In biblical lore, Aaron selected a goat on behalf of the entire tribe, cast upon it the sins of all members, and then banished it alone to the wild. The members of the tribe were then at great ease, having been freed from their cast-off sins—whatever those sins may have been.

Everyone felt better, though they had neither identified their specific sins nor atoned for them. They had simply agreed to hang them on the goat. If this spurious logic was obvious to anyone, it was not discussed. Why question an agreed-upon means of making everyone feel better?

Now about that goat. It was selected from the herd and sent forth into the wilderness for reasons having to do with the sins of others. The goat had done nothing to merit banishment. But once the ashes were cold on the rituals of dispatching it, the goat found itself alone in the wilderness, isolated from its herd, in unknown territory, suddenly forced to fend for itself. It faced dangers from predators; difficulty finding food, sustenance, and shelter; and it lived the constantly woeful insecurity of a herd animal without a herd.

This is the story of the scapegoat.

In dysfunctional families, for reasons similar to those Aaron devised, there can also be a designated person selected for the role of scapegoat. In a family system, the selection process is less overt than Aaron’s. It is done more by consensual and habitual shunning that becomes an unspoken code of behavior: one person is chosen to bear the brunt of any psychological discomfort experienced by the family as a whole. It is justified by repeating the stories that create and then reinforce the image of the scapegoat as being a person who is worthy of disdain and disparagement.

Like the strong goat Aaron selected, the target of family scapegoating is also often the strongest and healthiest member of the family. At first blush, this may sound counterintuitive. But think about it a little more. In Aaron’s case, there would be no group pleasure derived from banishing a weak animal who might easily die anyway, because that would not gratify the needs of the tribe to send off their sins on a robust vehicle, a strong goat who was up to the task of bearing the burden. So it is in families: the targeted individual is often the most accomplished. She—and for the purposes of narrative cohesion, our scapegoat is a female here—must be strong enough to withstand the weight of the shunning voices which might easily and quickly topple a weaker person. The scapegoating would fail if the weight of the sins killed the goat before it could even get chased out of town. Catharsis is the goal. The goat needs to be strong enough to suffer in order that the tribe members do not.

Just as the goat was blameless despite being sent to its lonely death, so is the human scapegoat innocent of all charges. She may not be a perfect human being, but she is no different from anyone else in her range of faults. It is not her character or her actions that have directly caused her banishment. It is the way her character and her actions, and often her accomplishments, have been experienced by the dysfunctional family members, who for their own unexamined reasons need to dispel this person from the family realm in order to avoid looking into their own consciences. They need to punish the scapegoat for provoking by her very existence the discomfort family members are feeling that is actually a result of their own unresolved issues.

If you are being scapegoated in your family, please seek professional help. You are not likely to be able to intervene in a dysfunctional system that treats one of its own members in this way. You may continue to experience the futile attempts at explaining yourself. You may fail to understand the way you are being treated. You may begin to doubt your own version of your life story. The price is too high.

Can a human scapegoat die like the goat of yore? Maybe. If not physically, certainly emotionally. It is difficult for the scapegoat to believe that her family would treat her in this unconscionable manner if she were not guilty of some grave sin. She wracks her brain and her heart to understand, but she cannot. The reasons she is given for being mistreated seem shallow, petty, and incomplete. It is difficult for her to believe these small transgressions could warrant such heavy condemnation.

She begins to doubt her own version of reality, since consensus in her own family supports a narrative different from her own about who she is and what she does or has done. She learns that if she tries to sort this out, she will be accused of “playing the victim” or being selfish, or being a “drama queen.” She is able to hold to her knowledge that this assessment and treatment are not right, until one day, utterly discouraged, she gives up. The full weight of the banishment settles upon her. She is alone. She doesn’t try to understand or explain anything anymore. She has moved into accepting a fate that makes no sense to her.

Good mental health at this point suggests she make her peace with leaving behind the family that fails her so completely. And if she is strong and well-supported with friends, she may be able to do this. She will pay a lifelong price for sins she did not commit, however, because it is difficult and painful to extract oneself from one’s family. It is counter to the most basic of human needs for home, shelter, affiliation. It is a cruel and inexcusable undertaking for a family to scapegoat a member.

If you look at the research regarding the fate of individuals who have been relentlessly bullied, you can draw conclusions about what happens to scapegoated family members, for scapegoating is bullying with focused and long-term intensity. Some bullied children go on to become bullies themselves. Some develop social skills to divert and challenge bullying, though the scars of having been bullied may insert themselves into their lives in many ways for many years to come. Others, however, do not survive, driven to suicide.

If you are being scapegoated in your family, please seek professional help. You are not likely to be able to intervene in a dysfunctional system that treats one of its own members in this way. You may continue to experience the futile attempts at explaining yourself. You may fail to understand the way you are being treated. You may begin to doubt your own version of your life story. The price is too high. Please find a counselor who can help you unravel the fictions that subvert the truth about your life and about who you are. Good counseling support can help free you from the binding ties of pain, guilt, and shame that you did not create and which are not justified.

You were not born to bear the sins of others any more than Aaron’s goat was born for such a fate.

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"Previously, on "arrow"..." "These waters' powers," "They have permitted me to live way beyond my time." "I want you to take my place." "I want you to become the next ra's al ghul." "If you insist on pursuing this path of resistance," "There will be consequences." "Is that a threat?" "I am the demon's head." "And very soon, your brother." "He will never take your place." "He will." "[thea groaning and gasping]" "[gasping]" "Thea?" "Oh, my god, thea!" "Hey!" "[groaning and gasping]" "I..." "Couldn't..." "Stop him." "I need an ambulance to 455 merced, unit 10." "Please hurry." "Ok, you're going to be ok." "You're going to be ok." "No, thea, stay awake!" "Come on!" "Up!" "Hang on, we're almost there." "How long was she like this?" "Uh, 20 minutes, 40, I don't know, I just found her." "You need to stay here." "I'm not going to leave her!" "You need to let us work." "Get me a set of vitals." "Lift." "Charge up to 200 joules." "Clear." "Draw up one milligram epi, charge to 200." "Clear." "[monitor beeping] [monitor flatlines] start cpr." "Come on." "Come on, stay with us." "[monitor beeping]" "[indistinct p.A. Announcement]" "We did the best we could under the circumstances," "But her injuries were extensive." "Even if she were to regain consciousness," "We can prolong her life for as long as you wish." "But..." "It might be best to consider other options." "I am truly sorry, mr." "Queen." "May I see her?" "[monitor beeping]" "Sorry." "Oliver..." "Thea." "What are they doing?" "Oliver:" "They're unloading the truck," "And, um," "Having dinner." "General shrieve is there." "[sighs] the alpha," "The omega, he's combining them." "[beeps] [beeping]" "The bio weapon is armed." "What happens now?" "Now he'll take it somewhere to be released." "Somewhere densely populated where it will have the most impact." "We need to stop that truck." "Where's oliver?" "[tires squealing] come on!" "You stole a car?" "I borrowed it." "Come on." "Oh, my god." "Oliver!" "Oliver, we came as soon as we could." "What happened?" "Oliver." "They just left her there." "To die." "Oliver, is--is she?" "It's bad," "What is it?" "Smoke." "The league?" "Screw 'em." "At least until we get our bearings, oliver." "Come up with a plan." "He's won." "There is no plan." "Stay here with thea." "Did you do this?" "You could still save her, oliver." "I don't understand." "Take your rightful place as heir to the demon..." "And ra's al ghul will bring your sister back." "Oliver, what's going on?" "Oliver, where are you going?" "Away." "You can't just leave like this." "You have to tell us what's happening." "There's a way for me to save her." "Thea?" "Come on, oliver, that's not possible." "Yes, it is." "There are waters in nanda parbat." "They've permitted ra's to live for over a hundred years." "And in rare instances," "Told in legend, those waters have been used to restore" "The dead to life." "He offered to use the lazarus pit on thea, didn't he?" "Lazarus, as in from the bible, lazarus?" "The pit's real." "I've seen it." "It can save thea." "Right." "But only if you become the new ra's." "Ok." "Well, even if a magic hot tub" "We're not going to let you go and join the league of psychotic murderers," "Even if it is to save thea." "Which this wouldn't." "The waters change a person." "In the soul." "Even if they work, the thea you get back" "Will not be the one you lost." "The one we lost because of you." "I'd prefer that we didn't do our usual "please don't go" dance." "Because I'm coming with you." "Assuming that this works," "Someone's going to need to bring thea back home." "We need a way to get to nanda parbat." "[computer beeping]" "Sorry to interrupt." "Everything all right?" "'cause you got that crease between your eyebrows" "That tells me things most definitely are not." "Something awful has happened." "And I-- I need a favor." "A really big favor." "Last time you asked me for a big favor," "I had to loan you my helicopter." "Don't need your helicopter." "I need your jet." "Well, oliver does, but both of us." "It's not what you think." "Well, if you're going to fly away with him," "Then..." "It's exactly what I think." "Last night was a bit of an eye-opener." "Insofar as realizing something" "I should have realized a long time ago..." "Can be considered eye-opening." "Last night, when you were saying good-bye to roy," "You and oliver... [sighs]" "You still have feelings for him." "When I..." "Told you that I loved you at the hospital," "You responded with jell-o." "Seeing you two together last night," "I realized..." "You don't love me because you're in love with him." "I'm sorry." "You're the last person I ever meant to hurt." "Take the jet." "I hope the something awful" "Stops being awful." "What did felicity say to dr." "Vaca?" "She thinks thea's being moved to central city." "You know, they do amazing things at s.T.A.R. Labs." "We might think about paying them a visit" "Before putting thea on a long flight to nanda parbat." "S.T.A.R. Labs can't save her." "Only the lazarus pit can." "Oliver!" "Look, I know what I have done" "Doesn't seem like the actions of a loving father," "But I do love her." "She's the only child I have left." "Having said that," "I would rather lose her forever" "Than--than subject her to the pit." "Get on the plane, malcolm." "You look chilly." "Yeah." "Thanks for letting me be here for you." "You're welcome." "I didn't have much of a choice, though, did I?" "No." "I wanted to make you think" "You had grown a little as a person." "I know that I've only told you a little" "About the five years that I was away." "Well, if by "little," you mean hardly anything." "I came back to starling city" "Before they found me on the island." "I don't understand." "It was complicated." "I saw thea." "She didn't see me." "She had a drug problem." "So I.." "Confronted her dealer." "And I broke his neck." "And I did it because I was angry, sure." "But I also did it because I felt like I was protecting her." "All I've ever wanted to do was protect her." "Malcolm told me." "He warned me what ra's might do." "But I never thought..." "I failed." "[tires squealing]" "Incoming!" "Maseo, take the wheel." "[tires squealing]" "You look like you can use a hand." "[all grunting] [screaming]" "It's not in here." "What is that?" "[sighs]" "It's the league." "Welcome home, al sah-him." "Maseo:" "I must prepare for the ritual." "I will treat her as my own family." "I hope you find the accommodations acceptable." "Such luxury is a small glimpse of the life that awaits oliver." "You trying to reassure me or yourself?" "I don't know what you mean." "You're oliver's friend." "Maseo, right?" "I am sarab." "Call yourself whatever you want." "If you gave a damn about oliver," "You wouldn't be working for the man who practically killed his sister." "Oliver will be spared grief" "In a manner that was denied to me," "And he'll receive an honor" "Greater than you can imagine." "[scoffs]" "League of assassins." "You're feared for your bravery" "And power." "Well, all I see are a bunch of weak men" "Running from their lives, trying to escape." "That's not powerful, maseo." "Or brave." "That's cowardly." "Do not presume to know me" "Until you've held your dying child in your arms." "Until you've told him everything would be ok" "So that the last words he ever hears" "Are a lie." "You know nothing." "I'm sorry." "What was your child's name?" "Akio." "You think akio would be proud of his father right now?" "[woman chanting in arabic]" "[all chanting in arabic]" "[chanting continues rapidly]" "[growling]" "[woman speaking arabic, thea gasping and groaning]" "Thea!" "Thea!" "I've fulfilled my end of our arrangement." "I suggest you begin to make peace with yours." "[gasping]" "Hey." "Hey." "It's ok." "You're ok." "I don't know you." "Thea, it's--it's me." "It's ollie." "Ollie's dead." "Dad?" "I'm here, thea." "It--it's me." "What's going on?" "Where am I?" "Some place safe." "You are safe." "Where's mom?" "Your mother's not here." "Just lie back, close your eyes and get some rest, ok?" "I love you, dad." "Ra's told me that the confusion would pass." "You have no idea what you've done." "What I've done?" "She's alive, malcolm." "What I did saved her life." "She thinks moira's still alive." "She'll be fine." "You don't know that." "Yes, I do." "Because you, diggle and felicity" "Are going to take her home and she will recover." "And what is that worth?" "Felicity..." "No, this is wrong!" "It is just wrong!" "I need to talk to you." "What is happening here is perverse!" "What you are doing to oliver." "What you are making him do;" "I am not going to let it happen." "You can leave us." "You have a great fire within you." "I can see now why oliver loves you." "If you knew the first thing about love," "You would not be ripping oliver away from his family." "I am merely helping him fulfill his destiny." "Yeah, I know all about the "survive my sword" prophecy," "And I am here to tell you that I could really give a crap." "Me and john and, god help me, malcolm," "Are not going to let this happen, and we have friends," "And we have resources and we will go to war to get oliver back." "You know, many lifetimes ago," "I loved a woman immeasurably." "And she loved me." "And we had a son and then a daughter," "And for many years, I felt I was the most fortunate man in the world." "My life was bliss." "And one night, a man came to my door" "And he gave me a horrible choice" "To leave without saying a word to them," "Or to stand and watch them tortured and then killed." "And I left without a farewell." "And to spare them pain," "I endured an agony worse than death." "Sounds like you gave up too easily." "If there's one immutable truth about life..." "It's often more cruel than it is fair," "And rarely provides an opportunity for any of us" "To find closure." "And all your posturing" "And all your threats of war" "Will merely delay the inevitable," "Causing you to forfeit" "The opportunity you have." "This was denied me." "You need to tell oliver good-bye." "Tell him how much you love him." "Tell him whatever it is your heart needs to express." "And do it now." "Before he is lost to you forever." "[knock on door] yeah?" "Hi." "Hey." "You ok?" "I'm sorry, that's a stupid question." "You..." "You just saw your sister come back from being mostly dead" "And you are about to give up everything you know and love," "So you're probably not." "I remember the first time that I thought" "I'd never see thea again." "When my father's boat went down." "But you did see her again." "And you'll see her again..." "Again." "This is different." "I've just been thinking about" "The..." "Person I was." "The brother that I was." "And how he probably died when that boat went down." "All this time, I've been struggling with who I am..." "And you think who you're supposed to be is..." "The next ra's al ghul?" "Honestly?" "I don't know." "The only thing that I can know for sure" "Is that everything that I've done;" "Every--everything that happened," "It lead me right here," "To this moment." "And felicity, if that's true," "Then..." "I don't know what it was all for." "I do." "I wish that I could change your mind about staying here," "But I know I can't." "Just like I know how leaving you here is going to destroy me." "I don't regret a single moment." "And you shouldn't, either." "You have done so much." "You have saved so many people's lives." "And you have changed so many for the better." "Including mine." "Knowing you has changed my life." "You..." "Opened up my heart" "In a way I didn't even know was possible." "I love you." "We searched the whole truck." "There's nothing in there." "Where is the bio weapon?" "[spits]" "Don't make me ask again." "[gasping]" "Food cart." "It's in the food cart." "Where is the food cart?" "We should split up to cover more ground." "What's wrong?" "I know you don't think we should be doing this." "You decided we should set an example for our son." "And for ourselves." "We can't just let people die." "If we do, we are just a shell." "Not really alive at all." "So..." "That happened." "I'm glad it did." "Me, too." "[chuckles] what's this?" "If there's any kind of god," "Something... [pours liquid] alcoholic." "[both chuckle]" "There's not much" "To toast to up here, is there?" "Well..." "There's us." "Yeah." "I'm sorry." "Hmm?" "Look, I know there's no way of avoiding this," "Going some place where ra's can't find us, but I..." "Oliver:" "Felicity... [cup falls on floor]" "Felicity." "What did you--[groans]" "I can't lose you." "I won't." "I stole some of that powder that that creepy priest lady used on thea." "Wasn't sure exactly what I was going to do with it when I got it, but I..." "Yeah, how did you know you were going to completely lose your mind?" "Or commit suicide." "Was your plan to sneak ra's prize out" "Unconscious without getting us all killed?" "No, it's an insane idea, but I couldn't do it." "I couldn't leave him." "We have to get him out of here." "Felicity, this place is like a fortress." "There's no way we're getting oliver out of here without ra's knowing." "We can't." "You can." "If we get caught, he'll kill all of us." "Then we can't get caught." "I have seen enough movies to know" "That the scary fortress always has a secret exit." "If there is anyone that knows the way out of this hellhole," "It's you, so don't even try to play games with me." "It's rough terrain." "It is not going to be easy." "How do we get there?" "Go get thea and meet us back here in five minutes." "Go!" "Which way?" "Temple's this way, come on!" "[all grunting]" "There's not much time." "Hurry." "Why are you helping us?" "Because if I don't, then I am just a shell." "You reminded me of that." "30 meters down." "The path forks." "Take the left passage." "Come on." "These tunnels are centuries old." "Stay close." "[thea gasping]" "Need to stop and rest for a minute?" "No, no, no." "Maseo says stay left." "[thea gasping] thea?" "We have to move, now!" "Too late." "Believe me, we do not want to be taken alive." "Hold!" "I am al sah-him." "Wahreeth al ghul." "Heir to the demon." "And you will obey." "Put your weapons down." "Now!" "Tell ra's I will return shortly." "[thea gasping]" "Oliver, please!" "Thank you for trying." "I love you all the more for it." "But this only ends one way." "This is as far as I go." "Oliver, the jet's less than a mile away." "Where are we going to fly to?" "Ollie, I don't understand." "Malcolm is going to take you home." "Aren't you coming with?" "Not now." "You go on, I'll catch up." "I love you, thea." "Don't ever forget that, ok?" "Oliver..." "Oliver, I don't know what to say." "I do." "John, you're the best man I've ever known." "And whatever happens," "You're my brother." "I am afraid I will never forgive myself for leaving you here." "You told me once that..." "Life is precious." "And that you wanted more from it than I could offer you." "Don't give up on that." "The only way that I'm going to survive this..." "Is if I know that you're out there" "Living your life, happy." "We're always saying good-bye to each other." "You'd think I'd be good at it by now." "Well, let's not say good-bye this time." "What's-- what's going on?" "Everything's fine, thea, you're--you're ok." "If you're here, then I'm not." "You're feeling a little more like yourself again." "[groans]" "What do you want?" "Where's ollie?" "Thea, do you remember" "Anything about the past few days?" "I..." "I was here." "And--and ra's, he" "He attacked me." "No, thea." "He nearly killed you." "Where is my brother?" "That is--that is what I'm trying to explain." "To save you, oliver had to join the league." "He--he traded his life for yours." "No." "No." "[sighs] I'm so sorry." "I know you hate me, and you have every right," "But I am going to make up for that, for everything." "That is not possible!" "Yes, it is!" "The league isn't after us anymore;" "I am free!" "I could go anywhere in there world," "But..." "This is where I want to be." "Here." "With my daughter." "You took care of me when I was injured." "Now, please, thea," "Please." "Let me take care of you." "[knock knock]" "Sorry to interrupt." "Is everything all right?" "No." "It's not." "[sniffles]" "Felicity..." "Oliver's..." "He's gone." "What are you talking about?" "He joined the league." "No." "He's not coming back." "Shh." "[sobbing]" "Blood was shed." "League blood." "And that is a transgression" "Punishable by death." "And yet you allowed your friends safe passage home." "You tell me why I should not hunt these people down" "And stain my blade with their blood." "Because I stayed." "I'm honoring our agreement." "Now no one close to me will ever be harmed again." "And your allies would not have been able to escape through the catacombs" "Without assistance, so you tell me who aided you," "Who this traitor is." "I don't know." "Me." "I struck down three of our number." "For my betrayal..." "I offer you my life." "You did not betray me tonight, sarab." "Maseo yamashiro did." "For a moment," "Your old life took hold." "But by coming here and kneeling before me," "You have proven your loyalty," "And I will not spill sarab's blood" "For maseo's actions." "Stand up." "Your service will be vital in the coming weeks." "Oliver will need you for his transformation." "And like sarab..." "Vestiges of your old identity still remain." "Oliver queen must be extinguished from memory." "Stop!" "[crowd screaming]" "No!" "Give me the vial." "[speaking chinese]" "The word "assassin" has fallen victim" "To many abuses of language." "Its real meaning hidden beneath the sediment" "Of lies and falsehoods." ""assassin" comes from hashishiyya," "Which means, "those who stand apart from society."" "Now, every man and woman in here" "Have renounced their past life" "And forfeited their identity in the name of something new." "And it is a cleansing" "Only achieved by fire." "[groaning]" "For it is your soul finally being unburdened." "Oliver queen is dead." "Eventually to be reborn as ra's al ghul." "But for now, only the arrow," "Al sah-him, shall remain." "Al sah-him." "Heir to the demon."


Photo: Peopleimages

There are several ways an undergraduate suitor can react when his anti-rape-activist date explains what she does. One of the most common is a sort of primal response — an instinctual, indignant protest: Not ALL men!

And, according to a number of women who have become campus leaders in the growing conversation around consent, that’s hardly the worst-case scenario.

These women have had their stories and their mission written up in student papers and in national ones; they’ve had bylines and quotes in the New York Times, interviews in the Huffington Post and on Dateline. They become poster children for sexual politics. And with their crusade often comes a weird social life: They’re out there fighting the good fight, but do they also get to hook up like normal college students?

Meghan Warner, a senior at UC Berkeley, serves as the director of the university’s sexual-assault commission and is part of a federal complaint against the school for its mishandling of assault cases. She’s appeared in a Glamour issue honoring college women who are about to change the world. And she says there were men who wouldn’t approach her or date her after recognizing her, or learning of her work.

“Nobody ever explicitly said, ‘Oh you’re a survivor, we can’t date,’” she told me. “But they’d assume that I was just doing this for attention, or more frequently they didn’t want to deal with it. It was too much. They assumed I’d have a lot of needs.”

Then there were those who were a little too eager to make it know that they would never, ever assault a woman. “Their first response is ‘I’m not one of those guys, I would never do that,’” she said. “I mean, what, should I be carrying gold stars now?”

Listen to New York Magazine’s ‘Sex Lives’ podcast for a weekly exploration of how we have sex now. On iTunes and Soundcloud

Chrissy Keenan, a UCLA senior, is the president of Bruin Consent Coalition, a campus group that works to raise awareness regarding sexual assault on campus. “When people know of me but they don’t really know the work, they hear the term ‘feminist’ or ‘sexual-violence prevention,’ they think, ‘super-extreme, bra-burning feminism,’” she explains, which often puts people on the defensive.

Keenan herself, though, sometimes finds it hard not to go on the offensive. She’s so used to laying down the nitty-gritty details of consent that she’s been known to open romantic interactions with a spiel that feels straight out of a student handbook.

She animatedly tells a story about a recent Tinder rendezvous: “One time, I agreed to meet with this guy at 8 or 9 at night. Before we met, I said to him, ‘This is the work I do, I know the chief of police … so, don’t try and get creepy; I know all my rights.’ And five minutes later, he was like, ‘Actually, I’m really not OK with how you just assume I’m a bad guy. And I get very bad vibes from that, so we shouldn’t hang out anymore.’”

“I was in a rage. He was a total fuckboy about consent,” she said.

“Honestly, even if they’re supportive, even if they say all the right things, and really want to discuss my job, it makes me feel weird about hooking up with them,” says Sofie Karasek, a recent UC Berkeley graduate and co-founder of End Rape on Campus. (She’s also involved with the UC Berkeley lawsuit and has a large part in the campus rape-culture documentary The Hunting Ground.) “It’s like, ‘Oh, we were just talking about rape, and now we’re going to hook up.’ It’s just weird.”

Maybe most insidious is an expectation that their advocacy — and their own experiences — put them somehow outside the realm of a normal social life.

“Just because someone wants to socialize and date doesn’t mean we’re bad victims or that our experiences haven’t been that bad,” Warner says. “When we talk about sexual assault or prevention, there tends to be the perception of ‘oh, you’ve ruined someone’s life.’ My life was not ruined.” Warner says. “People heal at different rates. Some people can’t date and they aren’t ready and they might never be ready. But we’re not broken. What happened to me had a lasting impact on my life, but I still enjoy my life.”

Still, Keenan can attest that sometimes — when she’s lucky — her advocacy and her dating life are perfectly compatible.

There was one time, one guy, knowing what I did and what I talked about, he made consent part of foreplay,” she reacalls. “You know, very intentionally asking, ‘Is this okay? Is this okay?’ It was cute. It was great.”


Struggling Grafton confident it can get to the playoffs

YORK — — No one has really landed a knockout blow on Bay Rivers District preseason football favorite Grafton High this year. But as in boxing, an accumulation of blows in football can leave you as dazed as a haymaker.

So it is with the Clippers (2-3, 2-2 district) as they ready for Friday's game at Bailey Field against current Bay Rivers leader Smithfield (5-0, 4-0). That their three losses were essentially decided on each game's final play — goal-line stands by Powhatan and Lafayette, and a blocked kick by Poquoson — doesn't make it any easier.

"We're pretty shocked," senior wide receiver/cornerback Jesse Santiago said. "You come out to practice and it's not as intense as it used to be.

"A big win Friday might jump-start us."

Senior tailback Dylan Bartells said, "There's a lot of frustration. From what I'm looking at, we've got to play four quarters of football."

There are some tangible reasons for the Clippers' slow start, including the program's quarterback curse. Grafton coach Jared Van Acker didn't decide on a starting quarterback until the third game, when he tabbed Jett Johnson, who broke his leg in game four. It's the fourth consecutive season Grafton has lost its starting QB to injury.

Perhaps a bigger loss to the offense has been Harvard commitment Marcus Spearman, the Clippers rushing leader in the season opener. He hasn't played an offensive down since the first game because of a shoulder sprain. An all-state defensive back, Spearman returned to the defense for Saturday's 15-7 loss to Lafayette, but is still far from 100 percent.

"They're a big part of the team," Bartells said. "It hurts to lose players like that."

But there is also a lack of urgency and execution that has dogged the Clippers, who have started slowly in every game. Even in victories against New Kent and Bruton, two of the district's weakest teams, the games were close well into the second half.

"We've got to get a mentality to get the job done for 48 minutes, especially against the good football teams," Van Acker said. "You give a team like Lafayette any room and they'll attack you.

"We're a different team when we kick it in."

For almost three full quarters, Lafayette pushed around the Clippers, limiting them to three yards total offense and building a 15-0 lead that could've been a touchdown bigger. Then the Grafton team many expected all along suddenly appeared.

The Clippers put together a 16-play, 91-yard touchdown drive, in which Jackson, 0 of 6 passing to that point, had six completions. His nicest was a 37-yard completion to Tyler Noel while rolling out. Jackson capped the drive with a 2-yard touchdown pass to Santiago on fourth down.

The momentum carried over. The defense, steady all season, forced a fumble. Five Bartells' runs for 51 yards moved the Clippers to the Rams 23, where a dropped pass on fourth-and-4 killed the drive.

After another stop by the defense, the Clippers drove 88 yards to the Rams' 2, Jackson completions of 27 and 37 yards to Bryan Mitchell being the big plays. On third-and-1 at the 2, Parker Kindley intercepted Jackson's pass at the goal line with 29 seconds left to decide the issue.

Were the final 16 minutes a season-changing punch or a desperate flurry by a staggering fighter? We'll know when the bell rings after 48 minutes against Smithfield, but the Clippers see hope in their strong finish.

"The execution and heart we showed at the end of the Lafayette game is something we need to carry through an entire game," Spearman said.

Bartells said, "Hopefully the momentum carries over to this week. We need to stick together and keep our cool."

Santiago said, "The second half against Lafayette showed our potential. We still have confidence that we can make the playoffs and do some damage."


Phenylalanine hydroxylation cofactor in phenylketonuria.
The enzymatic conversion of phenylalanine to tyrosine had previously been shown to require a nonprotein cofactor. It has now been demonstrated by direct assay that the cofactor is present in phenylketonuric liver samples. The lack of a functional phenylalanine hydroxylating system in phenylketonuria is not due to the absence of the cofactor.


Verification

What our clients say

Thank you so much for the amazing service provided by Christian and the use of your amazing vehicle! It was very comfortable and Christian made us all feel very welcome. Will never use anyone else when we return to GC.
Kind Regards,
Maria

Maria

Hi Joanne,
I wanted to let you know that the Chrysler 300C silver black car we had for our daughters wedding was just beautiful. It stands apart from other wedding cars and is truly magnificent. It certainly made the entrance at the wedding a talking point. Dean, our driver was absolutely fantastic and extremely polite, a credit to your business. He was very mindful that we had two little flower girls within the bridal party and catered to all their needs as well as the adults. The car was fully equipped with everything we needed and could expect from a limousine company with such high standards.
We will definitely be recommending your business to others. Thank you for helping to make our daughters day perfect.
Sue

Sue

Fantastic service. Shane was excellent. Thank you.

Ben

Cant say enough good things about the service yesterday, the car quality and everything was above and beyond expectation and the driver specifically made the experience.
Will be using again!
Until next time

Jake

Hi there,
Just want to say a big thank you for the great service we received last weekend.
We'll be sure to book your service again next time we return to the Gold Coast.
All the best,

Elisa

Thankyou for sevice, the girls loved it. Price was good , will definately use you again

Shane

Couldn't be happier with the service provided. Vehicle sensational, arrived on time, driver professional and polite, pricing very reasonable. I was very well looked after by Joanne who listened to my needs and recommended the right solution in a friendly and professional way. Her follow up was first class, as the first contact point she is a tremendous asset for your company.

Nick

Good morning,
I'm so sorry for our late feed back. While away I had the most terrible Internet service ... It drove me mad!! (First world problems I know!!

Suzy, Madi, Harry, Priyah, Baxter, Ruby & Archer!!!!!

Hi Joanne,
I have attached the receipt for that extra payment I made today. Thank you so so much again for helping us out with that :) I am so grateful for all of your assistance, you have been very patient with me :)
I look forward to next weekend - thank you so much again - I will make sure to recommend your company to all of my friends who are currently engaged and are planning weddings or functions.
Kind Regards,

Vanessa

Thank you for the transfers your team is the most professional drivers I have experienced. Well Done
Regards,

Renee

Can I just say that it was a great experience. The driver was there when I walked through the doors, he was courteous and helpful, and very informative. He was also very person centred in his approach to settling me in - re asking about any music etc I might like to listen to etc.
A thoroughly enjoyable experience.

Sam

We would just like to say a massive thankyou to you guys. Such prompt timing for pick up and Shane was a great host/driver. We thoroughly enjoyed it and and will definitely be recommending such an epic service to all our friends.
Thank you once again from some very very stoked customers

Brodie

Hi Joanne
Your very welcome.Wishing you all the best and if,or should I say when my wife and I visit the Gold Coast again. You can be rest assured you will be getting a call from me.
Regards

Darren

The service that was provided was excellent and we were so impressed with our driver David, a real asset to the company. We will be back on the Gold Coast in September and look forward to using Limousines in Paradise and meeting with David again.
Many thanks and kind regards.

Tori and Ian

To Bianca, Mal and all the team thank you for your friendly professional service!
Regards Anja and the girls from Melbourne and Gladstone.
We'll definitely be doing it again :-)

Anja

Thank you once again for your great service , we have used you twice now in 6 months on our holidays on the Gold Coast , great drivers , great vehicles , will be using you again when we come on our next trip , take care and thank you so much to your wonderful company ....

Rowan and Kylie

Thank you so much.
We had a wonderful experience with Limousines In Paradise.
Dean the chauffeur was amazing. Nothing was a problem.
The stretch was amazing.
Price was fantastic. We will definately be going straight to you for our next special event.
Thank you so much, you made our night.
Regards,

Denise M

Thank you, it was great. Driver was very friendly and patient. The kids loved it.
Thanks again

Treena

Thanks to Joanne and the team, for helping make my daughters 16th birthday very memorable indeed.
Will be recommending you guys everywhere!!!!
Can't wait to I need to once again need a limo on the Gold Coast!!
Regards,

Rob P

Thankyou so much for the wonderful service your company provided to us yesterday. Compliments to your drivers who were very professional and friendly, the cars were fantastic and your service was on time as promised(unlike another company that i have used in the past)
I have will be highly recommending your company to family and friends.
Thankyou once again

Gayel W

I would be very happy to give you my feed back as we were very impressed with the young gentleman who was our driver. He was punctual very pleasant to talk to you and couldn't be more helpful.
I may use your service again at the beginning of November as it is her 30th birthday and would be very happy to have the same driver. I would like to hire the hummer for her if possible.
Thank you again
Kind regards

Shirley M

Hi Jo,
What can say from booking the car with you on the phone to picking Casey and Libby up on Friday, you provide the best service I've seen in years. The car was immaculate, on time and the gentleman that picked them up was so professional in every aspect, he made there trip so enjoyable.
Thank you very much from all us, we will highly recommending your company in every way.
Kind regards Andrea

Andrea

I would like to put forward our compliments to our driver by the name of Simon who went the extra mile & was very courteous & welcoming. We were very happy with this service & will not hesitate to recommend your company.

Julie Stephens

Just wanted to say thanks for helping us out on the weekend, all your drivers were great, and you never let us down at all. All the Girls had a great time and cannot fault the service at all. Many thanks

The service, vehicle, punctuality, value and accommodation of our needs was
exemplary.
We were all impressed with every aspect of your company and will use you
again and highly recommend you to others.
Thanks so much Limousines in Paradise, well done

Nick Moore

We just wanted to let you know how pleased we were with the service you provided . We felt very special and not to sound silly we felt important so thank you for making our trip just that little bit more ,than 3 days on the Gold Coast .We will gladly recommend your services to everyone. Thank you

Yvonne Apostolou

Can you please pass on our thanks to Richard for his great driving and
looking after us.

Norval

Thanks for the amazing service provided by Mal on our recent trip to Surfers - on arrival and departure everything went smoothly and limousine travel is definitely the best means of transport after a long day of traveling! I would highly recommend Limousines in Paradise to anyone visiting the Gold Coast.

Tanya

Excellent service!!!
We will be back on the Gold Coast in November and I will book again.

Brendan Lee

Thank you always David
Hi. Folks. It was great. David was very pleasant and helpful. I will certainly use your firm again.
Many thanks. We'll done

Roy

Excellent service. Driver was on time, courteous and friendly. Car was well presented. Would definitely use again if we ever have another splurge.

Greg

Brilliant service - I use Limousines in Paradise for corporate travel about once a fortnight. The cars are always impeccable, the drivers are lovely and the service is excellent. Highly recommended. Thanks so much!

Just want to thank you for all you help. We had a fantastic time & great service.
Our bags did go in style!
We will definitely recommend you & use you again on our next visit!

Just a quick note to say how very happy we were with the service you provided recently. Our driver Roland was very pleasant and helpful. The vehicle was exceptionally clean. My family were delighted with the whole experience.

The Juffs Family

Thank you so much Limousines in Paradise, you have been just wonderful. Thank you, thank you, thank you x

Amanda Keys

Thanks for organising a surprise for my kids Trevor kept it a secret right up until they got to the limo. Thanks big time they had the ride of their life

Jo Brown

Hi I just wanted to send you an email to let you know how happy I was with the service you provided at the staff Christmas party this year. We had a wonderful time, the party started on a high with Trevor picking us up in the stylish black limo equipped with all amenities. Trevor paid particular attention to all our needs. He even allowed us to take photos of the team next to the car; we all wanted to thank him for exceeding our expectations. Limousines in paradise contributed to a memorable evening and we will be using your services again in the future.
With kind regards

Natasha Duncombe

I just want to say a big thank you, you guys made the start to our trip wonderful. My husband and son thought it was great and the 2 younger girls loved it too. It's funny we were calling ourselves the Beverley Hillbillies lol. I'm sure that's how we looked.
Anyway thanks again and I'll book you guys again next time we come to Qld if we didn't embarrass Trevor too much

Wendy D'Argo

I just want to say a big thank you to you for the successful booking for my son's Nick Year 12 Formal at Marriot yesterday.
You made everything so easy and smooth and it was such a great pleasure dealing with you.
Also please transmit to John, their wonderful chauffer, our Big Thank You too. John is a truly and kind professional that made Nick & Lauren feel so special and spoiled.
I am delighted with your Services which I will recommend to anyone I know and I am absolutely certain that I will be using again in the future ahead.
I would appreciate if you please forward this email to Limousine Paradise Management.

Delia

I would like to thank you both for making the hens weekend a day to remember. Jo thank you so very much for the providing the second limo on Saturday, the group was very excited and the Bride to be was over the moon!

Jess

Hi I just wanted to send you an email to let you know how happy I was with the service you provided at the staff Christmas party this year. We had a wonderful time, the party started on a high with Trevor picking us up in the stylish black limo equipped with all amenities. Trevor paid particular attention to all our needs. He even allowed us to take photos of the team next to the car; we all wanted to thank him for exceeding our expectations. Limousines in paradise contributed to a memorable evening and we will be using your services again in the future.

Natasha Duncombe

Thank you Limousines in Paradise for my first stretch limousine ride for my 80th Birthday with my family, I felt so special.

B Lean

Limousines in Paradise picked us up after a girls night out in their gorgeous black explorer - we felt like celebs!!! So much fun, so cool inside, great service, would recommend them to anyone who wants to be a star for a night. Thanks guys!

Kate Squires

Fantastic Reliable Service! We hired Limousines in Paradise for a product launch dinner and not only were the drivers prompt and friendly, the limos were clean and in great condition! The office staff (Lani) were also very helpful and patient in taking ou(...)

Rebecca

A big thank you to the friendly driver that turned around for us since we had a diabetic that had forgotten their medication, even tho he had a busy night he still showed patients and carried on with a professional manner, made our night rocking up in a s(...)

Ange Oconnor

You were fabulous and so accomodating! Did absolutely everything to make my mum's 60th birthday so special! I would totally recommend Limousines in Paradise to anyone living on the GoldCoast! : )

Chelsea Carland

Just wanted to say thank you for the lovely service. I'd actually proposed to my long term girlfriend earlier today, and having Warren take us to and from dinner was the perfect way to end our perfect day. Would happily use you(...)


Q:

SQL Server - Error 8623 Severity 16 State 1

Fist of all, let me clarify that I'm brand new to this whole DBA world so I apologize in advance.
One of the databases I'm (since a couple of days ago) in charge of is throwing an Error 8623: "The query processor ran out of internal resources and could not produce a query plan. This is a rare event and only expected for extremely complex queries or queries that reference a very large number of tables or partitions. Please simplify the query. If you believe you have received this message in error, contact Customer Support Services for more information."
This particular instance is running SQL Server 2014 SP2.
How do I find which query is causing this? I've been having a hard time finding documentation on this.
Thanks!

A:

You can use Extended Events to capture failing queries.
Here is a link to a detailed howto article.
https://www.brentozar.com/archive/2013/08/what-queries-are-failing-in-my-sql-server/
TSQL to create xevents and start:
CREATE EVENT SESSION what_queries_are_failing
ON SERVER
ADD EVENT sqlserver.error_reported
(
ACTION (sqlserver.sql_text, sqlserver.tsql_stack, sqlserver.database_id, sqlserver.username)
WHERE ([severity]> 15)
)
ADD TARGET package0.asynchronous_file_target
(set filename = 'C:\XEventSessions\what_queries_are_failing.xel' ,
metadatafile = 'C:\XEventSessions\what_queries_are_failing.xem',
max_file_size = 5,
max_rollover_files = 5)
WITH (MAX_DISPATCH_LATENCY = 5SECONDS)
GO

-- Start the session
ALTER EVENT SESSION what_queries_are_failing
ON SERVER STATE = START
GO

-- Trigger Level 16 error
select * from Person.Person where ModifiedDate='20aaa';

--Query the logfile
;with events_cte as(
select
DATEADD(mi,DATEDIFF(mi, GETUTCDATE(), CURRENT_TIMESTAMP),
xevents.event_data.value('(event/@timestamp)[1]', 'datetime2')) AS [err_timestamp],
xevents.event_data.value('(event/data[@name="severity"]/value)[1]', 'bigint') AS [err_severity],
xevents.event_data.value('(event/data[@name="error_number"]/value)[1]', 'bigint') AS [err_number],
xevents.event_data.value('(event/data[@name="message"]/value)[1]', 'nvarchar(512)') AS [err_message],
xevents.event_data.value('(event/action[@name="sql_text"]/value)[1]', 'nvarchar(max)') AS [sql_text],
xevents.event_data
from sys.fn_xe_file_target_read_file
('C:\XEventSessions\what_queries_are_failing*.xel',
'C:\XEventSessions\what_queries_are_failing*.xem',null, null)
cross apply (select CAST(event_data as XML) as event_data) as xevents
)
SELECT *
from events_cte
order by err_timestamp;


Q:

How to fill pandas dataframe with the same array

Trying to fill a pandas dataframe with an array (let's say np.zeros(200)).
If we want to fill a pandas dataframe with a value:
for col in df.columns:
    df[col].values[:] = 2

it works fine, but doing the same with:
for col in df.columns:
    df[col].values[:] = np.zeros(200)

will not work.
I don't understand why the error is thrown (could not broadcast input array X into shape Y,) since I thought I was populating each value of the dataframe individually, therefore the shape wouldn't matter in my opinion.

A:

Assuming I have an matrix of 200 rows:
df = pd.DataFrame(
    np.random.randint(1, 20,size=(200,3)),
    columns = ['one','two','three']
)
df.head()

    one two three
0   6   11  12
1   9   10  5
2   16  11  6
3   14  5   10
4   19  5   4

When placing: df [col] .values It does not refer to the column itself, it only obtains a series-to-array transformation, even more when putting [:] it is obtaining a memory copy of the obtained array.
The correct form would be:
for col in df.columns:
    df.loc[:, col] = np.zeros(200)
df.head()

    one two three
0   0.0 0.0 0.0
1   0.0 0.0 0.0
2   0.0 0.0 0.0
3   0.0 0.0 0.0
4   0.0 0.0 0.0

Assuming you want to do this work with other arrays that do not contain only zeros


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Business VoIP Providers

Residential VoIP Providers

VoIP Network Issues

VoIP Setup- Routers and Switches

Having more than one NAT/router in a network can
create issues that will affect your VoIP connection. You may experience issues right away, or they
may develop sporadically over a period of time. These issues can include
one-way audio and calls dropping for no apparent reason. Setting up your
network correctly can be beneficial for your VoIP quality and connection, as
well as the overall
stability of your LAN. We have added information on best practices,
including how to configure your second wireless router after adding a VoIP
Linksys RT31P2 ATA/router.

Routers, switches and other Devices:

Starting with the Internet connection you will usually find a DSL modem,
cable modem or another firewall/router device. These devices can have both
firewall capabilities and be NAT/routers, supplying DHCP addresses to the
internal LAN devices. The problem for VoIP is that they may use an
unfriendly NAT transversal or SPI (stateful packet inspection),
preventing the voice packets from making it back to the ATA correctly. (Netgear
routers use both a problematic NAT and SPI. You may need to place the ATA
into a
DMZ zone on a Netgear.

In most cases, if more than one NAT/router is between the
ATA and the Internet, this double NAT issue will create connection problems and
one-way audio. Add to that the fact that double NAT is not
considered good network practice, you will want to make sure to limit the amount
of devices supplying DHCP.

Many of our featured business VoIP providers below include a free setup and router with their service plans:

*Rates shown do not include taxes, surcharges and E-911 charges. Some providers offering promotions like unlimited calling have restrictions. Read individual providers terms and conditions before you buy.

Having problems with your VoIP connection then look at these possibilities:

More than one device with DHCP enabled between
the ATA and the public IP.

A device that uses NAT unfriendly SIP or SPI.

Firewalls blocking the UDP packets needed for the
ATA.

Most DSL modems act as a routers for the internal
LAN, while most cable modems do not. (This is the primary
reason why a cable VoIP installation will be much easier than a DSL install.) Some DSL modems and certain types of NAT/routers can use a SIP unfriendly
NAT. The result can be one way-audio, where the other party may hear you,
but you cannot hear any incoming audio. This is an issue caused by the
return voice packets stopping at the router, firewall or double NAT router and
never finding their way back to the ATA. If you suspect this, then:

Make your DSL a bridge and authenticate on the router or ATA/router using
PPPoE. If connecting a separate IAD device, then connect it directly to
the router.

Change any second NAT/routers to a switch and disable DHCP.

If you need to add a wireless or second router to your LAN and are using a
Linksys router/ATA, then the best configuration would be as follows.

Connect the Linksys/ATA (address 192.168.15.1) to the modem using the
Internet port of the ATA.

Connect a PC to your second router (address 192.168.1.1) and access the
internal setup pages. Disable DHCP and save settings.

Connect the second router via an Ethernet cable from a numbered port on
the ATA VoIP router to the second router's Uplink port (if this second router
does not have an uplink port then try a regular port).

One-way Audio:

VoIP protocols often will not function correctly in networks that include
some types of NAT transversal or firewall components. The result is usually
that calls can have one-way audio or fail.

One-way audio is usually the result from a double NAT issue or an
unfriendly NAT transversal being used on the network. VoIP itself
presents a problem for NAT, in that the normal protocols it uses deal with the
signaling and establishing of the connection. These packets are generally
identified by the NAT/router with the ATA device and will be forwarded
correctly. But, once the connection is established, the audio, which is an
entirely different protocol and can be on a port initiated from an outside
source, confuses the router. It does not recognize the incoming packets as
belonging to the ATA, and so they get dropped.

To alleviate some of these NAT issues some VoIP providers run Session Border
Control servers. These devices both handle traffic and shape the packets
to get past NAT transversal and firewalls.

How to troubleshoot one-way audio.

The first step would be to try to limit the devices between the ATA and the
Internet. To do this connect the ATA directly to the first device,
which would usually be a modem. (You may have to do a power cycle of the
modem to get the VoIP ATA to connect. more)
If after the ATA connects and establishes dial tone you have two way audio, then
you know that it works at that point in the network and you will need to locate
the device that is blocking the transmission further down line (usually a second
router).

If the VoIP ATA does not establish two-way audio connected directly to the
modem (first device), then look at these possible issues and solutions:

A DSL modem that uses an unfriendly NAT transversal and does not pass VoIP
protocols easily. In this case you should consider bridging the modem
and configuring the ATA/router or a VoIP protocol friendly router to use
PPPoE, with username and password.

Determine where the public IP address is sitting and if possible configure
the network so the ATA/router or IAD receives a public IP address.

Configure any firewalls to allow the correct ports needed to pass directly
to the ATA.

Place the ATA VoIP device in a DMZ or perimeter zone, where it will sit
outside the firewall.

networking tips

Linksys BEFSR41 Version 4
router

How to set QoS for VoIP
on this popular router. Although the QoS capabilities are not
as geart as we would like, having the ability to prioritize traffic to benefit
your VoIP connection is a big benefit.
[instructions]

One-way audio is usually the result of two or more NAT
routers or a router that uses a SIP unfriendly NAT.Other
possibilities can be a firewall that creates havoc with the voice packets and
they do not make it back to the phone.

VoIP itself presents a problem for NAT. The
normal protocols it uses (SIP) deal with the signaling and establishing of a
connection of the ATA. The router or firewall will not recognize the incoming voice
packets as belonging to the ATA, and so they get dropped.

Netgear routers can present problems for some VoIP
because of the type of NAT translations and SPI that it does. To
resolve issues with Netgear routers place the ATA into a DMZ zone.

If you are using two devices on the same network make sure that they do
not share the same IP address. This will lead to routing problems on your
network. For example if the two devices each have a LAN IP address of
192.168.1.1, the change the second device to 192.168.1.254, thus keeping it on
the same subnet, but stopping the IP conflict.

The most stable and best configuration for connecting a router/IAD would be
to have it connected directly to the modem, and any other router connected to it
with DHCP disabled. For full instructions see the following:

If you are using more than one ATA behind the same
router then you may want to set the port ranges on each ATA device so that they
will not conflict with each other.

Using multiple ATA's in the same location is usually not a problem if the
following conditions exist:

Each ATA/router has a public IP address.

Each ATA has a good Internet connection with sufficient bandwidth.

It's when multiple ATA's are sitting on the same internal network sharing one
Internet connection that special configurations and considerations are needed.
(For small businesses the first limitation will usually be sufficient
bandwidth.)


Introduction {#S0001}
============

Colorectal cancer (CRC) is the third most common cancer worldwide, with high morbidity and mortality in China.[@CIT0001],[@CIT0002] Because advanced colorectal cancer still lacks effective diagnosis and treatment, it is necessary to further explore and analyze the pathogenesis and regulation mechanism of CRC to determine effective biomarkers.

At present, a large number of studies have shown that long noncoding RNAs (lncRNAs) are important regulators in cells and are widely involved in the occurrence and development of human diseases.[@CIT0003]--[@CIT0009] LncRNAs are noncoding RNAs with a length of more than 200 nt, as ceRNAs, which further regulated the production of downstream proteins by regulating the miRNA/mRNA axis.[@CIT0010]--[@CIT0013] Some lncRNAs have been reported to be differentially expressed in colorectal cancer tissues and cells, but their functions have been verified.[@CIT0014] For example, lncRNA DLEU1 promoted CRC cell progression through regulating KPNA3.[@CIT0015] Moreover, lncRNA CRNDE induced cell proliferation and chemoresistance in CRC.[@CIT0016] lncRNA BANCR has been determined to relate to CRC poor prognosis.[@CIT0017] However, the regulation mechanism of lncRNA in colorectal cancer has not yet been elucidated, and further exploration and research are needed.

The lncRNA HNF1A antisense RNA 1 (HNF1A-AS1), as an oncogene, was involved in cell progression and metabolism in many cancers, such as lung adenocarcinoma, colorectal carcinoma, oesophageal adenocarcinoma, nonsmall cell lung cancer, bladder cancer and hepatocellular carcinoma.[@CIT0018]--[@CIT0023] Zhao et al demonstrated that induction of HNF1A-AS contributed to cell proliferation and metastasis through activating Wnt/β-catenin signaling pathway in osteosarcoma.[@CIT0024] In addition, lncRNA HNF1A-AS1 has been determined to be a novel diagnostic predictor for prognosis of cancers.[@CIT0025],[@CIT0026] However, the effect of HNF1A-AS1 on cell glycolysis has not been explained and the regulatory network HNF1A-AS1 remains to be further explored and analyzed.

In the present study, we investigated the functional effects of lncRNA HNF1A-AS1on the cell metabolism and glycolysis in CRC cells, as well as the potential regulatory mechanism.

Materials and Methods {#S0002}
=====================

Tissues, Cell Culture and Transfection {#S0002-S2001}
--------------------------------------

Forty pairs of CRC tissues and adjacent normal tissues were obtained from patients who were diagnosed at Xiangya Hospital Central South University. This experiment has been approved by the Ethics Committee of Xiangya Hospital Central South University. Informed written consents were acquired from patients.

CRC cell lines (SW620, HT-29, HCT116 and SW480) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The immortalized colon cell (NCM460) was purchased from Rongbai (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Sciencell Research, Carlsbad, CA, USA) and 1% penicillin at 37°C in a humidified atmosphere of 5% CO~2~.

Small interfering RNA (siRNA) targeting HNF1A-AS1 (si-HNF1A-AS1), siRNA scrambled control (si-NC), pcDNA-HNF1A-AS1 (HNF1A-AS1), pcDNA-MYO6 (MYO6), pcDNA, miR-124 mimic (miR-212-3p), miRNA negative control (miR-NC), miR-124 inhibitor (anti-miR-212-3p) and inhibitor negative control (anti-miR-NC) were purchased from GenePharma Co. Ltd. (Suzhou, China). These vectors and oligos were transfected into SW620 and HCT116 cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) {#S0002-S2002}
----------------------------------------------------------------------

Total RNA was extracted from CRC cells and tissues using TRIzol reagent (Invitrogen) referring to the protocols supplied by the manufacturer. 1 ng of RNA was reversely transcribed into cDNA using PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). For HNF1A-AS1 and MYO6 detection, One Step SYBR PrimeScript™ RTPCR Kit (Takara) was used to measure the relative expression of HNF1A-AS1 and MYO6. For miR-124 detection, TaqMan miRNA assays (Applied Biosystems, Carlsbad, CA, USA) were used to measure the relative expression of miR-124. All samples were performed on a CFX96 TouchTM Real-Time PCR system (Bio-Rad, Hercules, CA, USA). Glyceraldehyde phosphate dehydrogenase (GAPDH) and U6 were employed to normalize for mRNA and miRNA, respectively. The primer sequences were as follows: the primers for U6: forward, 5ʹ-CTCGCTTCGGCAGCACA-3ʹ and reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ. The primers for miR-124, forward, 5ʹ- GCCTAAGGCACGCGGTG-3ʹ and reverse, 5ʹ-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCATT −3ʹ. The primers for HNF1A-AS1: forward 5ʹ-TCAAGAAATGGTGGCTAT-3ʹ and reverse, 5ʹ- GATCTGAGACTGGCTGAA-3ʹ. The primers for MYO6: forward 5ʹ- GATGGAGCTGCACCCTGACA-3ʹ and reverse, 5ʹ- GCTCTCAATGGCGCTCTGAAG-3ʹ. The primers for GAPDH: forward 5ʹ-GGAGCGAGATCCCTCCAAAAT-3ʹ and reverse, 5ʹ-GGCTGTTGTCATACTTCTCATGG-3ʹ.

Western Blot Assay {#S0002-S2003}
------------------

Cells were lysed with RIPA Lysis Buffer (Thermo Fisher Scientific) and the protein concentrations were quantified by a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Protein samples were added onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies HK2 (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), MYO6 (1:2000 dilution; Santa Cruz Biotechnology) or GAPDH (1:1000 dilution; Santa Cruz Biotechnology) at 4°C overnight. After being washed in TBS, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The blots were detected by Clarity Western ECL substrate (Bio-Rad) and visualized with a ChemiDocTM MP Imaging System (Bio-Rad).

Glucose Consumption and Lactate Production Assay {#S0002-S2004}
------------------------------------------------

The dose of cloxiquine (CLQ, 50 μM) with/without GW9662 (10 μM, PPAR gamma antagonist) was used to treat SW620 and HCT116 cells for 24 h. Glucose content was determined using a Glucose Assay kit (Rongsheng Biotechnology, Shanghai, China) according to the supplier's instructions. The concentrations of intracellular lactate were analyzed with a lactate assay kit (Sigma, St Louis, MO, USA) following the protocols of the manufacturer.

Transwell Assay {#S0002-S2005}
---------------

For invasion assay, 2 ×10^3^ cells were seeded in the upper chambers with 8-µm pores Matrigel-coated (BD Biosciences, San Jose, CA, USA) and cultured in concentration in DMEM. The lower chamber was added with DMEM containing 10% FBS as a chemoattractant. After incubation for 48 h at room temperature, cells in the upper chamber were scraped off with cotton swabs. Cells in the lower chamber were fixed with methanol and then stained by crystal violet for 30 min. For migration assay, cells were added onto the upper chambers without Matrigel-coated, other steps were similar to invasion assay. The migrated and invasive cells were calculated using a Countess automatic cell counter (Invitrogen).

Luciferase Reporter Assay {#S0002-S2006}
-------------------------

The fragments of HNF1A-AS1 containing the wild-type or mutated miR-124 binding sites were amplified and cloned into a pRL-TK (Promega, Madison, WI, USA), namely HNF1A-AS1-WT-Luc or HNF1A-AS1-MUT-Luc. In addition, the fragments of MYO6 containing the wild-type or mutated miR-124 binding sites were amplified and inserted into pRL-TK, namely MYO6-WT-Luc or MYO6-MUT-Luc. Then, these vectors were co-transfected with miR-124 or miR-NC into SW620 and HCT116 cells using Lipofectamine 2000 reagent (Invitrogen). The renilla luciferase control vector pRL-ubi-63E was a control vector using transfection reagent Lipofectamine 2000 (Invitrogen). After transfection for 48 h, cells were collected and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega).

RNA Immunoprecipitation (RIP) Assay {#S0002-S2007}
-----------------------------------

The EZ-Magna RIP Kit (Millipore) was used for RIP experiments according to the manual. Briefly, cells were collected by centrifugation and lysed in RIP buffer. Then, cells were incubated with human anti-Argonaute 2 (Ago2) antibody (Abcam) or normal mouse IgG (negative control, Sigma-Aldrich) overnight. Nonspecific binding was removed with proteinase K buffer (Abcam), and then the immunoprecipitated RNA was isolated. RT-qPCR was used to analyze the enrichment of HNF1A-AS1 or MYO6.

Statistical Analysis {#S0002-S2008}
--------------------

Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). Comparison between the two groups was analyzed using Student's *t*-test and among three or more sets were calculated using LSD. All data were presented as means ± standard deviation (SD). *P*\<0.05 was considered to be statistically significant.

Results {#S0003}
=======

HNF1A-AS1 Was Upregulated in Colorectal Cancer Tissues and Cell Lines {#S0003-S2001}
---------------------------------------------------------------------

To explore the expression of HNF1A-AS1 in CRC tissues and cell lines, we collected 40 pairs of colorectal cancer tissues and adjacent tissues as well as purchased four CRC cell lines (SW620, HT-29, HCT116 and SW480) and immortalized colon cells (NCM460). As shown in [Figure 1A](#F0001){ref-type="fig"}, the expression of HNF1A-AS1 in cancer tissues was significantly higher than that in normal tissues. In addition, we also found HNF1A-AS1 expression was upregulated in SW620, HT-29, HCT116 and SW480 cells compared with that in NCM460 cells ([Figure 1B](#F0001){ref-type="fig"}). Moreover, we selected two CRC lines (SW620 and HCT116), which have a relative higher expression of HNF1A-AS1 for the following experiments. Collectively, these results showed that HNF1A-AS1 was upregulated in CRC tissues and cell lines.Figure 1HNF1A-AS1 was upregulated in CRC tissues and cell lines. (**A, B**) The expression of HNF1A-AS1 in colorectal cancer tissues and normal tissues (**A**), as well as CRC cells (SW620, HT-29, HCT116 and SW480) and immortalized colon cells (NCM460) was detected by RT-qPCR. \*\**P* \< 0.01. \*\*\**P* \< 0.001.

Knockdown of HNF1A-AS1 Inhibited Cell Migration and Invasion as Well as Glycolysis in Colorectal Cancer Cells {#S0003-S2002}
-------------------------------------------------------------------------------------------------------------

To determine the effects of HNF1A-AS1 on cell migration, invasion and glycolysis in CRC cells, we transfected two CRC cell lines (SW620 and HCT116) with siHNF1A-AS1 to construct CRC cells with lower HNF1A-AS1 expression than control cells ([Figure 2A](#F0002){ref-type="fig"}). As shown in [Figure 2B](#F0002){ref-type="fig"} and [C](#F0002){ref-type="fig"}, both relative glucose consumption and lactate production were obviously reduced in SW620 and HCT116 cells with HNF1A-AS1 knockdown compared with cells transfected with si-NC ([Figure 2B](#F0002){ref-type="fig"} and [C](#F0002){ref-type="fig"}). More than that, the expression of HK2 was also decreased in the si-HNF1A-AS1 group compared with that in the si-NC group of SW620 and HCT116 cells ([Figure 2D](#F0002){ref-type="fig"}). In addition, cell migration and invasion were significantly lower in SW620 and HCT116 transfected with si-HNF1A-AS1 than that in cells transfected with si-NC ([Figure 2E](#F0002){ref-type="fig"} and [F](#F0002){ref-type="fig"}). These data determined that knockdown of HNF1A-AS1 inhibited cell migration and invasion as well as glycolysis in CRC cells.Figure 2Knockdown of HNF1A-AS1 inhibited cell migration and invasion as well as glycolysis in CRC cells. SW620 and HCT116 cells were transfected with si-NC or si-HNF1A-AS1. (**A**) HNF1A-AS1 expression in transfected SW620 and HCT116 cells was monitored by RT-qPCR. (**B**) Glucose consumption was measured in transfected cells utilizing Glucose Assay kit (**C**) Lactate production was detected in SW620 and HCT116 cells with a lactate assay kit after transfection. (**D**) The protein level of HK2 in transfected SW620 and HCT116 cells was detected in by Western blot. (**E** and **F**) Cell migration (**E**) and cell invasion (**F**) in SW620 and HCT116 cells were examined with transwell assay. \*\**P* \< 0.01. \*\*\**P* \< 0.001.

miR-124 Was a Target of HNF1A-AS1 {#S0003-S2003}
---------------------------------

To further investigate the potential regulatory mechanisms of HNF1A-AS1 in CRC cells, we sought potential target miRNAs for HNF1A-AS1 through DIANA tools and found that miR-124 had a reverse complement to 3ʹUTR of HNF1A-AS1 ([Figure 3A](#F0003){ref-type="fig"}). Therefore, we constructed HNF1A-AS1-WT-Luc (binding sites of wild type) or HNF1A-AS1-MUT-Luc (binding sites of mutate) vectors. Luciferase reporter assay determined that luciferase activity of HNF1A-AS1-WT-Luc in SW620 and HCT116 cells were remarkably reduced by miR-124, while the luciferase activity of HNF1A-AS1-MUT-Luc has not changed ([Figure 3B](#F0003){ref-type="fig"} and [C](#F0003){ref-type="fig"}). Otherwise, RIP assay was applied to further validate the interaction between miR-124 and HNF1A-AS1, the results showed that HNF1A-AS1 was significantly enriched in the RIP-Ago2 than RIP-IgG in SW620 and HCT116 cells ([Figure 3D](#F0003){ref-type="fig"} and [E](#F0003){ref-type="fig"}). Meanwhile, we analyzed the expression of miR-124 in SW620 and HCT116 cells with HNF1A-AS1-overexpression or HNF1A-AS1-down-expression via RT-qPCR ([Figure 3F](#F0003){ref-type="fig"} and [G](#F0003){ref-type="fig"}). The results showed that upregulated HNF1A-AS1 inhibited miR-124 expression while downregulated HNF1A-AS1 promoted miR-124 expression in SW620 and HCT116 cells. Based on the above results, we ensured that miR-124 was a target miRNA of HNF1A-AS1.Figure 3miR-124 was a target of HNF1A-AS1. (**A**) The predicted wild-type and mutated type of miR-124 binding sites in the 3ʹUTR of HNF1A-AS1 were exhibited. (**B** and **C**) Luciferase activity was measured in SW620 (**B**) and HCT116 (**C**) cells co-transfected with HNF1A-AS1-WT or HNF1A-AS1-MUT and miR-124 or miR-NC through luciferase reporter assay. (**D** and **E**) RIP assay was performed to ensure the relationship between HNF1A-AS1 and miR-214 in SW620 (**D**) and HCT116 (**E**) cells. (**F** and **G**) the expression of miR-124 was detected in SW620 (**F**) and HCT116 (**G**) cells transfected with pcDNA, HNF1A-AS1, si-NC or si-HNF1A-AS1. \*\*\**P* \< 0.001.

The Effects of Downregulated HNF1A-AS1 on Colorectal Cancer Cells Were Impaired by Silenced miR-124 {#S0003-S2004}
---------------------------------------------------------------------------------------------------

To further understand the function of miR-124 in CRC cells, we analyzed the expression of miR-124 in four CRC cell lines (SW620, HT-29, HCT116 and SW480) by RT-qPCR and observed that miR-124 expression was significantly reduced in CRC tissues and cells compared to immortalized colon cells (NCM460) ([Figure 4A](#F0004){ref-type="fig"} and [B](#F0004){ref-type="fig"}). As shown in [Figure 4B](#F0004){ref-type="fig"} and [C](#F0004){ref-type="fig"}, we constructed SW620 and HCT116 cells with a lower expression of miR-124 through transfection with anti-miR-124. Furthermore, the si-HNF1A-AS and anti-miR-124 were co-transfected into SW620 and HCT116 cells for functional rescue experiments. We found that si-HNF1A-AS transfection significantly induced the expression of miR-124, which the promoted impact was alleviated by anti-miR-124 transfection in SW620 and HCT116 cells. Additionally, both relative glucose consumption and lactate production were inhibited by downregulating HNF1A-AS, while this inhibitory effect was weakened by anti-miR-124 transfection in SW620 and HCT116 cells ([Figure 4D](#F0004){ref-type="fig"}--[G](#F0004){ref-type="fig"}). In addition, the reduction of HNF1A-AS inhibited the expression of HK2, which was rescued by anti-miR-124 transfection in SW620 and HCT116 cells ([Figure 4H](#F0004){ref-type="fig"}). Strikingly, cell migration and invasion were suppressed by reduction of HNF1A-AS, while they were relieved by inhibition of miR-124 in SW620 and HCT116 cells ([Figure 4I](#F0004){ref-type="fig"}--[M](#F0004){ref-type="fig"}). Thus, these data determined that silencing of miR-124 could reverse the si-HNF1A-AS-mediated inhibited effects on cell migration, invasion and glycolysis in SW620 and HCT116 cells.Figure 4The repressed effects of si-HNF1A-AS1 on cell migration, invasion and glycolysis in CRC cells were impaired by anti-miR-124. (**A**) The expression of miR-124 in CRC cell tissues and normal tissues was detected using RT-qPCR. (**B**) The expression of miR-124 in CRC cell lines (SW620, HT-29, HCT116 and SW480) and immortalized colon cells (NCM460) was analyzed by RT-qPCR. (**C**--**M**) SW620 and HCT116 cells were transfected with si-NC, si-HNF1A-AS1, si-HNF1A-AS1 + miR-NC or si-HNF1A-AS1 + miR-124. (**C** and **D**) the expression of miR-124 was detected in SW620 (**C**) and HCT116 (**D**) cells using RT-qPCR. (**D** and **E**) Glucose consumption was measured in SW620 (**E**) and HCT116 (**F**) cells after transfection. (**G** and **H**) Lactate production was tested in SW620 (**G**) and HCT116 (**H**) cells after transfection. (**I**) The expression of HK2 was evaluated in transfected SW620 and HCT116 cells. (**J** and **K**) cell migration was detected in treated SW620 (**J**) and HCT116 (**K**) cells. (**L** and **M**) cell invasion was analyzed in SW620 (**L**) and HCT116 (**M**) cells after transfection. \*\**P* \< 0.01. \*\*\**P* \< 0.001.

miR-124 Directly Targeted MYO6 {#S0003-S2005}
------------------------------

We all know that miRNAs regulate the expression of downstream target genes. To further investigate the regulatory network of HNF1A-AS, we predicted the potential target genes of miR-124 by DIANA tools and found that MYO6 has a complementary sequence to miR-124 ([Figure 5A](#F0005){ref-type="fig"}). MYO6-WT-Luc or MYO6-MUT-Luc vector was constructed and co-transfected into SW620 and HCT116 cells with miR-NC or miR-124. Luciferase reporter assay analysis showed that luciferase activity of MYO6-WT-Luc was apparently decreased after co-transfection with miR-124, but no significant change was observed in luciferase activity of MYO6-MUT-Luc ([Figure 5B](#F0005){ref-type="fig"} and [C](#F0005){ref-type="fig"}). RIP assay also determined that MYO6 expression was enriched in the RIP-Ago2 than RIP-IgG in SW620 and HCT116 cells ([Figure 5D](#F0005){ref-type="fig"} and [E](#F0005){ref-type="fig"}). Additionally, the expression of MYO6 was inhibited by the upegulation of miR-124, while promoted by downregulation of miR-124 in SW620 and HCT116 cells ([Figure 5F](#F0005){ref-type="fig"} and [G](#F0005){ref-type="fig"}). The above results indicated that MYO6 was the target gene of miR-124 in CRC cells.Figure 5miR-124 directly targeted MYO6. (**A**) The predicted wild-type and mutated type of miR-124 binding sites in the 3′UTR of MYO6 were shown. (**B** and **C**) Luciferase activity was measured in SW620 (**B**) and HCT116 (**C**) cells co-transfected with MYO6-WT-Luc or MYO6-MUT and miR-124 or miR-NC by luciferase reporter assay. (**D** and **E**) RIP assay was performed to ensure the relationship between MYO6 and miR-214 in SW620 (**D**) and HCT116 (**E**) cells. (**F** and **G**) The protein expression of MYO6 in SW620 (**F**) and HCT116 (**G**) cells transfected with miR-NC, miR-124, anti-miR-NC or anti-miR-124 was detected with Western blot assay. \*\*\**P* \< 0.001.

The Effects Induced by Overexpression of miR-124 on Colorectal Cancer Cells Were Rescued by Upregulated MYO6 {#S0003-S2006}
------------------------------------------------------------------------------------------------------------

To further clarify the regulatory mechanisms of miR-124 and MYO6 in CRC, the rescue experiments were performed on SW620 and HCT116 cells. First, we measured the expression of MYO6 in CRC tissues and cell lines (SW620, HT-29, HCT116 and SW480), and the results showed that MYO6 was highly expressed in CRC tissues and cells ([Figure 6A](#F0006){ref-type="fig"} and [B](#F0006){ref-type="fig"}). In the following experiment, miR-124 mimics and pcDNA-MYO6 were co-transfected into SW620 and HCT116 cells. As depicted in [Figure 6B](#F0006){ref-type="fig"} and [C](#F0006){ref-type="fig"}, promotion of miR-124 inhibited MYO6 expression, while upegulating MYO6 could reverse this effect in SW620 and HCT116 cells. Furthermore, relative glucose consumption, lactate production and HK2 expression were obviously reduced by miR-124 transfection, which was impaired by the induction of MYO6 in SW620 and HCT116 cells ([Figure 6D](#F0006){ref-type="fig"}--[H](#F0006){ref-type="fig"}). More than that, the inhibited effects of high miR-124 expression on cell migration and invasion in SW620 and HCT116 cells were rescued by improving MYO6 expression ([Figure 6I](#F0006){ref-type="fig"}--[M](#F0006){ref-type="fig"}). These results showed that the induction of MYO6 could impair the inhibited effects of high miR-124 expression on cell metastasis and glycolysis in CRC cells.Figure 6The inhibitory effects of miR-124 overexpression on cell migration, invasion and glycolysis in CRC cells were rescued by upregulated MYO6. (**A**) The expression of MYO6 was analyzed by RT-qPCR in CRC tissues and normal tissues. (**B**) The expression of MYO6 was analyzed by RT-qPCR in CRC cells (SW620, HT-29, HCT116 and SW480) and immortalized colon cells (NCM460). (**C**--**M**) SW620 and HCT116 cells were transfected with miR-NC, miR-124, miR-124 + pcDNA or miR-124 + MYO6. (**C** and **D**) The protein expression of MYO6 in transfected SW620 (**C**) and HCT116 (**D**) cells was detected by Western blot assay. (**E** and **F**) Glucose consumption was measured in SW620 (**E**) and HCT116 (**F**) cells after transfection. (**G** and **H**) Lactate production was examined in treated SW620 (**G**) and HCT116 (**H**) cells. (**I**) The expression of HK2 in transfected SW620 and HCT116 cells was analyzed via Western blot assay. (**J**--**M**) cell migration and invasion were monitored in transfected SW620 and HCT116 cells by transwell assay. \**P* \< 0.05. \*\**P* \< 0.01. \*\*\**P* \< 0.001.

HNF1A-AS1 Regulated MYO6 Expression Through miR-124 in Colorectal Cancer Cells {#S0003-S2007}
------------------------------------------------------------------------------

In order to further explore the regulatory network among HNF1A-AS1, miR-124 and MYO6. Luciferase reporter methods and Western blot assay were conducted. We found that the luciferase activity was significantly reduced in MYO6-WT-Luc + miR-124 group compared with that in the MYO6-WT-Luc group. In addition, compared with MYO6-WT-Luc+miR-124 + pcDNA group, luciferase activity was notably increased in MYO6-WT-Luc + miR-124 + HNF1A-AS1 group in SW620 and HCT116 cells ([Figure 7A](#F0007){ref-type="fig"} and [B](#F0007){ref-type="fig"}). Not only that, the analysis of Western blot showed that induction of HNF1A-AS1 improved the protein level of MYO6, which was inhibited by the promotion of miR-124 in SW620 and HCT116 cells ([Figure=7C](#F0007){ref-type="fig"} and [D](#F0007){ref-type="fig"}). Therefore, these results indicated that HNF1A-AS1 regulated the expression of MYO6 through miR-124 in colorectal cancer cells.Figure 7HNF1A-AS1 mediated MYO6 expression through miR-124 in CRC cells. (**A** and **B**) Luciferase reporter assay was performed in SW620 (**A**) and HCT116 (**B**) cells transfected with MYO6-WT-Luc following introduction with miR-124 or miR-124+ HNF1A-AS1. (**C** and **D**) The protein expression of MYO6 was detected in SW620 (**C**) and HCT116 (**D**) cells transfected with pcDNA, HNF1A-AS1, HNF1A-AS1+miR-NC and HNF1A-AS1+miR-124 utilizing Western blot assay. \*\**P* \< 0.01. \*\*\**P* \< 0.001.

Discussion {#S0004}
==========

In this paper, we found that lncRNA HNF1A-AS1 was highly expressed in CRC tissues and cells, and further functional experiments showed that silenced HNF1A-AS1 reduced the cell invasion and metastasis abilities of CRC cells. And it was found for the first time that low expression of lncRNA HNF1A-AS1 inhibited intracellular glycolysis in tumor cells. In the research on the regulation mechanism of lncRNA HNF1A-AS1, luciferase reporter assay determined that miR-124 was a target of HNF1A-AS1 and MYO6 was a target gene of miR-124. Therefore, we speculated that lncRNA HNF1A-AS1 affected cell migration, invasion and glycolysis by regulating miR-124/MYO6 axis in CRC cells. Rescue experiments further confirmed our speculation. In this paper, we first put forward lncRNA HNF1A-AS1 regulates MYO6 expression by sponging miR-124 to affect the cell glycolysis process in CRC cells, which significantly improved our understanding of the regulatory mechanisms in CRC.

Cell invasion and migration are necessary biological processes for tumor cells, marking their abilities to migrate and spread.[@CIT0027],[@CIT0028] In clinical research, inhibiting and blocking cell migration and invasion are important processes in the treatment of cancer. Many noncoding RNAs, such as lncRNA and miRNA, as important regulatory factors of cell metabolism, were closely involved in cell progression for various cancers, including invasion and migration.[@CIT0029]--[@CIT0031] For example, lncRNA UCA1 impacted cell invasion and migration through regulating FOXO3 via sponging miR-96 in pancreatic cancer.[@CIT0032] LncRNA ZFAS1 was upregulated in rheumatoid arthritis and contributed to cell invasion and migration through the reduction of miR-27a.[@CIT0033] Moreover, lncRNA SNHG16 promoted cell migration, invasion and proliferation through modulating miR-520d-3p/STAT3 axis in hemangioma endothelial cells.[@CIT0034] Lin et al reported that HIFIA-AS2 promoted cell progression and EMT in CRC cells via regulation of miR-129-5p/DNMP3A.[@CIT0035] In our study, we also found that knockdown of HNF1A-AS1 or promotion of miR-124 inhibited CRC cell migration and invasion. Furthermore, we found that miR-124 was a target of HNF1A-AS1, and MYO6 was a target of miR-124. Accumulating evidence suggested that miR-124 had multiple roles in cell progression of cancers, such as brain tumor and CRC.[@CIT0036],[@CIT0037] A previous study determined that miR-124 was downregulated in CRC and inhibited cell proliferation and drug resistance.[@CIT0037]--[@CIT0039] More than that, MYO6 has been verified to highly express in many cancers, such as prostate cancer, CRC, breast cancer and gastric cancer, which also participated in cell progression.[@CIT0040]--[@CIT0043] In our study, consistent with a previous study, miR-124 expressed low and MYO6 expressed high in CRC tissues and cells, and rescue experiments confirmed the effects of HNF1A-AS1 on cell migration and invasion through targeting miR-124/MYO6 axis.

More than that, glycolysis provides energy for the metabolism of tumor cells and guarantees the proliferation and development of tumor cells. Lactate production and glucose consumption are important indexes for the determination of glycolysis. Additionally, the glycolytic enzyme hexokinase 2 (HK2) is one of the important aerobic glycolytic mediating factors in cells.[@CIT0044]--[@CIT0046] Increasing the expression of HK2 could promote cell glycolysis and release more energy for cell development.[@CIT0047],[@CIT0048] Various lncRNAs were related to glycolysis in cancers, such as lncRNA Ftx, lncRNA p23154, lncRNA MIF and lncRNA MALAT1.[@CIT0049]--[@CIT0052] The study of Li et al suggested that lncRNA UCA1 induced glycolysis in bladder cancer cells, and promoted lactate production and glucose consumption through upregulating HK2 via activation of mTOR-STAT3/miR-143 pathway.[@CIT0048] In this study, inhibition of HNF1A-AS1 or promotion of miR-124 decreased cell glycolysis. While inhibition of miR-124 or promotion of MYO6 could weaken the suppressive effect of reduction of HNF1A-AS1 or promotion of miR-124 on glycolysis, respectively. Therefore, lncRNA HNF1A-AS1 regulated cell glycolysis in CRC cells through targeting the miR-124/MYO6 axis.

Conclusion {#S0005}
==========

In this study, we first found that HNF1A-AS1 regulated cell migration, invasion and glycolysis via modulating miR-124/MYO6 in CRC cells, providing a new regulatory network and a new therapeutic target of CRC.

Disclosure {#S0006}
==========

The authors report no conﬂicts of interest in this work.


KTTZ

KTTZ may refer to:

 KTTZ-FM, a radio station (89.1 FM) licensed to Lubbock, Texas, United States
 KTTZ-TV, a television station (channel 25 digital/PSIP 5) licensed to Lubbock, Texas, United States


High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography.
Simvastatin (SS) is an effective cholesterol-lowering medicine, and is hydrolyzed to simvastatin acid (SSA) after oral administration. Due to SS and SSA inter-conversion and its pH and temperature dependence, SS and SSA quantitation is analytically challenging. Here we report a high-throughput salting-out assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for simultaneous LC-MS/MS analysis of SS and SSA. The sample preparation of a 96-well plate using SALLE was completed within 20 min, and the SALLE extract was diluted and injected into an LC-MS/MS system with a cycle time of 2.0 min/sample. The seamless interface of SALLE and LC-MS eliminated drying down step and thus potential sample exposure to room or higher temperature. The stability of SS and SSA in various concentration ratios in plasma was evaluated at room and low (4 degrees C) temperature and the low temperature (4 degrees C) was found necessary to maintain sample integrity. The short sample preparation time along with controlled temperature (2-4 degrees C) and acidity (pH 4.5) throughout sample preparation minimized the conversion of SS-->SSA to < or = 0.10% and the conversion of SSA-->SS to 0.00% The method was validated with a lower limit of quantitation (LLOQ) of 0.094 ng mL(-1) for both SS and SSA and a sample volume of 100 microL. The method was used for a bioequivalence study with 4048 samples. Incurred sample reproducibility (ISR) analysis of 362 samples from the study exceeded ISR requirement with 99% re-analysis results within 100+/-20% of the original analysis results.


#ifdef GL_ES
precision mediump float;
#endif

varying vec2 v_texCoord;

uniform sampler2D u_texture;
uniform vec4 u_left_color;
uniform vec4 u_right_color;

void main()
{
    //注意运算全部要是浮点型
    vec2 v_tex = v_texCoord;
    vec4 color = texture2D(u_texture,v_tex);

    vec4 new_color = color;

    //左边是u_left_color,右边是u_right_color
    if(v_tex.x <= 0.5){
        new_color *= u_left_color;
    }

    if(v_tex.x >= 0.5){
        new_color *= u_right_color;
    }

    //gl_FragColor = vec4(1.0,0,0,1.0);
    gl_FragColor = new_color;
}


Cutaneous silent period recordings in demyelinating and axonal polyneuropathies.
To investigate the cutaneous silent period (CSP), a spinal inhibitory reflex mainly mediated by A-delta fibres, in demyelinating and axonal polyneuropathy (PNP) and evaluate whether CSP parameters differ between patients with and without neuropathic pain. Eighty-four patients with demyelinating PNP, 178 patients with axonal PNP and 265 controls underwent clinical examination, DN4 questionnaire, standard nerve conduction study, motor-root stimulation and CSP recordings from abductor digiti minimi. We calculated the afferent conduction time of CSP (a-CSP time) with the formula: CSP latency-root motor evoked potential latency. In the demyelinating PNP group the a-CSP time was significantly longer; in the axonal PNP group, CSP duration was shorter than the demyelinating group (p=0.010) and controls (p=0.001). CSP parameters were not different between patients with and without neuropathic pain. The abnormality of a-CSP time in the demyelinating PNP group suggests the crucial role of A-delta fibres in the mechanism of CSP; the shorter CSP duration in the axonal PNP group supports the strong influence of the number of axons on this parameter. Our study suggests that neuropathic pain could be related to pathophysiological mechanisms differing from mere A-delta fibre loss. CSP evaluation is effective in detecting A-delta fibre dysfunction in axonal as well as demyelinating PNP.


Our Words Can Change Worlds

We need to realize that what we say can alter others drastically. For better of for worse.

Our words can change worlds. Those words that fill their hearts with the strength to continue. But the world chooses our words. The worlds with the kids with gashing scars in their brains and soft purple tattoos on their arms the words telling them they are worthless.

Those words that tell them they are beautiful. The worlds with the skeleton girl crying at the face in the mirror. The kids with caked on makeup to cover their true countenance.

Those words that tell them they're not loved. The worlds with the naked kids in showers, washing their scars. The kids who sew blooming purple flowers under red seams, all sewn together on ivory fabric. The self-scarred.

Those words that tell them they are alone. The worlds with the sobbing wracked souls on handmade quilts. The disabled, the fighters, the bullied, the diseased, the isolated, the believers, the minorities.

Those words that tell them they can never be good enough. The worlds with the fake teens trying to please and impress others. The cycle of covering up your true identity.

Those words that tell them to run faster from fear. The worlds with the kids who have to wake up to pain. Who must always be on their guard, hiding from their "loved ones" and "friends".

We are the world. We have power to change those words.

The words that tell them they're worthless. Let's change the world's words to telling them the truth: They're important to somebody out there.

The words that tell them they're the ugly duckling. Let's change those words to what they need to hear: they are stunning.

The words that tell them nobody loves them. Why can't we just tell them they don't need to hurt anymore? They can feel that existing love from us.

The words that declare war can be changed to freedom and peace. The words that can break your heart may bring closure. The words that hold their hands through affection less nights can become soft gloves always there.

Let's help them realize that they aren't alone. They are perfect the way they are. There is no need to be afraid. We can have solutions for these lost souls that were never fully lost.


Donald Trump's Best Twitter Haters

The Donald loves Twitter. Whether Trump is live-tweeting All-Star Celebrity Apprentice (its new season premieres this weekend) or taking pot shots at other celebrities or even inspiring Twitter memes that travel to Asia and back, his Internet presence covers just about everything imaginable.

And when you tweet so frequently and as freely as Trump does — and when the sheer magnitude of your narcissism is the size of Trump's — you're bound to attract some push-back. Not everyone can appreciate the Donald. Below are some of Trump's most notable opponents on Twitter and some of their acerbic, or simply enjoyable, comebacks. Let us know in the comments if there's one that stands out or that we forgot.

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Holy Taco, @holytaco

The L.A.-based comedy site loves nothing more than a Donald jab. In the past month, it's mentioned him in more than 40 tweets. Many are not family-appropriate, but the best involve speculations about Trump's possible birthplace. Taco Bell? Public restroom?

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Martian Donald Trump, @MartianDTrump

This bizarre parody account mimics typical Trump tweets by stringing together various famous Twitter handles and word "ack." The Donald tends to yack.

Angelo Carusone (@GoAngelo)

Carusone started the #DumpTrump hashtag to urge Macy's to stop carrying the Donald's clothing line. After Trump threatened to slap him with a $25 million suit, Carusone used the hashtag to rally soldiers.

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Very Frustrated Pete, @Arseburgers

As his Twitter name suggests, Pete has a lot of gripes with the general populace. But he reserves his most biting insults for all things Trump.

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Stoo Metz, @StooMetz

Many of Mootz's most recent tweets express bewilderment at those of his followers who come to Trump's aid in the midst of one of Metz's typical rants against the Donald. The Canadian burlesque performer recently tweeted: "I find it HILARIOUS the tweets i get for saying anything bad about @realDonaldTrump what a douche."

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Bill Maher, (@billmaher)

When Bill Maher alleged Trump might be part-orangutan, the Donald angrily responded with threat of a lawsuit. To which Maher responded by making the hashtag #Apers (a parody of the Birther movement Trump backed to get Obama's birth certificate) trend on Twitter. "We want proof in long-form," Maher said on his show.

Rosie O'Donnell, (@rosie)

Having put her TV feud behind her, Rosie now trolls Trump in the classy Twitter fashion — by regularly re-tweeting his enemies.

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Deadspin, (@deadspin)

Okay, so not an official Trump Twitter detractor, but no one will ever forget this most famous of Trump put-downs. After the Manti Te'o scandal broke, the Donald tweeted his congratulations to Deadspin for getting the story. Deadspin's response was curt, necessary, and beautiful.


Q:

Is the use of % in printf operator overloading? If not, what is it?

Please explain what happening here:
class Test{
public static void main(String[] args){
    int a = 43%2;
    System.out.printf("The test remainder is %d %s",a,"hello");
 }
}

In the above code i want to know is it operator overloading of the % operator?

A:

Nope. Java currently has very limited support for operator overloading, and this is not one of those cases.
The % in the string literal is handled by the implementation of java.util.Formatter. String#format and PrintWriter#printf delegate the formatting work to Formatter, which manually parses the string.
The only reason % has value there is due to how Formatter handles the string.
If you view the code, you'll find:
if (conversion == '%' || conversion == 'n')

followed by a switch statement which handles the different types.