be in geometry class, but they left that out.xiii
That he killed a cheerleader makes sensexiv; but killing some random 8th grader -- was he a first time pedophile?xv -- makes less sense.
Calling her a cheerleader certainly draws in the viewers, but at a huge societal costxvi. Most of us learn about murder through the news, and empirical evidence (news stories) tell us they're right, so we adopt their narratives. Narratives aren't necessarily bad -- unless they're wrong. I'm not saying he did/didn't do it; I am saying that when you put "cheerleader" in the headline, I am surer that he did it in spite of my attempts at being objective. I'm surer because you're surer.xvii
And narratives are hard to unlearn. Now that we know that she's an 8th grader and that he has no history of prior sex offenses, do we double back and give him the benefit of the doubt?
Filicides, the killing of your kids, is no different.
There are broadly five types: altruistic, psychotic, unwanted child, accident by neglect, spousal revenge. Although filicide is perpetrated in equal numbers by mothers and fathers, spouse + kid murderers are overwhelmingly men.xviii
That said, the media like to report on only three of these types of filicides: mothers who are psychotic (weak) ; women who are looking to get/please a new man (evil); and fathers committing "altruistic filicide" in which the father thinks he is sparing his family worse suffering by killing them (snapped).xix
The case of Hugh McFall (from the BBC):
A florist beat his wife and daughter to death with a rubber mallet before hanging himself amid fears he would lose a big customer, an inquest heard.
Hugh McFall, 48... was found hanged on 5 February, hours after wife Susan, 55, and Francesca, 18, were found dead.
An inquest heard how he left a note by his body saying "I hope I rot in hell".
We're about to get a standard altruistic murder story highlighting the role of the employment, which completely misses the important subtleties. Here are the more important ones, with some interventions.
1. He's not losing a job, he's losing his ability to keep up the lifestyle:xx
The self-employed flower salesman was facing accusations of invoice discrepancies from his main customer -- which had suspended his contract -- and feared a police investigation into his accounts... "His financial world had collapsed, his source of business income or at least 90 to 95% of it, had disappeared in a moment. Their lifestyle, as he knew it, would be over."
1b. The lifestyle often involves some kind of "soft" illegality (accounting irregularities, the use of drugs, etc). The news may cite jail as the main stressor; but the general fear is the irrreproducibility of the lifestyle (e.g. even if he doesn't go to jail, he'll never be able to make that kind of money again, legally.)
2. A sudden, temporary, but unshakable realization that there is no way out of this. "This is the end of me," "it's over," "I'm dead," etc.
Business associates told the hearing that Mr McFall had considered himself "finished"xxi after a meeting about alleged invoice discrepancies the day before his death.
If you hear a man say, "I'm finished", believe it. Especially if it doesn't seem as bad as he thinks it is. It's his inability to see alternatives (which would require another person's perspective) that makes him dangerous.xxii
3. While anyone can see how severe the problem is, no one else sees the problem as insurmountable -- except him. "Why didn't he just...?"
West Mercia Police said the case would probably not have ended up in court but an investigation would have been started if the owners of Stans Superstore had taken their concerns to police.
This is his inability to see things from another perspective except his own. What's obvious to you is not obvious to him, and opportunities to intervene can be missed if you think he "would have thought of that himself." Be concrete, be basic. "Look, are you a legal scholar now? Let's get a lawyer." A lawyer? Really? "Yeah -- I know a guy -- and let him tell you what he sees; if they can get OJ off, they can get you off. If nothing else, it's going to buy you some time..."
The longer he can experience his shame, the longer he will be able to live with his shame (or create a rationalization that will let himself live with it. The goal isn't to solve his problems, but delay him until he can think straight.xxiii
4. Media says family murders= financial problems, but the money is merely the cover for the real shame:
The coroner said Mr McFall's fears about his sexual health may have been "going through his mind" when he killed his family, as well as his business worries and concerns over his "social standing".
And from another article:
A computer expert and senior forensic investigator told the inquest he had examined two computers as part of the investigation, one from the McFall's home and the other from his business premises. [An investigator] said someone had been accessing pornography, escort and massage parlour sites and seeking advice for diagnosing HIV.
Illegal activity, affairs, drugs... the money is important because it hides those things, allows him to present himself as something he wants to be.
Sometimes the financial narrative is so compelling it seduces even experienced criminologists. Criminology professor David Wilson:
"They've previously had wealth, had possessions, they went on foreign holidays. The annihilator feels 'given that I can't give my family any of this any longer' as an act of almost mercy, as they would see it, 'I'll take their lives so as to prevent them having experiences of any hardship'."
Wrong, always. It is impossible to think your children are better off dead unless you are unable to see their perspective.xxiv If you asked them, what would they say? Why wouldn't you believe them? Why do you think you know better than they do?
They aren't better off dead; you're better off if they're dead. That's the secret that must be undone.
5. SHAME.
"I can't give them what they deserve" is a deflection from "I can't give them what they deserve." The panic is about them becoming aware of your failure.
[the criminologist] believes where they lived was a factor: "Oswestry is a face-to-face society. Those kinds of societies often provide a great deal of support, but if the wheel comes off in this type of society, then everybody knows your business... It's not like they are in a big city where they can simply disappear and become anonymous.
The same applies to honor killings.xxv
The money is most often the final straw; without the money, you can't keep up the appearances...
Murders happen before the exposure, before "everyone finds out." Once they've found out, there is no reason for the murder. So either tell his family, or make him think you have. But then:
6. Sometimes he kills himself, and sometimes he doesn't.
He kills his family because he can't face them knowing. He kills himself because he can't face that they know.
The other reason for the suicide is the sheer number of people who are going to know -- can't kill 'em all.xxvi
The likelihood of suicide increases as guilt increases, and decreases the more you can be convinced other people won't know/won't care.xxvii
7. A very fine line: it was both spontaneous, and premeditated.
Two years ago another family murder took place near Hugh McFall's town, and McFall was horrified:
I remember Hugh saying 'How could you do that? How can it get so bad that you could do that to your family?... It just doesn't make sense, surely there's other ways outxxviii however bad things get?'"
And certainly he wasn't thinking about killing his family before the financial problems hit. But as soon as he decides he's "finished," he starts planning the murders -- weapons/tools obtained, the biggest threat is killed first (i.e. wife before kids), etc.
7. Get out of the house, or at least the bedroom.
75% occur in the home, usually in the bedroom. It is extremely unusual (6%) for the man to kill only the children and not the wife. Since the purpose of this is to avoid shame, leaving the wife alive would be contrary to the point.
If you're the wife, don't go home, especially if he says he'll kill the kids if you don't.xxix
Holy god these people are fucking ugly.
How can these inbred fucks even stand themselves ? I mean... it's one thing for a misfortunate new life to come to its senses and find itself in the middle of fucking nowhere, complete orc shithole a la Belarus or Indiana or Argentina or whatever. But they get the fuck out just as soon as can attach some kind of wheels to some kind of plank, I guess, and that's that. At least there's trees to look at in the meanwhile or something.
Yet... to open your eyes to England ?! Just wake up one day for the first time and be surrounded by these ugly fucks ? Merely walking down the street's an exercise in unmitigated che va piangendo i suoi etterni danni ; that sack someone untalented but fucking insistent charcoaled a face upon "loves" you ?! The ugly can not fucking love ; as a matter of course I deem every single case of an Englisher not going postal and butchering everyone in sight a case of dumb-and-deafness, pretty much. They're too out of it to even notice what the fuck's going on around them at all, there's no other explanation fucking possibe.
How can these people be so ugly... [↩]
What do you call one ugly fuck rotting in hell ? [↩]
There's a few very good reasons :
Criminals do not kill people in their homes. Successful murder requires good control of the murder scene, and the one place any one ugly fuck likely controls better than a stranger is his own home. Who the fuck ever knows where he has a hidden can opener, where a floor plank gives way, where a neighbour drilled a hole in the wall to watch the family daughters change their stockings, etcetera. Whenever a murder's suspected in the home, a surviving resident's the first, second, third and last suspect for this most excellent reason -- really no more is needed. Indeed death can and does occur in the home -- it's by far the most popular place to die for the contemporaneous ugly fuck, as it happens -- but if it's not natural it's likely accidental, and if it's neither then the spouse/parent/children did it. Don't give me any of that "spies" bullshit, either -- Ogorodnik was arrested/killed at the entrance of his apartment. Not inside.
Criminals do not kill people with rubber mallets, or any other improvised instruments. Before guns, assassins used daggers and poison, but the principle remains : you need two things when setting out to kill someone, which are reliability and speed. A rubber ducky provides neither, and a rubber mallet is not much better.
Criminals also do not hang people, with the obvious exception of governments, I guess. This'd be self-evident if you ever killed someone, but anyway, for the lesser readership : what the fuck are you going to do ? Sit there and wait ? Waiting is hard, especially with a murder flag dangling in the wind ; people who want the victim to die strangled generally use their own hands ; everyone else picks a better tool than fucking rope. Hanging is however enduring on the preference list of suicides, and for very good (and well understood) psychological reasons.
Is three good enough ? [↩]
They're only interesting "in a pornographic way" recently ; before you gave up on your wife (by stopping beating her), and on society, and on everything else murders were interesting in a world-control sort of way. Are you aware how John Heath died, for instance ? Actually : are you aware how many deaths of reputed Wild West gunslingers were the result of "group of 100+ German miners did not see the humour in newspaper stories about the fellows' exploits" ? Well then.
Newspapers still report murders today because not so long ago -- seriously, this beauty that went on her knees for men because men made her go on her knees for them was born barely long ago to be someone's great-grandmother ; say it again with me, you useless "maga" twerps : not. so. long. ago. -- people actually read them. I don't mean "read" them, like you "read" right after "having sex". I mean read-read, the sort of reading that drives action.
What would you have them do, admit nobody reads anymore, because no, "reading" is no fucking substitute for reading ? And then what, deadline ? Hang themselves with a rubber mallet ? So then!
Come to think of it -- why are you still derping about ? Nobody cares about your feelings nor whatever other assorted crap goes on in that rotting pumpkin of yours, why are you still here to preciously inquire why the newspapers are still reporting your failure to exit in a timely manner on your own fucking power ? Hm ?
So then don't fucking ask about them. They're still here for the same reason you're still here : cancer. Things that used to be cells, people, interesting, worthwhile ; that meanwhile got damaged to the point where they aren't, nor could ever again be, but which also underwent damage so severe, not even apoptosis works anymore. The living dead, what. [↩]
This is a gem of a point. Go Ballas! [↩]
The important part is, of course, missing : [Impotent] Hundreds [impotently] Attend [pointless] Vigil for Slain Kansas [virgin] Cheerleader, aka everyone failed.
Life's an examination, you ever met this concept before ? [↩]
The implication he's not voiced, "you want to see her being fucked to death" is not nearly so indefensible, and especially not nearly as indicative of individual pathology as he (just as unvoicedly) implies.
Look up "alegoria moarte-nunta", [Eastern] Europe's (pro tip : what is "America", demographically ?) central, principal and most enduring folkloric conceit, wonder with me in bedazzled wonder as to why exactly these people'd bury young (="nelumit") men in wedding costume, fucking consider things for a moment past the cracking, maroon veneer of your neologistic puritanism! There's a very good reason the principal inquiry at the death of a young woman is whether her cooch had met the world yet or not really, and it has absolutely nothing to do either with the individual or with pathology, it's pure and strong (and yes, therefore beautiful) evolutionary biology.
But then again you [attempt to] live in a parasitic "culture" of ugly fucks, whose principal mechanism of parasitation consists of identifying poorly understood and therefore unfamiliar yet perfectly natural mental processes, declaring them "evil" and then asking for money.
Stop attempting that. It's stupid ; and no, it never did anything for you, either. [↩]
Aren't you starting to think just about now that this guy'd have been a whole lot better if he just got laid more ? Specifically, like a man, as opposed to "in a relationship" or whatever other bullshit. Or if you know, he had like... some kind of life experience, or... something. Whatever, anything whatsoever at all!
Well... in his defense, he sorta-kinda figured this out himself. [↩]
I do not suspect there's any kind of link between the two. Neither the fact that the man was reported to have gone into bedrooms uninvited before nor the fact that the man was never reported to have gone into vaginas uninvited before offers much basis for inference in the instant case. [↩]
By being sane. If you make the law stupid, a general expectation of stupid downstream is not unreasonable. [↩]
As far as I'm concerned, this is already a decent reason to terminate a 14yo girl -- which (very importantly) means that if you're starting a posse to find her Rocco, I most likely ain't going, which means further other things downstream.
Put it this way : if the only way Rocco's getting caught is by convincing some people to posse, no Kansas 14 yo vivacious female's ever gonna dare prefer flip flops over actual womanly attire ; whereas if you pretend like an imaginary "the government" can teleport alt-posses from thin air, all you get for your trouble is... your having to jack it to imaginary vivacious teens that don't, actually, exist.
Choices have consequences, in this life -- and the consequences unerringly end up of the same kind as the choices that spawned them. Now stop participating in pretend-posses that pretend to have been teleported from thin air by a pretender to sovereignity ; and in exchange you won't have to pretend you're fucking vivacious teens. Deal ? [↩]
It's fucking USistan, if you're not what you're going to be then what the fuck are you ?! That's how that place works, entirely. [↩]
They also left out how she was gonna be in theology. And her bra size. Sometimes some things get left out, as a matter of taste and deceny. [↩]
This "sense" is squarely of the virtual kind, which is to say nonsense. Consider the experience of that Costa Rican crow, and then your own : all the adult woman, sitting on her cunt the throne of her wonder has to do is laugh at you. It will send you packing, instantly, you'll run until you're out of breath ; as did the crow, as would have Rocco.
So, no -- that "he killed a cheerleader" makes not one whit or half a mote's worth of sense. To merely fuck her she'd have had to be drunk, and not a little drunk, either. "Carried her around like a side of beef", said Bayly, and for good reason : it's fearsome, cunt, it strikes terror in the heart of cripples, and even normal, boring bois avert their gaze.
It makes no sense whatsoever ; you wish to pretend it does, but that is another thing entirely. [↩]
8th graders, ie 14 year old females, ie girls that have been bleeding for years are eminently not the domain of pedophiles.
Fourteen year old girls are old enough to be people's mothers. Fourteen year old girls are old enough to marry in most jurisdictions, both currently and at all previous points in history. Fourteen year old girls are the same age the mothers of most US presidents had their first child.
A pedophile is interested in children, not young women. Children, you understand, six months old. Sixteen months old. As a perfectly useful rule of thumb, if you can't readily express the age in months, you can't talk of pedophilia. The pedo is the baby fucker, the "I can't even understand how anyone can even get it up with that, it's like trying to fuck a wine bottle" fucker, not the "omfg I wish I tapped that" fucker.
An ephebophile is interested in prepubescents (and generally boys, at that). So if the object of amorous maneuvers can walk, but still... hasn't grown any hair, or whatever exactly bothers you about the situation of people having perfectly satisfying sex with girls under 12, you can use that term. Almost nobody ever does, which explains why it's here you first encounter it. Why do you think everyone else doesn't use it ?
In the vein of which question, let's recount a story together. Two buddhist monks were walking through the countryside, alongside a river, looking for the bridge. Eventually they come upon the site of where a bridge once stood, but the river had destroyed the artefact. At the foot, a woman kneeled. Her worn through rags barely covered anything, but she was shapely and luscious, and she begged the monks to swim her across. One of the monks recoiled in horror at the notion of such close carnal contact, but the other swam her across on his back. Once deposited safely on the other shore, she offered herself in humble gratitude, for, as she modestly put it, lack of anything better to repay them with. One of the monks recoiled in horror at the notion of such close carnal contact, but the other fucked her, and then the pair went on their way.
A night came, and then a day, and then others. Eventually, the monks were old, but still, together in the same monastery ; and one day, the night finally came for good, for one of them. As he lay dying he gestured for his brother to approach, and then in his ear he whispered, "how could you make it, with that woman, at the river!"
The old monk looked into the dying eyes, confused. Had mind flown before spirit ? But then, in a flash, he remembered, and, taken aback, whispered in response : "My brother, I had left that woman on the side of that river. Why are you still carrying her ?"
Why are you still carrying that six year old or nine year old or twelve year old or whatever the fuck she was ? That girl, the first girl you wanted to... well... do something to, or with, the memory gets fuzzy, but that first one, that you wanted and then thought "shouldn't" or whatever, "can't", "it ain't fittin'", whatever it is you cripples tell yourselves.
Understand the important parts : that had you done what you wanted, you still wouldn't have done what you thought that may be, then (and absolutely not what you think it must've been, anachronistically, now). You'd just have found your own, natural limit, perhaps a little further out than the horrors your internalized notion of the Father seemed to you to have prescribed, but really not all that far ; and you'd have found them together with her, rather than by yourself, therefore discovering that what you seek and what's to be sought don't quite exactly coincide ; and, if you're any shade of lucky, also discovered that's okay.
Stop carrying your mental tars along ; it's not good for you, for one, and it's also not nice, to the people you've hijacked to employ as unpaid actors in the little diorama playing out in your mind. I'm not even talking of the girl here, but take your misfortunate father. What, you think he had any secret plans to castrate you ? You think it's his fault you've chosen to project his innocent existence into a ready explanation as to why you aren't sharing a bed with the nude object of your interest, nor doing anything overtly towards it ? Think of this poor fuck, your father -- he's the other monk, stuck with you at his bedside not because you love him, as you loudly yet so evidently falsely claim, but because you aim to ask him "how could you!!!" and don't "know" how. You know, the same "don't know how" at work in the "I don't know how to end up naked with the softest, most delicious girl in kindergarten". It's not fucking nice, you understand me ?
Start being a nicer person! [↩]
What does the author want to believe ?
Is it perhaps that "media" matters ?
Well... it doesn't. If all everyone ever saw from now on until the Sun expanded into red were nothing but back to back reels of fourteen year old cheerleaders riding the symbian machine, not one single extra erection'd occur in the entire known universe. It doesn't fucking matter, what! [↩]
Does this even work ? [↩]
Kid older than pedo-range is also overwhelmingly men. Female filicide generally disposes of infants (something mothers actually do a lot of -- leaving aside medical abortion and zygote-related failures to nest etc, lotta fecundated eggs still not reach first trimester. Mothers killed more children than Hitler! [↩]
It's true that I don't really follow "the media", but on one hand it stands to reason not all described types are equally likely to occur and on the other hand it stands to reason stories will follow the edge of the comprehensible, not the stolid portion of phenomena deeply inside that margin nor the unutterable portion of phenomena way out. Because... well, what can you do ? [↩]
This difference might indeed be too subtle for me. What's the substance ? Are jobs something mystically other than lifelines for maintaining lifestyles ? Ballas isn't expecting the man to have found his life's fullfillment in holding a can of spray paint at innocent flowers. Is he ? [↩]
Isn't it fucking scrumptious that the dude's name actually is Mc Fall ? [↩]
This is quite on point, yeah. [↩]
This is an eminently solid point, consider merely the case of Adrian Nastase, ex prime-minister of Romania. Like so many others, this schmuck (who also happens to be the picture-perfect closeted faggot, but whatever) let himself be used by the USGistani occupation government in Romania ; but tried to do it part-time only. As those stories always go, he was "unmasked" as an enemy of socialism, and well... got convicted to jail time. This was a very big deal for this peculiarly pretentious fellow, festooned as he found himself in cocoon after cocoon of alternable, plausible "lives" and personalities. Can you imagine why gay men in the closet might be affraid of going to jail ?
As the police came to arrest him, he tried to shoot himself. The competent officer present prevented him, and Adrian Nastase went to jail with a hole in his jaw. Then he came out of jail, and didn't want to kill himself anymore ; instead, made himself a blog, which he diligently maintains to this very day (even though he's not published his cock just yet). You see ? Ballas is exactly right : force him to live with his shame for long enough, the female in him will find a way to survive. It's what they do, and, evolutionarily, what they're even for, because it's how babies are begot in the first place.
PS. Is the fact that this process works the fuck better on faggots than on, say, actual women any indication to you of anything in particular ? [↩]
Deeper than that : in order to rather kill your children than say
well... de, dragii moșului, așa o fi. N-om fi știind nici românește… Dar de acuma mult au fost, puțin au rămas, și în locul nostru voi veți stăpâni lumea și veți orândui-o cum vă va plăcea vouă". Și, luându-și ziua bună de la lume ca de la cel codru verde, s-au strecurat pe rând acolo unde nu le mai zice nimenea că au fost răi români"
in their general direction you must actually believe they do not in fact exist. The others reduced phenomenologically to mere figments of one's imagination, as (for instance, and inter alia) the very heavy societal cost of not permitting the beating of children. This is what happens, you realise. Do you ? If society forbids the father beating his children, the price society pays is the father no longer regarding those children as things. This is a good thing, you think, with your rotting pumpkin, but if they're not things then therefore they're figments. If he beat them in time, he'd have found how much they can carry, and why, and therefore wouldn't have killed them. Certainly not with a rubber mallet, you fucktards that there are no other, seriously, you look at the world around like the cat at the calendar.
Psychopathy, you understand, is the magical quality of being completely confused by the banal ; and shockingly underperforming even mongoloids. Check and check, yes ?
Well, what now ? Do you find a way to "live with" the shame of it all, maybe ? There's only one way, really, as Scott Adams well points out. [↩]
Speaking of which,
That unpleasant moment when "principles" you were supporting ~strictly~ because "of the principle of the thing" and pointedly not because of the convenient manner in which they worked upon a restricted set of circumstances decide to move on to another set of circumstances and work consistently but against your (inconsistent) preference. The only thing to do is look away. [↩]
Can sure as fuck try, which is probably a major driver of "mass shootings". [↩]
Everyone taking a tumble / being fucked in public imagines everyone else saw up their snatch, when in fact most people in attendance are so very busy with their self-secreted inconsequential minutia, they don't even notice. [↩]
As it turns out, the other ways out aren't nearly as much fun.
It's what it is, after all, isn't it ? A contemporary aspie fuck's responsibility is first, second and last to himself ; he must make sure he's having a good time at all junctures, measure of mankind as he finds himself and all that. No ?
Psychopathy is... [↩]
But... that wouldn't be nearly as much fun, which is why they always do go home.
See ? If you stop to think about it, everything makes perfect sense. All you have to do is shake the monkey off your back for a moment first. But...
well, it's not nearly as much fun without the monkey on your back, is it now. [↩]
Category: Adnotations
Christos Ballas ("TLP", thelastpsychiatrist.com) archive on Trilema - A blog by Mircea Popescu. (via Pingback)
[...] 8 Characteristics of Family Annihilators [...]
thelastpsychiatrist.com - Abusive Teens Force Their Girlfriends To Get Pregnant! (Don't Let The Truth Get In The Way Of A Good Story). Adnotated. on Trilema - A blog by Mircea Popescu. (via Pingback)
[...] and would really really like some ; but she won't put out (read : eat cunt) because "reasons" aka goange in her own head, preferring instead to fritter her life away pointlessly making up transparently [...]
thelastpsychiatrist.com - A Case Study On Why Policy Changes Fail: Pharma Paying Docs. Adnotated. on Trilema - A blog by Mircea Popescu. (via Pingback)
[...] can't decipher this guy's stance anymore (or, for that matter, his culture's). So, on one hand, "we all know what cheerleader means" would require "young women getting a few more rectal exams is all the better for everyone, loosen [...]
thelastpsychiatrist.com - According to Time, The World's Most Influential Person Is.... Adnotated. on Trilema - A blog by Mircea Popescu. (via Pingback)
[...] Note how transparently and self-obviously it is an utopia (ie, a system nobody actually lives in, notwithstanding the loud organized groups pretending otherwise) -- not even for a second and not even in the most minute and inconsequential crinkle of his daily life does even the loudest Jehova "witness" believe Jehova's a thing. Ballas doesn't go "they voted and therefore m00t's boss", he goes "they voted and therefore the election was rigged" for the very fucking obvious reason : democracy isn't a thing, exactly in the way and exactly for the reasons your "relationship" with your favourite pornstar isn't a thing. [↩]Except not. A "search" ie, asking Google to tell you what to think will produce more of the same, in the author's own words, [...]
[...] no use for Google and "rig" polls, well... they're not Time readers anyway, certainly not in any real sense. They may "read" Time with their whores, to laugh at it, but they sure as fuck aren't about [...]
thelastpsychiatrist.com - A Generational Pathology: Narcissism Is Not Grandiosity. Adnotated. on Trilema - A blog by Mircea Popescu. (via Pingback)
[...] Just kidding. She ain't going anywhere. She's been a slave for a year and she ain't going anywhere, what the fuck now ? Because I constantly ask the girlies how long did their longest relationship last, and they always break out the month as a unit of measure, like true pedos. [...]
thelastpsychiatrist.com - A Primer on Pedophilia. Adnotated. on Trilema - A blog by Mircea Popescu. (via Pingback)
[...] to be sourced from the group, somehow, not self-reported. [↩]And there is entirely nothing medically wrong with this. [↩]No. They're [...]
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text:
Costa Rica on lookout for fugitive Mexican ex-governor
By AFP Dec 23, 2016 in World
Costa Rican authorities said Friday they were on the look-out for a fugitive Mexican ex-governor suspected of embezzling hundreds of millions of dollars after Mexican counterparts said he was last traced to a luxury zone in their capital.
Security Minister Gustavo Mata told a news conference that there was no sign of the former head of Mexico's Veracruz state, Javier Duarte de Ochoa, entering Costa Rica under his own name.
But he didn't rule out that Duarte was traveling under false papers, as reported in Mexico's press.
"I have ordered authorities with the migration service to investigate, and if he is located to deport him immediately to Mexico," Mata said.
He added that the last time Duarte used his Mexican passport to enter and leave Costa Rica was six years ago.
According to an online report by Mexico's El Financiero media group citing security officials, Duarte disappeared from Veracruz with his family in October shortly after stepping down from his job amid corruption allegations.
He was traced to neighboring Guatemala and then, last week, to a five-star hotel in the chic western Escazu district of Costa Rica's capital from which he was said to have made an intercepted phone call to relatives.
Duarte is being probed for the suspected embezzlement of $1.6 billion from public accounts through a network of shell companies, among other crimes.
Mata confirmed that Duarte's former treasurer, Vicente Benitez, owned several properties in Costa Rica.
But he said a search of them had not turned up any leads.
More about Mexico, costarica, Politics, Corruption
Mexico costarica Politics Corruption

redpajama_set_name: RedPajamaCommonCrawl


The Season 6 finale was the show's most exciting episode in a long time. But it demonstrated that 'Game of Thrones' is all too eager to pander to our fantasies of unchecked power.
Sunday's Season 6 finale of Game of Thrones was nothing if not satisfying. "The Winds of Winter" sees Daenerys finally crossing the Narrow Sea to stake a claim to the Iron Throne; Jon Snow's parentage is finally revealed, which doesn't stop him from being anointed King of the North; and somehow Arya has finally managed to travel from Braavos back home to Westeros, where she gleefully avenges the murder of her family by feeding Walder Frey his own sons baked in a pie before slitting his throat. Apparently revenge is a dish best served in the form of your victim's offspring.
When the season began, I complained that the show seemed to be stalling — that it was clearly setting up major conflicts to come but that the early episodes in and of themselves were just meh. "The Winds of Winter" certainly fulfilled the promise of those early, boring episodes in an action-packed 60-plus minutes. But the finale's emphasis on the satisfaction of revenge left me feeling more than the usual Game of Thrones-induced queasiness.
In a stunning opening sequence, Cersei uses the Red Keep's store of wildfire to blow up the sept with everyone inside: The High Sparrow and the rest of the Faith Militant, Margaery, Loras and Mace Tyrell, and a whole whack of bystanders. The slow, deliberate pacing of the scene —the juxtaposition of each character getting dressed for the momentous day; the suspense-building cuts between Lancel inching closer and closer to the green globules of wildfire and Margaery's dawning realization that something's not right — lent the opening an eerie sense of inevitable disaster.
Cersei may be an unequivocal villain, but for the past couple seasons the series has pitted her against an even more menacing figure in the form of the High Sparrow and his army of hardline religious fanatics. With Cersei, viewers can at the very least sympathize with a leader trying to preserve her power in a world that offers most women very little of it — not to mention a mother who's now lost all of her children.
In the sixth episode of Season 6, Arya watches a troupe of performers reenact Joffrey's wedding-slash-death. Unlike the rest of the goofy cast, the actress who plays Cersei is an affecting performer; when she leans over the actor who plays Joffrey, lamenting the death of her son, the crowd is rapt. Her feigned grief even wipes the grin off Arya's face. "All hope is lost," the actress moans. "All joy is gone. And there is no tomorrow." The scene is a reminder that every death has meaning for someone.
In the morally complex world of Westeros, Cersei is easy to hate. But in the High Sparrow and the Faith Militant, Game of Thrones created a foe even more loathsome than Cersei. The Faith Militant are for the most part nameless, identical automatons with a severe and unforgiving moral code that leaves no room for individual desire or choice. It's hard to imagine any of them weeping for anyone; they seem to lack basic human impulses like joy, desire, or fear. I doubt they'd even partake in a lemon cake.
Certainly Septa Unella wouldn't. The rest of the High Sparrow's followers died anonymous deaths, but the joyless, bell-ringing, shame-calling clergy member was treated to a special kind of agony courtesy of Cersei's enforcer, Gregor Clegane. Apparently fans cheered the demise of Septa Unella, who is promised a slow, torturous death at the hands of Gregor; thankfully, the camera stays on the outside of her cell door, with only her spine-chilling scream to indicate her fate.
But this death and others like it that are meant to be satisfying conclusions to long-brewing conflicts left me empty. Arya — one of few characters that fans have unequivocally cheered on since the series began — unknowingly takes a page from Cersei's book when she kills Walder Frey. "I said my face would be the last thing you saw before you died, remember?" Cersei tells Septa Unella before she brings in Gregor. "The last thing you're ever going to see is a Stark smiling down on you as you die," Arya promises Walder Frey before slitting his throat.
In the previous episode, Sansa orders Ramsay Bolton's own dogs to devour their owner, grinning with pleasure as she walks away to the sounds of Ramsay's flesh being torn from his bones. Again, viewers applauded the demise of this brutal man who had spent so many episodes torturing others — first Theon, then Sansa, with a few minor characters, like Osha, tossed in for good measure. Like Walder Frey and the Faith Militant, Ramsay was made out to be such an unabashed villain for so long that it wasn't hard to get excited about his death.
Maybe I'm just feeling burned out by 2016 so far, but I'm not in the mood to get excited about anyone's death. Cheering on the brutal, graphic murders of characters like Septa Unella, Walder Frey, the slave masters in Slaver's Bay (now the Bay of Dragons, thanks to Daenerys) and even Ramsay Bolton feels somehow too easy — particularly when those deaths come at the hands of characters whom we've been conditioned to root for, like Arya and Dany. It reminds me of Breaking Bad fans who eagerly praised Walter White's latest "badass" stunt even though the character was so clearly becoming a selfish, egomaniacal, violent man who put his whole family in danger.
I realize that a major point of Game of Thrones is to demonstrate how morally compromising it is to live in the world it depicts. But other shows do that, too, and they manage to remain on far more solid moral ground. Orange is the New Black is premised on the fact that almost every character has done something so bad as to warrant her imprisonment. And yet through flashbacks, the show demonstrates that these women aren't evil — they're just imperfect people, often in the wrong place at the wrong time. Even the prison guards are portrayed not as straight-up villains but as people under pressure who make mistakes.
The antagonist of Outlander, Jonathan "Black Jack" Randall (Tobias Menzies) brutally rapes and tortures one of the show's protagonists, Jamie Fraser (Sam Heughan) in the first season. But so far, the show refuses to do to him what he did to Jamie. In the second season, Outlander deepens the character even more, revealing him to be a man with harmful impulses that even he knows he can't control. That doesn't mean he's absolved of his crimes, but it makes it that much harder for the viewer to look at Black Jack and see a projection of society's evils; instead, we see a flawed human being. And on The Americans, murder isn't performed with glee but weary resignation, each death sapping the killers of their sense of humanity. The show makes the act of killing look not like awesome fun but awful work.
Of course, as a fantasy series, Game of Thrones exists in a different world than OITNB, Outlander, and The Americans. But as viewers, we're all living in the same morally complicated world. Game of Thrones —a show that continually captures the attention of a record-breaking amount of viewers — is all too eager to pander to our fantasies of unchecked power.
It's not that the show presents a world without consequences. But while other dramas never let us forget that a life is a life, Game of Thrones takes so much pleasure in punishing its characters it's easy to forget they're all human.


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With yesterday's announcement that Arizona Gov. Doug Ducey has appointed Congresswoman Martha McSally to fill the Senate seat currently held by Senator Jon Kyl of Arizona, Navajo Nation President-Elect Jonathan Nez and Vice President-Elect Myron Lizer are requesting McSally and Senator-Elect Kyrsten Sinema to work across the aisle for the benefit of the Navajo Nation and all Arizonans.
Gov. Ducey also stated that Sinema will become Arizona's senior senator when she is sworn into office. As a democrat, Sinema narrowly defeated Republican candidate McSally in November's general election to become Arizona's first woman senator.
Vice President-Elect Myron Lizer also congratulated Martha Mcsally and commended Gov. Ducey on this important appointment.
"We wish incoming Senator McSally the best as we will endeavor to join her and Senator-Elect Kirsten Sinema to work on the challenges and the hopes we all have for the future to get 'positive things done' for Arizona's tribal nations," Vice President-Elect Lizer said.
Senator-Elect Kyrsten Sinema will be sworn into office on Jan. 3, 2019, while McSally will take the oath of office at a later date.


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text:
Home > Public Diplomacy > human rights > Non-Parent: Documentary...
Photo: Embassy of the Czech Republic
Non-Parent: Documentary Movie Screening and Human Rights Discussion
20.09.2019 / 15:02 | Aktualizováno: 09.05.2020 / 16:30
Embassy of the Czech Republic in Islamabad organized a documentary movie screening at the Residence of the Ambassador in Islamabad on September 18, 2019. The screening of the movie "Non-Parent" directed by Ms. Jana Počtová was followed by discussion on human rights and gender equality in Pakistan. The event focus on students from Islamabad and Rawalpindi and representatives of Pakistani civil society.
The documentary movie "Non-Parent" (2017, 83 min.) which was presented at the human-rights film festival One World in the Czech Republic in 2017 where it received the price for the best Czech documentary movie, represents a time-lapsed social probe into lives of six different families representing six alternatives to what is considered as a traditional family. Even though some of the issues raised by the movie, such as the question of the right of an individual to adopt a child, a right of a same-sex couple to raise children or a right of a baby to know his/her biological partner may not be that pertinent for the Pakistani society, the overarching question of a freedom of choice of whom a family consists, is very relevant for the Pakistani society as well. Chargé d'affaires a.i. Jaroslava Bobková who introduced the movie and moderated the post-screening discussion with audience, raised two main issues related to the overarching question specified above, namely the issue of arranged and forced marriages (sometimes also related to honour killing cases), and of the family planning, the absence of which leads to overpopulation of Pakistan, representing a dangerous demographic problem related also to economical and social problems and a slowdown in the development of the country.
The discussion was very lively with spectators actively involved and reacting to each other. Among other things, the discussion revealed the main reasons for which the youth in Pakistan supports the tradition of arranged marriages, namely reluctance of the young people to take on the responsibility for results of their own choice, an excessive dependence of young people on their parents but also the lack of opportunities for girls and boys to interact between themselves and get to know each other. A reputed women rights' activist has proposed an alternative term for the opposite of arranged and forced marriages, "own"choice marriage", which was warmly welcomed by the rest of the audience as an alternative to the discredited lable of "love marriage". The participants to the discussion also agreed on the need to solve the uncontrolled population growth in Pakistan. Besides lack of awareness, they considered the lack of relevant facilities to be an obstacle to family planning in the country.
Discussion with the Audience
Post-Screening Discussion
economy/trade
visa/consular
study/travel
education, compatriots


This pokemon papercraft is Zygarde a dual-type DragonGround Legendary Pokmon based on the vector game Pokemon the paper model was created by vectorierini. Zygarde is not known to evolve into or from any other Pokmon. Zygarde has a serpent-like body with a green and black pattern on it. There are black frills on the back of its head like a frilled lizard.Zygarde is a dual-type dragonground Legendary Pokmon. Pokemon Zygarde Papercraft117 SEADRA - Pokmon Papercraft Name Seadra Type Water Species Dragon Pokmon Height 1.2 m (311) Weight 25.0 kg (55.1 lbs.) Interesting Facts Seadra is a blue seahorse-like Pokmon with a long snout and a two fin-like ridges curving over either side of its head. Its belly is covered with rough cream-colored scales which have sharp pointed cream-colored tips that exude venom.


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Crisp and crunchy cucumbers are great in a salad or as a DIY spa treatment, but there are more reasons why you should always have a few in your crisper.
Cucumbers are rich in potassium, which has been shown to help lower blood pressure, and vitamin K, which is essential for calcium absorption and bone health. Cucumbers also contain fisetin, an anti-inflammatory substance that plays an important role in maintaining cognitive function. What is more, cucumbers can help lower the inflammatory response in the body and reduce the risk of certain cancers and cardiovascular diseases. They also happen to be delicious. From vibrant salads to spicy stir-fry, here are nine ways to take advantage of the cucumbers' potential health benefits.
Instead of serving cucumbers cold, try this tangy stir-fry featuring mushrooms spiced with ginger, garlic and jalapeño.
This salad celebrates the humble cucumber in its raw and pickled state.
This fast pasta salad is reminiscent of Japanese soba-noodle dishes.
Cucumbers combined with sugar, salt, red wine vinegar and thinly sliced onion make a supereasy, delicious side dish.
Chef Paul Qui's light crêpes are filled with a crisp and refreshing julienned salad of cucumber, carrot, daikon and green mango.
This terrific take on fried rice features cucumbers, salami and arugula.
Chef Bill Kim marinates these cucumbers for just two hours, so they're still crisp.
These incredibly simple pickles have just the right amount of garlic and dill.
Olive oil-marinated cucumbers are fantastic served with this airy feta spread.
This refreshing, unsweetened cocktail makes a great digestif.


text:
La Sonata per pianoforte n. 1 in fa minore Op. 6, di Aleksandr Nikolaevič Skrjabin, è la terza delle dodici sonate da lui composte. Fu completata nel 1892. La chiave della sonata è quella oscura di fa minore. La musica è emotivamente carica poiché gran parte di essa è stata scritta dopo che Skrjabin si era danneggiato la mano destra per aver suonato eccessivamente il pianoforte.

Storia
Prima della sonata op. 6 che porta il n° 1, Skrjabin ne aveva già composte due. La prima, un unico movimento in sol maggiore (movimenti associati non sono stati trovati) è stata pubblicata solo postuma. La seconda, in mi bemolle minore, fu condensata e riscritta per diventare il singolo movimento Allegro Appassionato op. 4.

Secondo quanto riferito, Skrjabin si stava esercitando in modo eccessivo sulle Réminiscences de Don Juan di Liszt e sull'Islamey di Balakirev, quando questo super lavoro gli danneggiò la mano destra. Fu informato dai medici che non avrebbe mai più suonato. La sonata n° 1 è stata il grido personale di Skrjabin contro Dio: la tragedia della perdita di un pianista virtuoso a causa del destino capriccioso, il disegno di Dio. Durante questo periodo di disabilità, scrisse Preludio e Notturno, op. 9 solo per la mano sinistra; tuttavia, a tempo debito la sua mano destra si riprese.

La perdita temporanea dell'uso della mano destra è anche attribuita al fatto di spingersi a sviluppare schemi ritmici complicati per la mano sinistra in molta parte della sua musica. Questa qualità aiuta a spiegare, in parte, perché molti dei suoi pezzi sono considerati piuttosto impegnativi da eseguire.

Struttura e contenuto
La sonata è in quattro movimenti e richiede circa 18-20 minuti per l'esecuzione:

Allegro con fuoco
Adagio
Presto
Funèbre

Il primo movimento, "Allegro con fuoco", inizia con un tema di apertura molto oscuro e appassionato. Questo cresce in un culmine leggermente più ottimista, ma scende di nuovo in un clima desolato vicino al tema. Continua con un secondo tema malinconico in la bemolle maggiore che si incrementa fino alla fine molto maestosa dell'esposizione del 1° movimento. C'è una sezione turbolenta di sviluppo, seguita da una ricapitolazione dei due temi principali, in forma leggermente varia e con le modulazioni modificate per riportare il secondo tema in fa maggiore. Il movimento termina in modo molto silenzioso, vacillando incerto tra fa minore e maggiore, prima di stabilizzarsi in fa maggiore nell'ultimo accordo sostenuto.

Il secondo movimento, in do minore, è un "Adagio" molto triste in forma ternaria, che termina tranquillamente in do maggiore.

Il terzo movimento, "Presto", di nuovo in fa minore, è in una forma rondò piuttosto condensata e compatta. Il movimento è aspro e agitato, alleviato solo brevemente dal tema centrale più tenero in la bemolle maggiore e martella con rabbia una fine irrisolta, che si risolve nel movimento lento finale, il "Funèbre", sempre in fa minore e simile nell'umore alla marcia funebre della sonata per pianoforte n. 2 op. 35 di Fryderyk Chopin. L'oscurità è inalterata fino al desolante finale in fa minore.

La sonata è stata pubblicata dalla prestigiosa casa editrice di proprietà di Mitrofan Petrovič Beljaev nel 1895 inizialmente come Sonatina, solo più tardi come sonata.

Note

Bibliografia

Approfondimenti
Skrjabin, Alexander. Complete Piano Sonatas. 1964 Muzyka score republished in 1988 by New York:  Dover Publications. .

Collegamenti esterni

Sonate per pianoforte di Aleksandr Nikolaevič Skrjabin

redpajama_set_name: RedPajamaWikipedia


text:
Q: Python обработка списка со словарями через map, lambda Мое почтение. Задача такова:
Имеем список словарей, надо из каждого словаря, получить значение по ключу и добавить в новый список(Который будет хранить, только эти значения). Размер списка со словарями всегда известен.
Через циклы по старинке проблем нет реализовать это дело. Но было бы интереснее реализовать через map, lambda или подобное.
Пример списка со словарями:
response_oi = [{
    'symbol': 'BTCUSDT',
    'sumOpenInterest': '109555.40300000',
    'sumOpenInterestValue': '2235729975.64190000',
    'timestamp': 1657847400000},
    ......... и т.д.
]

Обработать один словарь, ок, но как получить доступ к элементу словаря когда он не один через lambda или map уже голову "сломал".
Заранее благодарю за помощь.

A: list_dict = [
    {'a': 1, 'b': 2},
    {'a': 3, 'b': 4}]

result = list(map(lambda x : x.get('a'), list_dict))

print(result)

Взято отсюда

redpajama_set_name: RedPajamaStackExchange


Ovarian cancer (OvCa) most often derives from ovarian surface epithelial (OSE) cells. Several lines of evidence strongly suggest that increased exposure to progesterone (P4) protects women against developing OvCa. However, the underlying mechanisms of this protection are incompletely understood.
To determine downstream gene targets of P4, we established short term in vitro cultures of non-neoplastic OSE cells from six subjects, exposed the cells to P4 (10-6 M) for five days and performed transcriptional profiling with oligonucleotide microarrays containing over 22,000 transcripts.
We identified concordant but modest gene expression changes in cholesterol/lipid homeostasis genes in three of six samples (responders), whereas the other three samples (non-responders) showed no expressional response to P4. The most up-regulated gene was TMEM97 which encodes a transmembrane protein of unknown function (MAC30). Analyses of outlier transcripts, whose expression levels changed most significantly upon P4 exposure, uncovered coordinate up-regulation of 14 cholesterol biosynthesis enzymes, insulin-induced gene 1, low density lipoprotein receptor, ABCG1, endothelial lipase, stearoyl- CoA and fatty acid desaturases, long-chain fatty-acyl elongase, and down-regulation of steroidogenic acute regulatory protein and ABCC6. Highly correlated tissue-specific expression patterns of TMEM97 and the cholesterol biosynthesis genes were confirmed by analysis of the GNF Atlas 2 universal gene expression database. Real-time quantitative RT-PCR analyses revealed 2.4-fold suppression of the TMEM97 gene expression in short-term cultures of OvCa relative to the normal OSE cells.
These findings suggest that a co-regulated transcript network of cholesterol/lipid homeostasis genes and TMEM97 are downstream targets of P4 in normal OSE cells and that TMEM97 plays a role in cholesterol and lipid metabolism. The P4-induced alterations in cholesterol and lipid metabolism in OSE cells might play a role in conferring protection against OvCa.
Ovarian cancer (OvCa), with a lifetime incidence of approximately 1%, accounts for more deaths than all other gynecologic malignancies combined . Approximately 90% of OvCas originate from the ovarian surface epithelium (OSE), a single layer of cuboidal cells covering the ovaries . Although many somatic gene defects have been detected in OvCa, genetic alterations unique to OvCa have been difficult to identify. Consequently, the molecular mechanisms leading to OvCa remain amongst the least understood of common cancers. Certain epidemiological variables such as advancing age, low parity, infertility, and family history are associated with increased risk; whereas oral contraceptive use is associated with decreased risk of OvCa . Several biological models have been advanced to explain the mechanisms of these risk-modifying factors. The incessant ovulation hypothesis postulates that repetitive wounding and healing of the ovarian surface where the OSE cells proliferate to repair the rupture during ovulation predisposes to OvCa by leading to accumulation of mutations . The gonadotropin theory postulates that increased levels of pituitary gonadotropins during ovulation and sustained high levels during menopause stimulate production of estrogens and other hormones to increase risk of OvCa. Although incessant ovulation or chronic gonadotropin stimulation could contribute to the etiopathogenesis of OvCa, it appears that other hormonal factors such as androgenic and progestogenic stimulations also play important roles .
Risch  first proposed a protective role for progesterone (P4) against OvCa on the bases of multiple lines of evidence. First, the protective effect of pregnancies, and especially of twin pregnancies, against OvCa has been attributed to the elevated levels of P4 in addition to the suppression of ovulation , because the degree of protection conferred by pregnancies seems too high to be explained simply by the pause in ovulation. Second, P4 reduces the proliferation rate in normal OSE cells both in a primate model and in tissue culture [6–8] and suppresses the transformed phenotype in vitro . Third, P4 is a potent inhibitor of proliferation in cultured human OSE cells at concentrations similar to the levels reached during pregnancy . One potential mechanism for this protective effect is that high does of P4 could reduce invasiveness of OvCa by reducing epithelial membrane fluidity . Accordingly, it has been observed that pretreatment of mice with P4 reduced the numbers of OvCa implants in the abdominal cavity, whereas P4 treatment had no effects once the tumors were implanted .
Despite evidence for an anti-carcinogenic role for P4 in OvCa, it has been difficult to fully understand the underlying mechanisms. The intracellular effects of P4 are mediated primarily by intracellular P4 receptors (PR) that are expressed as two protein isoforms, PR-A and PR-B, encoded by the same genetic locus . The implicated mechanisms underlying the protective effects of P4 against OvCa include induction of cell cycle arrest or apoptosis, possibly through activation of the extrinsic apoptotic pathway and Fas/FasL signaling, alternative expression of transforming growth factor beta isoforms, and alterations of the fluid dynamics of plasma membranes in OvCa cells . In a recent study, that analyzed ~2400 genes in OvCa lines, Syed et al. found four suppressed genes that were derepressed upon P4 exposure. Depression of these genes suppressed the transformed phenotype in OvCa cells. Although these studies have provided clues for potential mechanisms of P4's anti-carcinogenic effects on OvCa cells, little is known about their relevance for P4's prophylactic role against OvCa. In other terms, mechanisms regulated by P4 that could prevent the neoplastic transformation of normal OSE cells are unknown.
To the best of our knowledge, no study has systematically addressed the transcriptional impact of P4 on normal OSE cells. In the light of strong epidemiological evidence implicating P4 as a protective hormone against OvCa, it is plausible that certain downstream effectors of P4 in normal OSE cells could be involved in protection against OvCa. Here, we conducted a global survey of the expression changes induced by high concentrations of P4 exposure for five days on normal OSE cells obtained from six women. Our analysis, using Affymetrix oligonucleotide microarray chips, revealed a coordinate and highly significant up-regulation of multiple genes in the cholesterol and fatty acid biosynthesis pathways in response to P4.
Primary cultures of OSE cells were obtained following informed consent from cases who underwent hysterectomy and oophorectomy for various clinical indications other than OvCa. In all cases the ovaries showed benign alterations upon postoperative histological examination (Table 1). Ovarian cancer samples were obtained from Magee-Womens Hospital Tissue Bank. The research protocols were approved by the University of Pittsburgh IRB review committee.
The OSE cell cultures were established by brushing the epithelial lining of the fresh ovaries. The epithelial content of the OSE cells were confirmed by immunofluorescence staining (Figure 1). Briefly, primary OSE cells were spun onto slides using a cytospin centrifuge and fixed in cold methanol at -20°C, and subjected to immunofluorescence staining as described previously . A mouse monoclonal anti-cytokeratin antibody (clone K8.13, 1:500 dilution, ICN, Irvine, CA) was used for cytokeratin staining. Secondary antibody used was FITC-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL). The nucleus was counterstained with 4',6'-diamidino-2-phenylindole (DAPI). The purity of the epithelial component in each OSE cell culture for the six cases were 100%, 100%, >90%, 90%, 100% and >80%, respectively.
Ovarian surface epithelial (OSE) cells in culture. The epithelial origin of primary OSE cultures is demonstrated for one responder (case 2) and one non-responder cultures (case 5). a, cytokeratin staining, b, DAPI counterstain, c, merge of cytokeratin and DAPI staining. Magnifications: 10 × 20. The majority of OSE cultures expressed keratin, confirming their epithelial origin.
For P4 exposures, OSE cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 15% fetal bovine serum (FBS) and streptomycin/penicillin. 48 hours before the hormone treatment, the OSE cells were plated in four T25 flasks, the medium replaced with DMEM supplemented with 15% charcoal-stripped FBS and the cells allowed to reach 70–75% confluence. The medium was then replaced with fresh media containing 10-6 M P4 (Sigma) or vehicle (ethanol). The final concentration of ethanol in the media was 0.03% both in the P4 and control experiments. The hormone-treated cells were exposed to P4 for a total of 5 days, with three fresh additions of media containing P4 (or vehicle) every 48 hours for stable bioavailability as described . At the end of the fifth day, the medium was removed. The cells were washed with PBS, collected following trypsin treatment and immediately frozen for RNA extraction. Cells from duplicate flasks were combined. To test bioavailability of P4 in tissue culture, we used breast cancer cell line MCF-7 to confirm upregulation of human DLG5 gene by P4  and found 5.2-fold and 2.2-fold gene expression increases by real-time RT-PCR at 8 and 24 hours, respectively.
The total RNAs were extracted following a commercial protocol (RNA-Bee, Tel-Test, Inc., Friendswood, TX) that used phenol and guanidine thiocyanate. The total RNAs were resuspended in DEPC-treated distilled water and further purified using RNeasy mini-columns (Qiagen, Valencia, CA) in preparation for microarray analyses. After column purification, the total RNAs were quantitated by absorbance at 260 nm using a Beckman DU-64 spectrophotometer. Approximately 10 μg of total RNA were provided for microarray analysis of global gene expression patterns. After the RNeasy™ purification step and demonstrating an OD 260/280 ratio of 1.8 or higher, The University of Pittsburgh Microarray facility confirmed RNA integrity via Agilent Bioanalyzer 2100.
The high-density microarrays (Affymetrix gene chip type U133A) used in this study contained 22,283 unique human transcripts derived from the RefSeq database and were commercially available from Affymetrix, Santa Clara, CA. The hybridizations to microarrays were performed by The University of Pittsburgh Microarray Facility. All RNAs passed quality control tests before they were processed further for microarray hybridizations. Afterwards, approximately 5 μg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that used an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the poly T (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription by T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns and hybridized to gene chips following the manufacturer's protocol. cRNAs derived from the control unexposed cells and the P4 exposed cells were individually hybridized to chips using identical experimental protocols to reduce sources of technical variation.
Each transcript is represented on the chip as a set of 16–20 probe pairs with each pair containing a perfect match and a mismatch. The mismatch is the same oligonucleotide as the perfect match but with a single base substitution at the central position. The amount of non-specific hybridization is corrected by comparing the hybridization level between the perfect match and the mismatch. Affymetrix 418 Array Reader was used to scan the fluorescently tagged microarrays. Global scaling techniques were used for all probe sets, to make the average intensity of each image equal to an arbitrary target intensity, set to 150. The expression data (signal intensities) across the chips were normalized so that the expression levels of a gene were directly comparable across the chips. The normalization of all 12 chips was performed by D-chip software to the probe level in the P4-exposed chip for sample#3, which was arbitrarily chosen as the standard . Results of the genome-wide gene expression microarrays are submitted to the public repository database, GEO: Gene Expression Omnibus .
qRT-PCR was used to validate the up-regulation of genes identified by microarray analysis using an ABI Prism 7700 Sequence Analyzer according to the manufacturer's recommendations (Applied Biosystems, Foster City, CA). The comparative threshold cycle method was used for the calculation of relative transcript amounts as specified by the manufacturer. Complementary DNA was synthesized with SuperScript II reverse transcriptase (RT) using total RNA. qRT-PCR analysis of cDNAs for each gene included a negative control template generated without addition of reverse transcriptase during cDNA synthesis. Average fold changes of target genes were determined relative to the internal control genes. Each target and control gene was amplified in separate wells in triplicates. The control genes were chosen from among the housekeeping genes whose expression levels did not change upon P4 exposure in the microarray data. The probes for qRT-PCR were purchased commercially from Applied Biosystems. PCR efficiency tests were performed before the transcript quantification assays to confirm that the amount of input cDNA was inversely proportional to the threshold cycle number for each gene (i.e. increased cDNA input reduced the detection threshold in a linear fashion).
PR gene polymorphisms are screened by standard PCR amplification and restriction enzyme (RE) digestion. We screened the V660L polymorphism by PCR amplifying PR gene exon 4 by PCR oligonucleotide primers 5'-TTCTCTGTAGGTCGAAAATTTAAAA-3' and 5'-TATTATTACCTGGCAATGATTTAG-3' at an annealing temperature of 59°C and digesting with the BsrI RE. The allele for leucine destroys the BsrI RE site. Similarly, the +331 G/A SNP in the promoter region was screened by PCR amplification using oligonucleotide primers 5'-CACTACTGGGATCTGAGATC-3' and 5'-TTTATCTCCCGACTTTTTCTC-3' at an annealing temperature of 55°C and NlaIV RE digestion. The A allele destroys the NlaIV RE site. RE enzymes were purchased from New England Biolabs (NEB). To investigate the expression status of PR, we conducted real-time qRT-PCR analysis using PR primers amplifying the transcript portion common to both the PR-A and PR-B isoforms and specific to the PR-B isoform in the P4-exposed and baseline control cultures in samples 2,3 and 6. This analysis showed that both isoforms of PR were expressed in the responder and non-responders cell cultures without significant changes upon P4 exposure (Additional File 1).
The normalized expression data from twelve chips were analyzed jointly to detect genes and/or pathways regulated by P4. The signal intensities of the transcripts in the chips provide a quantitative measure of their relative abundance. To assess gene expression changes upon P4 exposure, for each transcript, we calculated signal log ratio (P4/control) in each of the six experimental pairs and plotted the average of the six signal log ratios on the y-axis against the average signal intensity in twelve chips on the x-axis (Figure 2). The signal log ratio is a measure of fold change in expression level for a transcript in the P4-exposed sample relative to its unexposed control. The change is expressed as the log2 ratio. A signal log ratio of 1 is the same as a 2-fold increase and signal log ratio of -1 is the same as a 2-fold decrease in signal intensity of a transcript. We assumed that signal intensity of a transcript that is not regulated by P4 would randomly fluctuate around a mean value on repeated measurements. Therefore the average signal log ratios of the six pairs would randomly distribute on the y-axis close to zero. We also assumed that signal intensities of transcripts expressed at similar levels would show similar levels of variations, as strongly suggested in Figure 2, on repeated measurements and therefore their average signal log ratios would also be comparable. However, if a transcript is regulated by P4, its average signal intensity would be displaced towards higher (up-regulation) or lower (down-regulation) mean values on the x-axis and its average signal log ratio would be dispersed further away from zero on the y-axis relative to the other transcripts of similar signal intensity, which are not regulated by P4. We refer to the transcripts with the most extreme average signal log-ratios (on y-axis) compared to those transcripts expressed at similar levels as outliers and evaluate them as candidates for regulation by P4.
Genome-wide view of fold-change in gene expression by P4 exposure. Average of raw signals in 12 microarrays (x-axis) are plotted against the average of log2-based fold-changes in expression in 6 microarray-pairs (y-axis) in the presence of 10-6 M P4 for five days. 224 transcripts were statistically determined to be outliers (shown by triangles). Eleven of the outliers encode cholesterol biosynthetic enzymes (shown by plus signs). No transcript showed more than 2-fold average change (i.e., no points are dispersed beyond y = 1 or y = -1).
Different approaches to identify the outlier transcripts are conceivable. For example, distribution of signal log ratios for all transcripts can be approximated by continuous curves that define significance boundaries . Here, we first ranked the transcripts on the basis of their average signal intensities in all 12 chips and generated a group from every consecutive 500 transcripts. The grouping of transcripts with similar expression levels safeguarded against preferential selection of lowly expressed transcripts which showed greater variations in their signal log ratios. As expected, the signal log ratios of all transcripts distributed symmetrically around the x-axis (Figure 2). Then, starting with the most abundantly expressed transcripts, we found the transcripts whose average signal log ratios were most extreme within each group of 500 transcripts. We found the transcripts whose average signal log ratios were located more than 3 standard deviations (SD) away from the mean of the group and whose fold changes were located more than 2 interquartile regions (iqr) away from the first and third quartile of the group. We defined these transcripts as outliers. In a normal distribution, 99% of the data points fall within three SDs of the mean. Whereas the 2iqr limits around the median delimits moderate outliers in any distribution. These two measures of dispersion were jointly used to detect the outlier transcripts at a moderate stringency level even if the distribution of signal-log ratios may not be normal in a group.
The outlier transcripts were then submitted to the Affymetrix web site  to identify biological pathways and clusters listed in the Gene Ontology database associated with the outlier transcripts. No particular pathway or cluster was expected to be significantly enriched by the outlier transcripts under the null hypothesis. To test this hypothesis, we conducted one-sided two-by-two Fisher's exact test  for enrichment of each of the pathways or clusters by the outliers. The total number of annotated (i.e., assigned to a pathway) outliers and the total number of annotated non-outliers were constant entries in each Fisher's exact test, whereas the numbers of outlier and non-outlier transcripts in a specific biological pathway/cluster were entered as variable values. We implemented two conservative measures to the serial significance testing of pathways for enrichment. First, the number of outlier transcripts belonging to a pathway was reduced by 1, as the first outlier transcript hit to a pathway was designated as the "ascertaining transcript" which could be a chance ascertainment. This step eliminated from further testing all pathways represented by only one transcript. Second, because some pathways were expected to show significant enrichment by chance as a result of multiple testing, we performed a Bonferroni correction and multiplied the nominal p-values by the total number of pathways ascertained by the outliers before declaring significance.
The OSE cultures derived from six women (Table 1) were exposed to P4 (10-6 M) for five days and gene expression changes were profiled in P4-exposed and baseline control cultures using U133A Affymetrix chips. These exposure conditions were chosen because comparable serum P4 levels (~552 nM) are reached at term in normal pregnancies  and because five days of exposure to 10-6 M P4 slows down proliferation in OSE cells in vitro . Thus these conditions were likely to mimic the physiological effects of long-term P4 exposure. The average signal intensities obtained from 12 normalized chips plotted against average signal-log ratios of the expressional changes from six experimental pairs are shown for 22, 283 transcripts in Figure 2 (also see methods). Transcripts expressed at lower levels showed more variation than the ones expressed at higher levels (Figure 2). The dependence of signal-log ratios, which is a measure of fold-change, on signal intensity was previously recognized , and is attributed to small random changes in signal intensities which could inflate fold-changes in less abundant transcripts than they would do in highly expressed genes. To lessen the impact of this artifact, 853 of the most-weakly expressed transcripts (~3.8% of all transcripts), which fell below an arbitrary signal intensity cutoff level of 50, were removed from further analysis. The remaining 21,430 transcripts were evaluated further for gene expression changes upon P4 exposure.
The average of the signal log ratios in six experimental pairs revealed that no transcript showed a change in gene expression level more than two-fold upon P4 exposure (Figure 2). To determine smaller but statistically significant gene expression changes, we evaluated 224 transcripts (outliers) that showed the greatest fold-changes in gene expression upon P4 exposure relative to the transcripts expressed at similar levels. Next, we tested whether these outlier transcripts were enriched for any of the biological pathways/clusters listed in The Gene Ontology database (see methods). One hundred sixty seven of the outlier transcripts were annotated to a cellular pathway or cluster. These annotated outlier transcripts belonged to 551 biological pathways (Table 2). Although no particular pathway is expected to be enriched by the outlier transcripts (null hypothesis), we found that several metabolic pathways involving lipid biosynthesis were significantly overrepresented by the outlier transcripts (Table 2). The most significant enrichment was found in the cholesterol biosynthesis pathway: eleven of the 36 transcripts in this pathway were present among the outliers, although none (0.4) was expected under the null hypothesis (Figure 2). These eleven transcripts, encoding seven enzymes in the cholesterol biosynthesis pathway, were also responsible for the significant enrichment of other lipid metabolism pathways (Table 2).
a Fisher's exact test, corrected for multiple pathway testing (n = 551).
b Fisher's exact test, corrected for multiple pathway testing (n = 542).
c No significant enrichment for any of the 449 pathways.
An inspection of the gene expression changes in the eleven outlier transcripts in the cholesterol biosynthesis pathway in each experimental pair revealed that whereas the cultures derived from cases 1, 2 and 4 (responder group) showed consistent up-regulation of these transcripts, the other three cultures (non-responder group) did not show appreciable changes in the transcript levels. This finding prompted us to analyze the outlier transcripts of the two groups separately. The responder group had 18 upregulated outlier transcripts encoding 11 genes in the cholesterol biosynthesis pathway, whereas the non-responder group had no significant enrichment for any biological pathway (Table 2). A full list of outlier transcripts detected in the three responder cases is provided in Additional File 3.
TMEM97, encoding a transmembrane protein of unknown function (MAC30), showed the highest fold-change (1.94-fold, Additional File 3) among the upregulated outlier transcripts in the responder group. TMEM97 was first identified as a downregulated gene in meningioma  and was later found to be transcriptional target of the BRCA1 gene . TMEM97 is strongly expressed in the pancreas and other gastrointestinal tissues and is downregulated in pancreatic cancer .
The presence of 18 transcripts encoding 11 genes in the cholesterol biosynthesis pathway among the outlier transcripts of the responder group further prompted us to evaluate the expressional changes of other genes in the cholesterol biosynthesis pathway (Figure 3). We found that transcripts encoding lanosterol synthase, sterol-C4-methyl oxidase-like, and sterol C5 desaturase were also among the outliers (Additional File 2) although they were annotated as part of steroid or sterol biosynthesis, but not cholesterol biosynthesis in the Gene Ontology database. Inclusion of these genes brought the total number of upregulated genes in the cholesterol biosynthesis pathway to 14. In addition, transcripts for other cholesterol biosynthesis genes such as squalene epoxidase were also upregulated in the responder group, although they were not detected as outliers (Additional File 2).
An overview of the cholesterol biosynthesis pathway. The enzymes whose transcripts are upregulated upon P4 exposure are shown by asterisks. The full names of the gene symbols are listed in Additional File 2. The cholesterol is synthesized from acetyl-CoA. HMGCR is the rate limiting enzyme because reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate is the committed step in cholesterol formation. Mevolanate is converted to isopenthenyl pyrophosphate by three sequential reactions requiring ATP. Then squalene is synthesized from six molecules of isopenthenyl pyrophosphate. Then squalene epoxide is cyclized to lanosterol which is converted into cholesterol. The carbon numbers of some intermediates are shown in parenthesis. P4 upregulates transcripts encoding enzymes located throughout the complex biosynthetic pathway. Whereas PRKAA1/2, PRKAB1/2, PRKAG1/2, the genes for subunits of AMP-dependent protein kinase (AMPK), which inhibits HMGCR, appear not to be regulated by P4 (Additional File 2).
The profound impact of P4 on cholesterol metabolism was also evident by transcriptional regulation of other important genes in cholesterol metabolism. Insulin induced gene-1 (INSIG-1), low density lipoprotein receptor (LDLR), and ATP-binding cassette transporter G1 (ABCG1) were upregulated; steroidogenic acute regulatory protein (StAR) and ATP-binding cassette transporter C6 (ABCC6) were downregulated (Additional File 2). In addition, several genes involved in fatty acid metabolism including fatty acid desaturases 1 and 2 (FADS1 and 2), stearoyl-CoA desaturase (SCD), endothelial lipase, phospholipase A2, group IVA, long-chain fatty-acyl elongase, cytochrome P450, subfamily IIC, polypeptide 18 were among the outlier transcripts (Additional File 2).
To confirm the transcriptional up-regulation of outlier transcripts in the microarray data, we selected TMEM97 and three upregulated genes in the cholesterol biosynthesis pathway: HMG CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthesis, isopentenyl-diphosphate delta isomerase (IDI1) and 7-dehydrocholesterol reductase (DHCR7) for additional analyses. We used two genes as controls, small nuclear ribonucleoprotein 70 kDa polypeptide (SNRP70) and ubiquitin carboxyl-terminal esterase L3 (UCHL3), which were expressed at similar levels to the tested genes but whose expressional levels did not change upon P4 exposure in the microarray data. For all four tested genes, the real-time quantitative RT-PCR analysis showed expressional changes similar to the microarray results in two responder (cases 2,4) and one non-responder sample (case 6) (Table 3).
*: RNA used in the microarray hybridizations was not available for this test.
We explored whether genetic differences in the PR could explain the differential response to P4 in the responder and non-responder OSE cell cultures. It has been suggested that certain genetic polymorphisms in the PR gene could modify the transcriptional activity of the PR [28, 29]. These SNPs were V660L, which tags a distinct haplotype known as PROGINS, and +331 G/A, a SNP located in the promoter region. The responder case 2 was heterozygous for both SNPs, whereas the other five cases were homozygous G/G-V/V. Thus, the genotypes of the two SNPs were not significantly different between the responder and non-responder cases. To investigate the expression status of PR, we conducted real-time qRT-PCR analysis using PR primers amplifying the transcript portion common to both the PR-A and PR-B isoforms and specific to the PR-B isoform in the P4-exposed and baseline control cultures. This analysis showed that both isoforms of PR were expressed in the responder and non-responders cell cultures without significant changes upon P4 exposure (Additional File 1).
To determine whether co-expression of TMEM97 and cholesterol biosynthesis genes also occurs in other normal tissues, we analyzed tissue-specific expression levels in the GNF Gene Expression Atlas2 database  using UCSC Genome Browser's Gene Sorter utility . The Gene Sorter ranks 20,161 human genes based on their difference in tissue-specific expression levels relative to a selected gene, which was ranked at number 1, using data from multiple tissues including fetal brain, whole brain, amygdala, thymus, bone marrow, CD4+ peripheral blood cells, adipocyte, pancreatic isles, heart, lung, kidney, liver, ovary and testes. When TMEM97 was ranked at number 1, farnesyl diphosphate synthase (FDPS), farnesyl-diphosphate farnesyltransferase (FDFT1) and acetyl-CoA acetyltransferase 2 (ACAT2) genes were ranked, at numbers 10, 34 and 43, respectively, among the top 50 genes that have the closest tissue-specific expression levels (last column in Additional File 2). The ranking of 3 of the 23 cholesterol biosynthesis genes among the top 50 genes is highly unexpected (expected number = 0.06, observed number = 3; Poisson p-value = 0.00003). Alternatively, the ranking of 18 cholesterol biosynthesis genes among the top 5000 is also highly unexpected (expected number = 6, observed number = 18; Fisher's exact test = 0.00047). These results support our conclusions derived from the microarray analyses of OSE cells in that the expression of TMEM97 is co-regulated with cholesterol biosynthesis genes in normal tissues.
Because somatic alterations in downstream targets of P4 in OSE cells could play a role in the pathogenesis of OvCa, we compared the expression of TMEM97 in normal OSE cells and OvCa cells that are maintained in the short-term culture and harvested for RNA extraction under identical conditions. We evaluated the expression of TMEM97 in OvCa samples and in normal OSE cells relative to the control genes using real-time quantitative RT-PCR. Compared to the normal OSE cells, the OvCa samples showed 2.71- and 2.22-fold reduced expression of TMEM97 when SNRP70 and GAPDH were used as control genes, respectively (Table 4).
To test for somatic mutations in the TMEM97 gene, which is located on chromosome band 17q11.2, we sequenced all of the coding nucleotides (528 base pairs), that are distributed to three exons, in 39 ovarian cancer samples. We found an intronic single nucleotide polymorphism (IVS1 +12 C>T) in a single sample but no coding sequence variations.
To determine the intracellular gene targets of P4 in non-neoplastic OSE cells, we analyzed changes in gene expression patterns in six independent short-term cultures using genome-wide transcript arrays. We found a highly statistically significant regulation of transcripts involved in cholesterol metabolism. For example, multiple transcripts representing cholesterol-homeostasis genes including HMGCS1, HMGCR, IDI1, FDPS, FDFT1, NSDHL, EBP, DHCR7, INSIG1, FADS1 were upregulated by P4 exposure (Additional File 2). Examination of each experimental pair revealed that the transcriptional activity induced by P4 exposure originated only from three of the six experimental pairs. Re-analysis of these three responder pairs uncovered a functionally uncharacterized gene TMEM97 as the most-responsive transcript which showed a 1.95-fold increase upon P4 exposure. Examination of genome-scale, tissue-specific gene expression levels in the GNF2 database uncovered a strong correlation between TMEM97 and cholesterol biosynthesis genes. This finding and our current microarray analyses suggest that TMEM97 plays a role in cholesterol metabolism. We also found that the expression of TMEM97 was downregulated ~2.4-fold in OvCa samples. Collectively, these findings suggest that P4 is involved in cholesterol metabolism in OSE cells. To our knowledge, P4-regulation of cholesterol homeostasis genes in OSE cells has not been previously suggested. An earlier work has shown suppression of a subset of the cholesterol biosynthesis pathway genes by PR receptor inhibitors in rat periovulatory granulose cells suggesting that P4 regulation of cholesterol homeostasis could be, at least in part, receptor-mediated .
The lack of significant gene expression changes in three of the six experimental pairs suggests that the transcriptional response to P4 may not be universal among OSE cells, at least under the experimental conditions described herein. The reasons for this differential response are currently unclear. Analyses of two genetic polymorphisms and the expression of PR gene did not reveal appreciable differences between the responder and non-responder groups. Certain clinical characteristics such as history of oral contraceptive use, serum gonadotropin, estrogen or progesterone levels at the time of surgery might have influenced the responsiveness of the OSE cells to P4. However, because the OSE cells remained in culture under identical conditions for at least two weeks before the experiments, it is unlikely that any reversible in vivo effects of these hormones would persist when the cells were treated with P4. Alternatively, certain constitutional or somatically-induced genetic changes might affect the P4-responsiveness of certain OSE cells. Notably, one of the three non-responder OSE cells was derived from a subject (case # 3) whose ovaries were removed because of her sister's history of early-onset ovarian cancer (Table 1). A second non-responder sample (case # 5) was derived from a subject with endometrial cancer, which sometimes co-occurs with OvCa . Thus, whether mutations increasing susceptibility to OvCa could blunt responses to P4 remains to be investigated. Although hormonal and/or genetic factors might affect the responsiveness of certain OSE samples to P4, our data indicate that at least certain OSE samples clearly show a broad transcriptional regulation of cholesterol homeostasis genes upon P4 exposure. Thus, potential impact of cholesterol metabolism on the pathogenesis of OvCa should be considered.
Our data suggest that the impact of P4 on cholesterol metabolism is profound and involves broad transcriptional regulation of many genes regulating cholesterol and lipid biosynthesis and transport. The P4-induced transcriptional changes in most genes in cholesterol metabolism predict an increase in intracellular cholesterol levels. These changes include (a) up-regulation of 14 enzymes catalyzing both early and late steps of de novo biosynthesis (b) up-regulation of the low density lipoprotein receptor and ATP-binding cassette (ABC) transporter ABCG1 genes , which predict increased cellular uptake and (c) reduced conversion to gonadal and adrenal steroids by down-regulation of the StAR gene . It is conceivable that increased cholesterol synthesis could compensate for the downregulation of StAR and lead to increased OSE steroid synthesis. Similarly, the P4-induced transcriptional up-regulation of genes involved in unsaturated fatty acid metabolism predicts an increased synthesis of unsaturated fatty acids. Whereas the up-regulation of stearoyl-CoA and fatty acid desaturases predicts an increased de novo synthesis , up-regulation of the phospholipase A2 gene PLA2G4A and endothelial lipase  may promote release of unsaturated fatty acids such as oleic acid and arachidonic acid from membrane phospholipids. Similarly, down-regulation of cytochrome P450 gene CYP2C18  might help reduce conversion of arachidonic acid to other biologically active eicosanoids.
Cholesterol and fatty acids are essential components of the cell membrane . Thus, the P4-induced changes in cholesterol and lipid metabolism might influence such physicochemical characteristics of the OSE cell membrane as fluidity. OSE cells remain physically intact during pregnancy and the secretory phase of the menstrual cycle, when P4 levels are high and no ovulation occurs. Perhaps, in these physiological states, an increase in the cholesterol content of OSE cell membrane induced by P4 might decrease membrane fluidity and help maintain the physical integrity of the OSE cells that cover the ovary. Notably, inhibitory effects of P4 on membrane fluidity of SKOV-3 ovarian adenocarcinoma cells has been independently suggested and linked to decreased tumorigenic behavior of P4-treated OvCa cells . The present results suggest that increased cholesterol biosynthesis might underlie the decreased membrane fluidity observed in SKOV-3 cells. The current findings might be further interpreted to suggest that defects in the P4-induced cholesterol biosynthesis pathway might increase vulnerability of OSE cells to inflammatory or physical damage and increase the risk of neoplastic transformation. Accordingly, we found that TMEM97, the most upregulated gene by P4 in our dataset, was coordinately expressed with cholesterol biosynthesis genes in other tissues and was suppressed 2.4-fold in the OvCa samples relative to normal OSE cells. Further experiments including evaluation of the expressional status of other cholesterol biosynthesis genes and functional studies of cholesterol metabolism genes by employing such experimental approaches as siRNA inhibition of the candidate genes in OvCa cells might help to understand whether alterations in cholesterol homeostasis plays a general role in pathogenesis of ovarian cancer.
The present results suggest that TMEM97 and cholesterol and lipid homeostasis genes are transcriptional targets of P4 in normal OSE cells. If confirmed, these results could provide new insights on the molecular steps involved in the development of OvCa.
We thank Dr. Y. Lin for normalization of the Microarray data by dChip software and J. A. Patterson for help in tissue culture. This work is supported in part by grants from Department of Defense Gynecologic Disease Program, Scaife Family Foundation, Anne L. and Irene V. Caplan Philanthropic Fund, and The Ladies Hospital Aid Society.
BEB and JAD designed the study. BEB, CBW, GOF, JEWB and LCH performed the experiments. BEB and CBW performed the statistical analyses. BEB and JAD obtained funding. CWB and BEB drafted the manuscript. GOF, JEWB and LCH revised the manuscript for important intellectual contribution. All authors read and approved the final manuscript.


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2019-20 Rivalry Series
The Rivalry Series was introduced by USA Hockey and Hockey Canada last season and comprised three games between the women's national teams of the two countries. Two games of the 2019-20 Rivalry Series took place in December and the final three games were played in February. The U.S. defeated Canada, 4-1, in the series opener in Hartford, Conn., on Dec. 14 before earning a hard-fought 2-1 victory over the Canadians in Moncton, N.B., on Dec. 17. The U.S. dropped a 3-2 overtime decision to Canada Monday (Feb. 3) at Save-on-Foods Memorial Centre in Victoria, British Columbia, but responded with a 3-1 win two nights later (Feb. 5) in Vancouver, British Columbia.
The U.S. won the final contest on Feb. 8, 4-3, in overtime in front of a record-breaking crowd in Anaheim, Calif. The two teams played in front 13,320 fans, which set a record for the most-attended women's national hockey team game ever played on U.S. soil. The previous record was 10,158, which was established on Jan. 6, 2002, when the U.S. faced Canada at Joe Louis Arena in Detroit.
The U.S. and Canada have battled in the gold-medal game of every IIHF Women's World Championship and Olympic Winter Games staged, with just two exceptions (2019 World Championship, 2006 Olympics).
2019-20 Series Schedule
TIME (ET)/Result
Tickets/U.S. Player Of The Game
Sat., Dec. 14* Canada W, 4-1 XL Center
Hartford, Conn. NBCSN Alex Cavallini Stats
Tue., Dec. 17 Canada W, 2-1 Avenir Centre
Moncton, N.B. NHL Network Alex Carpenter Stats
Mon., Feb. 3 Canada L, 2-3 (OT) Save-On-Foods Memorial Centre
Victoria, B.C NHL Network Hilary Knight Stats
Wed., Feb. 5 Canada W, 3-1 Rogers Arena
Vancouver, B.C. NHL Network Katie Burt Stats
Sat., Feb. 8 Canada W, 4-3 (OT) Honda Center
Anaheim, Calif. NHL Network Megan Bozek Stats
*The December 14 game in Hartford was part of the 2019 Team USA Champions Series, presented by Xfinity.
February Roster
December Roster
Rivalry Series History
Women's Teams News
Family Matters: Support Helps Matheson in Golden Career
By Dan Scifo 07/30/2020, 9:45am EDT
A four-time world champion, Matheson is looking forward to making her second Olympic appearance
An Olympian as a Teenager, Cayla Barnes is Just Getting Started
By Steve Drumwright 06/06/2020, 10:15am EDT
Barnes is eager to resume her career at Boston College and with the U.S. Women's National Team
Next World Championship Opportunity will Have to Wait for Savannah Harmon
By Dan Scifo 05/14/2020, 10:15am EDT
Pandemic cancelled what would have been the defender's first world championship appearance
Abby Roque Caps Memorable Career at Wisconsin
Roque finishes collegiate career as one of the greatest players in a storied Badger program
U.S. Closes Out Rivalry Series with 4-3 Overtime Win Over Canada
By USA Hockey 02/08/2020, 10:30pm EST
USA wins Series 4-1 before record crowd