Back in March, Twitter suspended the account of the massively popular Christian satire site, the Babylon Bee, after it awarded Man of the Year honors to Rachel Levine, a trans woman serving in the Biden administration's Department of Health and Human Services who had been named one of USA Today's Women of the Year. The Babylon Bee's editor-in-chief, Kyle Mann, says that the article was intended to satirize media treatment of identity politics, not demean trans people. We love trans people and we love, you know, we don't, we don't consider people like that beneath us. You know, the Christian world do is that everybody, everybody has the opportunity to be saved, you know, and we can love everybody and I'm no more deserving of God's grace than a transgender person is. But when the culture bows down and starts handing out trophies to people for stuff like this, it's when we say, hey, wait a minute, we need to protect women in our society as well. The Bee's Twitter account remains locked because the publication steadfastly refuses to delete the tweet and acknowledge it contravene Twitter's policy against hate speech. In response to the Twitter ban and persistent demonetization and minimizing of the reach of its content at Facebook, the Bee has created its own social network and subscription model, both of which are flourishing. The episode shines a light on how contemporary culture wars are waged online and illustrates the specific travails that evangelical Christians face in a country that is increasingly secular and socially liberal. Reason caught up with Man at Freedom Fest, the annual gathering in Las Vegas, to talk about why he loves making fun of Donald Trump and Joe Biden, but saves his deepest burns for mega church pastors such as Joe Lasting, why he believes that the left and Gen Z can't deal with humor that makes fun of them, and why he loves personal liberty and personal freedom, even if he creates a culture that is deeply hostile to his faith. Kyle Mann, thanks for talking to reason. Yeah, thanks for having me. You've been editor-in-chief of the Babylon Bee since 2016. I've been with the Babylon Bee since 2016, but I first was just part-time. I was in construction sales and writing headlines before I went into my soul-sucking job. And the soul-sucking job is at the Babylon Bee? No, no, no. I have my construction sales job.  That is dead end, right? Yeah, yeah, for sure. So when did you become editor-in-chief? In 2018. 2018, I think. Two years later, yeah. You recently tweeted something to the effect of you love your job and you did a screencap of the Babylon Bee from like a day or two ago. And I just wanted to go through some of the headlines and get your commentary on that because I think it's a good way to kind of sum up where the Babylon Bee is right now. And like the main headline is in the well of the page, Up Yours, Woke Moralist Cries Jordan Peterson While Attempting to Ride the Ostriches at the Zoo. That's one story. Also near the top is Americans Offer to Trade Lebron to Russia for Brittany Griner. Time Travel and then subscriber headlines. Time Traveling, Joel Osteen Gives Job, Job, as in the biblical book of Job, a copy of Your Best Life Now. Trump's Children Change Their Last Name to Biden so that media will ignore them. Why do we start with those types of things? Like, how, you know, you said, I love my job. Like, what what's great about the Jordan Peterson? Well, that morning I had spent an hour photoshopping Jordan Peterson onto an ostrich. Right. And and there's these surreal moments when you're running the Babylon Bee where you're sitting there like, am I really getting paid for this? How does this work? You know, how how did I get to this point?   So I got to this point. I'm like, I write, I photoshopped Jordan Peterson on an ostrich and I get to talk to John Cleese. So what it was a moment of the surreal realization that I that my job is is so awesome, like we get to create this alternate world and then write the newspaper about it. Right.  Remind me, what does the Babylon Bee respond? What is that reference as? Because you guys are Christian. You're Bible based evangelical Christians. Yes. Yeah. And and you believe the Bible is the inerrant word of God. OK, so this is all as a backdrop of saying you you're being funny and you have tied five hands behind your back because this just adds to the level of complexity. But what what does the title of the Babylon Bee refer to? Yes, so Babylon is a biblical reference to a time when Israel was in Babylonian captivity, Babylonian exile. And, you know, there was this sense where Israel had to maintain their identity as the people of God, despite no longer having the promised land. Right. And so that's where we are as Christians in America. You know, we don't America is not the promised land. It never was the promised land. And yet we're grateful for obviously all the freedoms we have in America. And yet we have to go. But we're exiles or strangers in a strange way, right? But there's a sense of humor that comes with that. You know, there's a you have to have a sense of joy about that, I think. What is the what is the bee in that? Because the bee or the beehive is has biblical dimension. So but it's also a very Mormon. The Mormon. Yeah, there was no. There was no Mormon reference there that we were at them. I don't think so. Yeah. It was a it's got a stinger, which is great for satire. OK.  And Babylon Bee has that great. Yeah, alliteration and there's the Sacramento Bee. So I mean, it's not an unknown name for it. So I realize this is the worst thing that I can be asking. And you talked to John Cleish yesterday. I did. But so I'm going to do the worst thing possible and say, what is funny about a headline that says up yours, what, more or less, cries Jordan Peterson while attempting to ride the ostriches? Well, yeah, because Jordan Peterson is riding an ostrich. So that's that's what your sense of Peterson is.  They find him intolerable and just every extra book he sells, it makes their head explode a little bit more. But do you also do you think he is because he's certainly not a Christian?  Or is it mostly just that he is a flash point for the people that you dislike?  Trump was similar. Trump was great to write jokes about because you write quotes in Trump's voice and people are like, it's exactly how he would say it. So he's great for comedy. I never really followed Peterson that much until, I don't know, a year or two ago, I picked up 12 rules and I thought it was good wisdom. You know, it was interesting as from a Christian perspective that he he hasn't embraced the Christian faith, but he draws a lot of wisdom and applications from and probably knows the Bible better than a lot of Christians do. So it's been interesting to see a mind like his kind of go on this journey. He's come close to Christianity and then move and back the other way. And, you know, I think about if I was a contemporary of someone like C.S. Lewis, who was converted later in life, you know, he was this brilliant philosopher in his own right and brilliant professor in his own right, brilliant author and then comes to faith. You know, and I see a similar kind of. Jordan Peterson is going to join your team at some point, right? Well, if we ever get to interview him, we'll try to convert him. OK, C.S. Lewis, I want to get into your theology a little bit later, but isn't he a little too Catholic for you? Well, he's Anglican.   And then that's a whole problem. You know, yeah, maybe some of the traditions are a little more Catholic. But that's, you know, J.R. Tolkien is my favorite author and he's very Catholic. Oh, my God, that's why I was raised half again. I can't read the Hobbit.  What about the LeBron James, you know, American offers to trade LeBron to Russia for Brittany Griner? And she's the women NBA star who's being held in a Russian prison for having weed vapes. You know, again, why is it so funny to trade LeBron to Russia? Because, you know, we're offering to trade him for Brittany. It's a great joke. And you always say that like you'd never explain the joke, but it's obvious that they're not equivalent in terms of basketball skill and what like whatnot, but or or presents. Well, LeBron is kind of represented, you know, for jokes.  And I hate America and yet America has given me everything that I have. So those kinds of personalities are great punching bags.  And to the extent that that matters, you've got to kind of admire that and give that. And then, you know, he was a rank hypocrite in terms of when Black Lives Matter took place. He said, you know, the NBA should allow or foreground protest by players about George Floyd and Black Lives Matter. And then when it came to people saying, like, hey, we want to protest, you know, the mistreatment of Uyghurs and other people in China.  Yeah, is that I mean, even as he was refusing to do that, when other people were saying, is that the nut of why LeBron bugs you? I don't even know that he bugs me that much. I don't get to I try not to get too passionate about the stuff that we're joking about. Like it's a great joke.  And that's another area where that's great for humor and anybody who's a hypocrite. And that's why Christians have been a great punching bag for years, because people hone in on hypocrisy within Christians really easily, which brings me to the headline time. Time traveling, Joel Austin gives Joe a copy of Your Best Life Now. Explain, I mean, if you follow the Christian world at all, everybody knows who Joel Austin is, but some secular people might not. Who is Joel Austin and why is this funny? Yeah, so he's a soft prosperity, gospel preacher, you know, that kind of has this idea, it's a very associated with the word faith movement and, you know, the whole of the secret and everything that comes with that, that you can speak wealth into existence. You can speak health into existence. Your words have creative power, which is also kind of if you're right with God, you're going to do extremely well in this world. It takes the proverbs to the extreme, you know, it takes the proverbs which say like, work hard and you'll be blessed. But the proverbs are a general principle that say in general, if you work hard, then you're going to reap the rewards of that. But it takes that as this kind of like divine promise that if I put in the work, God is going to reward me with health and wealth. And it's really, it's really devious because it's, it tells people who have cancer, it's your fault. You don't have enough faith. You haven't given enough money. You haven't done enough. And so we've from day one really punched at the prosperity. At the bright shining teeth of Joel Osteen. He's got great teeth. You had to admit that. I don't know where they came from. I don't think they're natural. That might they might have come directly from God or Satan. Satan has great teeth, right? I'm assuming he's got a really good smile. But Osteen, yeah, you you've mocked him. Like whenever there's a natural disaster, Joel Osteen in a Babylon B headline shows up, giving out his own books, right? And then Job, how, you know, Job is obviously, you know, the one of the more interesting characters in the Old Testament. Just briefly recap, you know, why Job. Yeah, Satan basically gets permission from God to beat up Job for a while. And he loses all his livestock and his family and his farms. And he's left with just his nagging wife and boils on him, so it's almost a tragic comedy, you know, one of those dark comedies. It really reflects well. Well, that's the Old Testament God, right? I mean, yeah, so. Right. But so Job just needs to book up. Trump's children changed their last name to Biden so that the media will ignore them. Yeah. And that's another example where hypocrisy, especially in the media, is a great target for us because everybody's sitting there. See, we all see it. We all see what's happening in the media, how they treat one side completely differently from the other side. And when you can just shine a light on that as our satirical newspaper does.  And you guys, you were hitting Trump pretty hard and things like that. A lot of people said as Trump kind of went on, you got a little bit softer on him and you started going after liberals more. Do you think that's a fair criticism? Or did your or did your focus change for other reasons or your focus did not change? I think we went after Trump harder than we've ever gone after anyone on the left. And we still do a lot of good jokes about Trump. You know, I think there's I think there's a lot of humor to be found in Trump. The problem with Trump humor is that the left was doing it so much and so poorly that it just kind of didn't interest me anymore.  So when Trump humor can really play with his character and realize that he already he's already funny in his own right, you don't need to you don't need to add anything. Yeah, and you can just get out of the way that you got a role. You got a role with his character. Once we realized that, I think that Trump humor really became more fun like that. But we've called we had a joke, you know, that said that people who vote for Trump are like voting for the Antichrist or something, you know, so so it's like we never done that kind of savage stuff on the left. So there was something where we were punching our own a little harder. Do you consider Trump a Christian? I don't think so. Yeah. I mean, he says he is there was a and he held the Bible.  And if he touches the Bible, his hand would burn or something. I think we did a big joke on that, that he he was rushed to the hospital with with severe burns. Yeah. And when he was asked, like, what's your favorite book of the Bible? And he was like, all of them. It's like he obviously could not name a saint. He could not say they Genesis or a single guy. Well, then he said two Corinthians, which is how we say it. But I heard him speak. I heard him speak at the Faith and Freedom in Nashville. And he he was making fun of himself for not knowing the Bible. He says, I know, I don't know the Bible as well as people know the Bible. And I think he realizes like, oh, wow, I'm in the space where I'm a complete noob. And why did why did evangelical? I mean, they supported him incredibly strongly. And I realized maybe outside of your expertise, but you're an evangelical. You must know people who were like, yeah, I voted for Trump.  You know, he seems to be a despicable character as a businessman.  His personal life is a mess. He is a kind of horrible character. Why and he is patently not a Christian, but got the Christian vote. And he was one of the few politicians who actually did a lot of the things he said he was going to do. Yeah. So you think they knew that? Well, yeah, I think maybe they got that sense from him. But but also like some people just hated Hillary. I mean, there was a real revulsion to Hillary across the board. And, you know, was that left versus right thing in the binary choice that, you know, I don't know if if we didn't have that binary choice that Trump would. No, it was a trider. There was a man named Gary Johnson who died a criminal's death. Yeah, you know, he was the Trump stuff led to one of the most bizarre moments. I think in my lifetime in a lot of ways, where I like to think every once in a while, you know, that we're living in a Philip K. Dick universe of science fiction writer who, you know, just, you know, where the world is insane in ways we can't fully appreciate. But you guys in I think it was 2018 or there about you published a headline saying that Trump takes credit for doing more for Christianity than Jesus. And then literally a couple of years later, I mean, I guess it was this year or last year, Trump actually said I've done more. And he said it a couple of times in different contexts. I have done more for religion or for Christianity than anybody in history. I mean, how do you know how do you deal with that?  And I think that he doesn't. I don't think Trump takes himself as seriously as his haters think he does. I think he says these big boisterous things because he knows it gets a reaction. He knows it gets applause and laughter.  And I think he leans into that.  Do you I don't I don't really care one way or the other. I mean, it'd be great for comedy. So I'll take that. OK. Although it's kind of bad for comedy, right? And you've pointed this out in other people work with comedy when you don't understand him, but it was great for us. Yeah. That allowed us to have this meteoric rise where we were doing great Trump jokes because the late night liberals didn't know how to do it. Yeah. They were going very, I mean, the easy obvious. It's not even low hanging fruit. It's like Trump. His name is not Trump. It's Trump. Yeah. Like that's that's comedy gold in the late night comedy world. What do you why do you think late night comedy, even places like the onion, which is still doing well, but seems to have lost something of an edge? Is it is it just simply that as liberals retake power, you know, because Trump came in and, you know, the Republicans were running, you know, Congress and the White House. They have the Supreme Court and things like that. That's gone. And does comedy suffer when your team, for lack of a better term, is is kind of running the show? It can. I think you have to be more careful. I don't really buy into the punch up, punch down paradigm where comedy can only punch up. And so if you're if you're out of power, then you're funny. And if you're in power, then it's not funny because it's much down. I don't really buy into that. I do think like I was talking earlier about how Christians were such a great target because Christians are self serious. Christians are dour Christians have this obvious hypocrisy in a lot of areas. Well, I think that describes the woke liberal far left right now.  And they say they were against racism and yet a lot of their policies are racist. They take themselves very seriously because politics is their religion, you know, they believe in salvation, but they believe in salvation through wokeism and they believe in salvation for our country, but only by voting for the right policies. And so if you make fun of them, you're making fun of their religion. Yeah. You, the Babylon be published, a guide to wokeism or wokeness. What do you think define wokeness briefly? And then what is driving it?  Yeah, well, I don't pretend to be a cultural expert or anything. But in my limited experience of what I've observed, it has been this like flashpoint in the last year or two that is just, you know, you heard the word woke kind of being thrown around five years ago, but it wasn't it wasn't this big cultural thing where I can talk to my neighbor and say, welcome to like, I hate woke people or I love, you know, there's now everybody on the street knows what it's right. But yeah, wokeness is a very it's a tough thing because it is a moving target. It is I think at its core, it is kind of the Marxist ish. And I won't say it's exactly defined with classical Marxism or anything, but it is the Marxist ish idea that there's oppressors and oppressed, you know, and they just kind of take that and apply it to different areas, whether that's gender or race or nationality. And and that's where you find all your misery from. Why do you think that viewpoint has captured if if it hasn't become if it's not something the masses believe in it, something that the elite particularly on the left side of the political spectrum really is going going deep on. Yeah, I'm not sure. I mean, I think there's probably a lot of white guilt going around and it's a way to deal with it. You see that because they pay, you know, Robin D'Angelo, $50,000 speaking fees or whatever to show up and tell them how evil they are. But I mean, ultimately, I think I think people need a I think people need a way to salvation and redemption and they need that narrative in their lives. And if they can say, well, here's my sin, I was I mean, my ancestors were evil and then they can find a way to atone for it by doing better. I think that helps them, you know, in their mind, at least. Do you think it is?  Yeah, yeah, absolutely. You know, there's but but I think there's. Well, and I was going to ask if that's the case. So maybe people, you know, maybe we assume that there is a fixed amount of people always want to find salvation, the phrase you use. And it used to be you found salvation through religion. And if religion is gone, then you look, you seek for it and you seek it in other secular passions. Well, I mean, I believe people are created to worship. I believe people are created to find meaning. And if you don't find meaning in the church, you're going to find meaning somewhere else. So people aren't leaving the church. They're going and finding their own church. And it happens to be the religion of woke ism on the left. Right. So one of the things, you know, that is interesting is that you've been, you know, for years now, you guys have been in this interesting kind of war. And I think you you all must own snopes.com because when, you know, the minute that they started fact-checking you as a bad source, as misinformation, because people, they said that people were uniquely taking the Babylon be as truth, you know, and that needs to be fact-checked.  Can you talk a little bit about that? Why do you think there is a concerted effort? And it's not just snopes. It's also CNN and whatnot. There is this interest in kind of delegitimating the Babylon be as, you know, I mean, it's a satirical site that can't be missed. But there seems to be this attempt to say you guys aren't legitimate because people are mistaking you seriously, which is not really true to begin with. But then and that means you shouldn't be doing satire. Yeah, it's an interesting question. And I don't fully know the answer, but it does it does seem like the left is very confused by comedy that makes fun of the left. They don't understand it. They don't get it because they're used to controlling these cultural institutions. They're used to controlling the late night shows and the satire sites and all of that. And so when all of a sudden we come along and we have a hopefully a good sense of humor and we're writing comedy that people are finding funny, they think there's some like nefarious motive there. And that's what they always say in these fact checks. They're intentionally muddying the waters of a current event, news story to confuse people. Like that's the accusation that's made, which is so insane. Like we're writing very similar jokes to the jokes that the other guys are writing on the other side and we're just writing them from our perspective. But I really think that they don't understand that we we just want we just want to put humor out into the world. You know, we want to put laughter out into the world. Yeah, a lot of it's going to come from our world viewer or our perspective, but who's isn't? So I think there's a there's a real confusion over it. You know, that we'll write articles that will make fun of ourselves.    And people on the left freaked out and said, this is such a bad joke. You know, we're kind of sitting there and snickering like, yeah, that's the point. You know, but they really just thought that we didn't have a sense of humor about ourselves because they couldn't they can't comprehend how conservative that sense of humor. Right. One of the in an interview with the Atlantic that you gave last fall, you talked about how, you know, if you have to, you will you you identify more your humor with the baby boom generation or you'll you'll take the you'll side with boomer humor over kind of zoomer humor, Gen Z humor.   And you I think in that interview, you said that, you know, a lot of your readers are boomers and they kind of get it in a way that younger people don't. Yeah, there's a well, you know, I think there's a split. Most of the time I'm at a conference, people will say my college son loves your stuff or my grandpa loves your stuff that's always always. They never like my stuff. No, of course not. Yeah, they're holding a gun on you when they're like that. But, you know, so that so I think there's an interesting thing in that it does appeal to multiple people. But the to me, boomer humor was this kind of classic Seinfeld cheers, you know, and I know that's during the Gen X era. Yeah, it's a 90 or 80s and that's when that's when boomers with your 40 and we're watching that stuff and tuning into it. There's a classic, you know, just just good old fashioned men do this. Women do this. Isn't that silly observational humor about life? And now it seems like, you know, with Gen Z's humor, they subvert that and they say, no, there's no punch line. You watch a TikTok video, there's no punch line. It's something crazy happens. And then the video cuts off. There's there's like a nihilism to the humor. And I think absurdity can be great in humor, of course. But, you know, then they go and they attack everybody that builds all these great cultural institutions for comedy, you know, whether that's Saturday Night Live or all the classic sitcoms or Calvin and Hobbes. I mean, these are things that like these are heights to which, you know, I don't think Gen Z humor can ever aspire to be, you know, and we'll see what comes of it. But yeah, is part of it that older forms of humor and boomer humor. And I'm thinking like early Saturday Night Live, which is, you know, made by the early boomers were parroting, you know, TV shows from the 50s and the 60s and kind of broadly held, you know, cultural templates, people had a common culture. So you knew what the form of a presidential address was, you knew what the form of a sitcom was, etc. And like you can satirize that kind of thing as we get into an age where people have less and less in common or consume fewer forms in common, it becomes hard to do satire. I agree with that. And I also think social media is, you know, it creates all these little subcultures, it shortens people's attention spans, you know, even even you look at the format of comedy on TikTok, where if you don't make the joke in three seconds, people flip to the next video, you know, people used to have a longer attention span where they could they could track with a sketch that was six or seven minutes long, you know, you look at Monty Python sketches that just go on and on and are absurd and don't have a point. But but go on and you're willing to let the joke develop and you're willing to let the little narrative arc build. And that's great. I also think that reporter was asking me about boomer humor. I just got a little annoyed. Yeah, because she she was like, she was saying a lot of boomers like your stuff.  Yeah, do you want to come out? Okay. I mean, I'm a I'm a boomer and I'm kind of ashamed to be associated with that. But you know, you talk about social media and fragmentation from a kind of religious point of view or from a Christian point of view, isn't it kind of good, though, that you live or we live in a world where the barriers to entry are pretty low and that a lot of different groups. I mean, this is part of the certainly the Protestant Reformation. And as it came out of England in the 17th century, it was kind of great that anybody who wanted to could start a church and had the freedom to do that. Isn't that somewhat analogous to, you know, the landscape we're in where it's like, if you believe this thing, you know, whether it's religion or media or whatever, you can kind of create that world and try to draw adherence to your community. Well, I think it's a double-edged source. I'm not completely down in social media. Obviously, the Babylon Bee wouldn't exist without it because we were able to say, Hey, you know, do you like the onion? But hate when they are really bad at humor because they're too, you know, absorbed in the leftist worldview or whatever. Here's something that here's comedy that doesn't hate you. You know, that was kind of the pitch. Yeah, we'll make fun of you too.  You know, that was kind of the way that it got popular. So obviously, we were able to find a subculture using that that we wouldn't have been able to launch the Babylon Bee on, you know, network TV or something, getting some deal there. Yeah, or just through traditional print. Our traditional print would be hard to find that audience too. Yeah. Let's talk a little bit about social media because you've been in a kind of fight or a standoff now with Twitter. You still are on Facebook and you have a massive presence there, right? Well, it's very suppressed by Facebook. Okay. What does that mean? We've been demonetized and de-platformed several times by Facebook and they... When you say demonetized, that means... Well, like, we'll get either videos demonetized. We've had, when we get those fact checks that come up, we get dings on our account where we can't monetize until we take certain steps. And we also have been told that our site is rated low for sharing false information. So now, whenever you see a Babylon Bee dot com, even if you share it from your personal page, Facebook flags, that is like, this is that information we can't spread. Although you have a large presence on Facebook. We have a large following, but it's just our followers don't see our content. Yeah. Okay. But you are on Facebook. You're active on Facebook. On Twitter, you have been... You were banned when you named Rachel Levine, who's an assistant secretary of health, who's transgender. She transitioned from a man to a woman, I think, in 2010 or 11. And you named her Man of the Year. And that triggered a Twitter response. Can you walk us through what happened? Yeah. So we gave Rachel Levine our prestigious Man of the Year award, and we were sent a message a few days later that you're locked out of your account, you know, you have to recant. You have committed hateful conduct, and you are calling for violence against somebody and acknowledge our hateful conduct policies and click this button to delete the tweet and acknowledge that you've committed hateful conduct. And is that the actual language? Yeah, I'm paraphrasing. I could find the original if you need. And did they say that this was committing violence or leading to violence or something? If you click on it, please review our hateful conduct policy, you can click on it, and it says calling for violence against people calling for... So it's part of the list, but they're not necessarily saying that tweet that you were calling for. It wasn't typed custom for us, but it was part of that language, yeah. OK, so they said, you know, you got to get rid of this tweet, and then we'll unlock the account. Right. OK, and you guys have chosen not to. Right. OK, can you walk us through the logic of that? There wasn't a ton of discussion. It was very much we got together and said, we're not deleting this, right? And it felt, it just felt there's a humiliation like bow before Twitter and apologize and acknowledge. You know, you're calling someone who is a biological male no matter what Rachel Levine identifies as. And really, we weren't even making fun of Rachel Levine so much. We were making fun of the fact that you had said today named Rachel Levine one of their women of the year. And so this had kind of blown up on social media, you know, that transgender people have started to take awards away from biological women and starting to erase that category in our society. And we said, well, you know, we're not going to delete this tweet. And we just kind of announced that on all the channels that we were still able to access, you know, on our personal Twitter profiles. And you have your personal Twitter account is still up and running. Mine got deleted shortly after that. I got locked out as well. And then I was able to get back on though. How did you get back on? I appealed it and I was I had made a joke about something how like if the Babylon B had only committed genocide against Uyghurs, then we would be allowed on Twitter. Right. And they flagged that for violence. Yeah. Did you retweet the headline about Rachel Levine? I don't think I did. Personal account, because would that have bounced you or whatever? I think pretty quickly we were like, OK, let's not share it on our personal pages, because if we do that, all of a sudden we're going to have no ability to access anything. But we'll leave the main account locked. So you're talking about the Rachel Levine joke. It's I mean, you're kind of this is there was a South Park episode shortly after Caitlyn Jenner transitioned. And the punchline of that was that everybody, no matter what, at every moment in the show, would stop and talk about how Caitlyn Jenner was an incredibly brave, you know, an courageous person. You're saying that that's the joke, really, that you're making with Rachel Levine, that it's kind of it's the larger cultural response that you're parodying or that you're making comment. Yeah, it's the it's the absolute, you know, obviously we we love trans people and we love, you know, we don't we don't consider people like that beneath us. You know, the Christian world do is that everybody everybody has the opportunity to be saved, you know, and we can love everybody. And I'm no more deserving of God's grace than a transgender person. But when the culture bows down and starts handing out trophies to people for stuff like this, is when we say, hey, wait a minute, we need to protect women in our society as well. And do you think do you think trans people have are they mentally ill that they want to transition? I mean, probably nowadays, there's a lot of social a lot of social pressure as well as we've seen among a lot of trans transgender teens, especially women. So, you know, one of the things that's interesting is the your response, the Babylon Bees response to Twitter, like you're saying, OK, we're not we're not going back on Twitter. And you seem to be holding pretty firm to that. That's one thing. And do you do you agree that Twitter, you know, has a right to kind of throw people off if they want? Like you might disagree with that, but I'm I'm of two minds about it because I see both sides of the argument. You know, on the one hand, yes, they are a private company. You know, and I don't know that I want to see a world where the government is sending regulators into bureaucrats into Twitter. And what are you guys doing and give us reports on censorship?  On the other hand, if they are acting as de facto sensors for the federal government, you know, how are they doing that? Well, because they're talking with the government, you know, we know that they're communicating with the White House about who they want censored and and and, you know, and all that. So when we had it, we had a person who was an ex CIA agent come after the Babylon Bee for sharing misinformation, you know, talking about how she had been monitoring the Babylon Bee for sharing misinformation. And so then she's talking about how we just use satire as a shield to prevent ourselves from getting blocked up. But she's an ex CIA agent. So what does that mean? Like what's her connection to the government? The thinking is that that had been a pattern of thought within that agency, you know, is that people like the Babylon Bee are sharing misinformation. At what point? I mean, because people used to this is kind of faded, but it'll come back.  But I mean, because you would say nobody wants the government to come to the Babylon Bee or to reason and say, you have to carry this speech. Right. But at what point then what what makes Twitter different that you might be OK with saying, OK, you know what, this should be treated like a public utility. It's like the old phone system where anybody can make a call about anything they want and you can't block. Yeah. Well, and I don't I don't know that I entirely agree with that argument either that that, you know, the publisher versus platform distinction. I think that whole thing is a little misunderstood from a legal perspective. Yeah, well, it's invented. It's made or it's not anywhere in the like it's like it's like it's like it isn't the protection for social media that people think it is. Right. I think so. I don't know that I entirely agree with it either. So I don't know what the answer. But my but my my feeling is culturally, no matter what you think legally, culturally, we're in a really bad place when the place where 90% of speech occurs is online and it's all controlled by the left and big tech. Yeah. What's the answer? I don't know. I think we do need to build alternative platforms. I think we do need to build our own stuff. But as soon as we do that, they kick us off Amazon server, you know. So well, let's talk about that, because I think one of the most interesting things from a from a libertarian or from a market perspective is your response to the Twitter stuff is not simply to say, OK, well, Twitter's closed off to us and we'll just drop it. You have really robustly built out your online infrastructure. You have like more subscription plans and you're pumping out more material in a place in an environment that you control, including creating a social kind of social media network. Right. And that's part of not to be social. Can you talk a little bit about that because this is, you know, it's kind of like you got kicked out of one city in a valley and you're building a kind of shining city on a hill for yourselves. Right. It's true. Yeah. Yeah. We built a little social network on the back of our not to be website, which is also kind of connected. And it's a back end for both sites. You know, so that's what we're building out is that you log into Babylon B and now you can share Babylon B articles with other Babylon B subscribers.  Right. I used to be that we didn't all go to Facebook to talk about things. You know, if you were a hotty car enthusiast, you went to the car forum. You know, it was like use that in that very certain to see that fragmentation as people are doing all this stuff. So I do think it's exciting to see that because of the pressure that we're facing on social media, we just said, OK, well, we're going to move over here. Right. But at some point that breaks down because, like, you know, we could get cut off by payment processors or banks or has that has that happened?  I mean, this is I agree with you broadly, you know, it's disturbing.   That's totalitarian or it's totalist. It's very disturbing for sure. Yeah, we've had stuff like MailChimp suspending for a while.   Like, I got to go get subscribed. Are you like what about, I mean, but then we see a proliferation of places like Substack that are at least for the time being explicitly committed to not being MailChimp in terms of, like, when we don't, as long as you are not breaking the law or, you know, we don't really care what you're writing about. Yeah. I mean, that's good, right? I think that's a great thing. And I think that's a great adaptation that the market has made. I don't know if it's going to completely solve the problem. Yeah. But I also don't have government involved. So I don't know. I just write jokes on the internet. Yeah. Can I ask a kind of final broader question?  You're a stranger in a strange land, right? Because, you know, what what maybe 20 percent of America is evangelical or no, smaller than that. Right. Yeah. But is there a real tension? Because, like, you know, you believe you come from a belief system that believes in revealed truth that is absolute, that is unchanging. And that calls upon you to act in a particular way. And it's like, you're going to render unto Caesar what is Caesar's. But there comes a point where it's like, no, you're not going to participate. How does that feel?  Well, the Christian is is prepared for this because, you know, Jesus already told us like the world hates him. So imagine how they're going to treat you. You know, but he also says, let's face it, you're not Jesus. And it's true. But he says, take heart, because I have already overcome the world, you know. And I think I think there's a there's a there's a little bit of being based to being a Christian nowadays, and I'm OK with that. Like, I'm OK to not be like everybody else. Right. And so I don't think I don't think there's a fear or worry. I think there's almost an excitement. The Christian church is always flourished under persecution. Right. You know, the Christian church is going much faster in China than it's growing here, despite it being banned in China. And, you know, I was thinking of that because we're mere steps away from Caesar's Palace, the Casino in Vegas and they have the Colosseum. And I was I was talking with my son about I was talking about the early Christians. You know, it's but do you feel like American culture in your lifetime? You know, there was a period in in the 70s and in the 80s, especially, where Christianity, the religious right, evangelical Christianity, seemed to be a growing and almost dominant cultural force. It would rarely admit to that. But it really was, you know, it moved politics in a big way. It it created a lot of cultural phenomena on that where became broadly popular. Are those days over? Or do you do you think, you know, is Christianity evangelical Christianity shrinking as a source of cultural power in America? Well, I hope so, because we made a lot of bad Christian art in those days. Yeah, that's true. And I think the you know, I think there's a bit of a culling, you know, you're having a lot of people, especially during the pandemic that stopped going to church. But, you know, we kind of wonder how committed were they anyway? You know, and I don't, you know, if we're attracting big crowds, but these people's lives aren't being changed and they're just there for cultural power, then we don't really we don't really mind, you know, that now we're kind of seeing who the true believer. Now you're sounding like the certain aspects of the libertarian movement. You know, it's like we don't need big numbers. We just need hard people, right? That are we need true believers. Is there a way, you know, one one story that we tell about America? And I say this to someone who's raised Catholic, you know, this was a story I was told is that America ultimately was a place of religious toleration and that that spread out into different, you know, into a secular form of toleration, where the genius of the country really is that we have enormous numbers of people who come from very different backgrounds and believe very different things, some religious, some secular, whatever, and that kind of the genius of the country is that we can create a, you know, a governance system, not government, but just like a kind of operating system where as many of us can live peaceably together without killing each other.  I do the same. Is that, you know, is that a version of America that is attractive to you? And then if it is, are we failing to do that? Yeah, I mean, I think that's great. I love I love personal liberty and personal freedom. And I think a lot of those values were thought up in the West, you know, because of Christianity. Absolutely. I mean, freedom of conscience, the right to believe, you know, what you believe and act on that is like central to, you know, 17th century British to the Reformation, ultimately to America.  But but I do think there needs to be a kind of a common appreciation of some traditional values. And I wonder how we how we manage that when the country is so far divided and polarized right now, you know, when we can't agree on what a man is. Can we have that kind of tolerance and freedom? What is a man? I don't know.  You know, that's OK. That doesn't get us very far, though, right? But what would what, you know, looking forward to the next couple of years, you know, certainly in the political arena, nothing, nothing's going to get better, right? Like we are the only thing we know is we're falling. And we're, you know, we're we're not near the bottom, probably where we're going to hit. What are the types of things that might might allow for, you know, a kind of settling of saying, OK, here are the three, four or five basic things that we all agree on as Americans.  Yeah, I mean, and the new tribes may be the way to go. You know, I don't know. I increasingly don't see a way for America to reconcile beyond, you know, secession. Yeah, which is not necessarily, it doesn't have a great track record. It doesn't have a great track record. You know, the onion wrote a joke about peace in the Middle East, where they said that that a new peace treaty was going to be three hundred and eighty million little, you know, as each person. Right, right. Yeah.  And I don't know. I mean, I don't know practically how we see that in our lifetime. But I do feel like G.K. Chesterton had a quote that another Catholic. I know. You're going soft, man. The Catholics have the best quotes. Protestants don't do as well. But he had a quote about how all politics should be as local as possible. And he said that that the politician who keep your politicians close enough where you can kick them. Yeah. And he says a shame. How many politicians, how few politicians are hanged? Yeah, that could get you banned from Twitter. I think that's up. Yeah. Yeah, I tweet that occasionally and I hasn't gotten me banned on Twitter yet. But but it is, you know, I do believe that what we've seen through the pandemic is local elections, local politics, keeping things as local as we can is one of the solutions. OK, well, we're going to leave it there. Thank you, Kyle. Man of the Babylon Bay. It was great talking. Awesome. Thank you so much.


My name is Yaman Shira. The purpose of this project is to utilize the catcher residues to produce biofactorizer. What we have here is a converter and we have a catcher residues which is rice husk and we have firewood which will be fed or loaded on our base. So now our first stage will be to pack those firewood into our base. Then after packing them we light up and we wait until the ignition is well distributed in our firewood. Then we cover our base with rice husk along and as you see rice husk along this is 100 kg as you can see. After this we are waiting for no less than 4 hours for our char to be ready. Yes this is our char manufactured from rice husk and through using our converter it takes 5 to 6 hours for the process to be complete.


Given that the speed of light in a vacuum will equal the light's frequency times its wavelength, all we need to do is to use a laser to create monochromatic light and measure its frequency and wavelength. To fully understand how this is done, we need to take a little deeper look at the laser that creates the light. The name laser is an acronym for light amplification by stimulated emission of radiation. The key is stimulated emission. You may recall from the How Small Is It segment on the atom that electrons exist in discrete energy levels around the nucleus. When a photon with an energy E hits an electron in a shell around a nucleus that has a higher shell it can reach with this same exact energy, the photon's entire energy is transferred to the electron instantaneously. This jumps the electron to the higher energy level and the photon is eliminated. When the electron drops from this excited state back to a lower energy level, a photon with the exact difference between the energy levels is emitted in a random direction. In 1916, Einstein theorized a photon with its electromagnetic field and the right energy could stimulate an electron to drop to a lower energy level and emit a photon with the exact same energy, trajectory, polarization, and phase as the incident photon. Where there was one photon, there are now two. They will travel off in random directions. Probabilities have it that every once in a while a trajectory will be parallel to the tube they are in. Lasers have one mirror that reflects 100% of the light that reaches it and the other reflects 99% and lets 1% through. So these photons will travel to a mirror and be reflected. As they pass back through the tube they will stimulate the production of additional duplicate photons as we just described for as long as the pump keeps energizing electrons to higher energy levels. In short order, there will be trillions of identical photons leaving through the 99% mirror. This is the laser light. By the 1970s, the development of methane stabilized helium neon lasers with high energy spectral stability and accurate cesium clocks made measurements to within an error of plus or minus 1 meter per second possible. To measure the frequency, a technique called infrared frequency synthesis was used. It is the same idea we see with sound wave beats. We start with a known frequency, mix it with a higher unknown frequency, and count the beats created by the combination. Here we see a 400 cycle per second tone mixed with a higher frequency that produces a 5 cycles per second beat. This gives us the frequency of the second wave at 405 cycles per second. Using an iterative process, this can measure still higher sound frequencies. For light frequencies we use an oscillator with a known frequency and mix it with a higher frequency using a mixer diode to count the beats. We iteratively get to the frequency of the laser light's output. They found that the laser produces light with a frequency of 88.376181627 trillion cycles per second. To measure the wavelength, we use an interferometer. First we set up equal path lengths for the light by getting the maximum constructive interference. Then, as we shorten one of the paths slowly to get, say, 10,000 friend shifts, the wavelength of the light is the distance that we had to use to get this many shifts divided by twice the number of fringe shifts. The results showed that the wavelength is 3.392231376 micrometers. This measured wavelength, along with the measured frequency, gives us the speed of light at 299,792,456 meters per second. As of 1983, the speed of light in a vacuum is defined to have an exact fixed value when given in standard units. In fact, the meter has been defined by international agreement as the distance traveled by light in a vacuum during a time interval of 1 over 299,792,458 seconds. This makes the speed of light fixed. In the next segment on special relativity, we'll cover why this value also represents the fastest that anything can travel.


Hello good morning everyone. We're gonna go ahead and get started with today's grand rounds. Today we have a guest speaker and what I'm gonna do is slightly a little different from what was expected. I'll introduce Brian Jones who will then introduce Dr. McCall. Thanks. Not sure how this usually works but Dr. McCall is actually gonna give two lectures today as usual for a basic science person so I'd encourage you also to attend the noon lecture where you'll get a different story. So it's my distinct pleasure actually to introduce my friend Maureen Dr. McCall and we've known each other for a while. Maureen Dr. McCall got her bachelor's in psychology at the University of Maryland and then did something very cool. Got a master's degree in psychophysics and psychophysics is not taught really much anymore which is a tragedy especially given all of the clinical therapeutics that are going in into play. It's actually one of the great shortcomings. I'll leave that editorial there. And then she got her PhD in neurobiology at Albany in New York, did a postdoc in Wisconsin and then started shortly thereafter a little while after at University of Louisville where she's been for a number of years now. So Dr. McCall thank you. So thank you for inviting me and in particular because it's snowing in Louisville and the university's closed and also thank you for having grand rounds at 8 a.m. instead of at 7 30 the way we do in Louisville. And lastly you know I talk fast and I get going and so sometimes I don't see a hand raised so please feel free to say hey slow down and explain something to me as I go through my seminar this morning. So my lab has two arms to it and this morning I'm going to talk to you about the work that we do that's more translational and related to retinal photoreceptor degeneration. And so just to give you an idea of where we're going to go with where I'll go with this lecture is I'm going to briefly touch on the retinal circuit and then the changes that occur in retinitis pigmentosa and so most of you probably are more of an expert in this than I am. And then I'll talk a little bit about the genetic nature of retinitis pigmentosa because all of this leads into why why this is a disease that can be addressed in terms of potential therapies with gene therapy which is where I will end up. And then in between I'm going to tell you about the creation and characterization of a pig model of RP because the pig is a very good model for the human disease. And then finally I'll tell you a little bit about some of the data that's coming out of our gene therapy approaches in this model. So this is just a schematic of a retinal circuit and from an electrophysiologist point of view the photoreceptors are on the top and I know that anatomical views usually put the photoreceptors down here and so I'm just going to tell you that most of the time in my slides you'll see photoreceptors on the top and the ganglion cells on the bottom but occasionally when we are collaborating with our our anatomists the slide will be flipped upside down and I'll try to remind you that that's the case. Okay so we all know that the way this works is light comes in through the transparent retina and then that light energy is transduced into a signal within the photoreceptors and then that results in a change in glutamate release from the photoreceptors which then leads to a vertical information flow through the bipolar cells to the ganglion cells and out from the ganglion cells to the rest of the of the visual system. So in in retinitis pigmentosa because of mutations that occur in proteins in the rod photoreceptors you get the first part of the first step in the sequence of neurodegeneration is that the rod photoreceptors die and they die over a certain length of time and then it because of reasons that we don't understand but are active areas of investigation the cones can't be sustained in their normal morphological and functional normally and normal morphology and function and what we end up with at end stage disease are cone photoreceptor somas that don't have their outer segments or their pentacles so these structures have have retracted but even late in in in disease in both the in both the pig model that i'll talk about in the rodent models that we know about in humans there are these cone photoreceptor nuclei that remain and so this is exciting from the point of view that if those cells are still there there's the potential that they can be rescued and remake their connections but in this in this stage when the cones now have retracted and no longer make connections with the second order neurons we get blindness and and so in in the end stage disease in humans they are legally blind and don't have any any real light perception at all so this occurs in a lot of patients and in as an autosomal dominant disease 20 to 30 percent of the of the patients that you see will have an autosomal dominant mutation that means that they only need one mutant copy and that and then they get the disease manifestation and out of those 20 to 30 percent eight and a half percent of these autosomal dominant rp arise from mutations in the rhodopsin gene which is shown here and the rhodopsin gene is this transmembrane protein here's the extracellular side and here's the cytoplasmic side and you can and there are mutations in a bunch of different places the one that i'm going to focus on today is a proline to histidine mutation here at position 23 but there's another there's several other models there's a there's a model out here that has a mutation at position 347 and then there are a bunch of others in between so what happens is that because this protein is mutated it doesn't traffic properly and to the outer segments of the of the photoreceptors and it becomes toxic and the photoreceptors die as a consequence of this so it's a little bit like the neurofibrillary tangles that you hear about in Alzheimer's and they gum up the works and because of that the cell goes into into stress and the cell dies in rp most of the it's a progressive disease and as you all know we're our visual system is really good at fooling us you don't see patients in a lot of cases until they're very well progressed into either rp or glaucoma any of these things because our visual system fills in and and is redundant in some respects and so these patients really don't come in until they experience night blindness is usually the first complaint when they're seen in the clinic so once they get patients have come in they've lost a lot of their rod photoreceptors already and so we don't know what the early sequences are in these patients it might become more obvious now that we have with that we're better at diagnosing because these are autosomal dominant and so that we can do genetic testing and then we could track children as they start to progress into the disease but that hasn't become one of the things that's that's that's currently done a lot and so what we've done is to turn to animal models to try to study the early steps in in the sequence and and then and in these models to try to intervene early mid and late in the disease to see what what our therapies can do so the classes of animal models there there are two one are spontaneous mutants and these are animals that are found usually by mistake we found a mouse model for congenital stationary night blindness because one of our collaborators was using mice from a particular colony and the control animals didn't have an ergb wave and so all of a sudden that opened up a whole set of ways of looking at congenital stationary night blindness other mouse models are that are common for rp are rd1 and rd10 and again these have mutations that are part of not the rhodopsin gene itself but part of the photo transduction cascade there's a rat model with a the royal college of surgeons rats that that's a mutation in the rpe is important because it fagocitizes the outer segments and if you don't fagocitize the outer segments then that becomes toxic to the photoreceptors themselves and then they die again from a slightly different point of view and then there are several inbred dog models and these arise because because of the inbreeding in in particular strains of of dogs and so then the puppies come up blind and then those puppies are usually donated to a facility at the University of Pennsylvania where they study them and then more recently we've been able to molecularly manipulate the genome of mice and a little bit in the rat and then a little bit in the bunny and most recently in the pig and so what we can do is we can create either one of two kinds of molecularly manipulated animals we can create transgenic animals and they mimic the human defect because they they can carry actually you can actually make them carry the human a mutant gene but they don't mimic the gene complement so in a in a retinitis pigmentosa patient as I said if it's autosomal dominant you'll have one copy that's a mutant copy and one copy that's a wild type copy in the transgenic animals you get two normal copies and then the trans gene that's that's inserted into the genome as an extra protein and and examples of this are the p23h redoxin mutant mouse that's not spelled right rat and then the pig that i'll tell you about today there's another mutation in redoxin at the at 334 this is a searing to a to a termination stop codon and this is a rat model and then the original pig model that was that was made in in john and uh rom petters lab was a pro lean to lucine at at at redoxin 347 this was a full-size pig the pigs i'll tell you about today are mini pigs so now more recently we've been able to knock in the mutant gene so you replace the one copy of the wild type gene with the mutant gene and part of this uh the ability to do this is because of the work of mario kopecki who i you probably all know as well as oliver smithy's who who died just recently and in here this now you can get the human defect because you can knock in the human uh the human mutant gene and and you knock out and and and they knock out one copy of the of a wild type gene at the same time so now you have the human defect that that is mimicked and the gene complement that's mimicked we have not been able to make a knock-in pig yet but but the but there's a recent recently developed p23h knock-in mouse that's been very useful made by uh chris paulchevsky as well as by ted ted wensel so these are the animal models that we have available to us and like and as i said i'm going to tell you a lot today about this p23h rhodoxin mutant pig that we developed and we did this in collaboration with the national swine research resource center uh that's located at the university of missouri and they are the they are the cutting edge of of making transgenic and knock-in uh pig models and so they have been successful in making knock-in pig models but not for rp yet this is one of the goals that we are continuing to work with them on and randy prather is the is the lead at the national swine research center and we work with both eric walters and and and jason ross to create founders of the p23h mutant pigs so this is a mini pig uh and the reason that the pig is such a good model is that its eye size is similar its developmental program is similar to humans it's vision it has a visual streak so here's the fundus image of one of our our transgenic pigs here's the optic nerve head the blood vessel pattern is somewhat similar to to humans we have a a blood vessel free uh what what is called the visual streak it's not a phobia it's not macula but it's the next best thing given that the only other organisms that have phobia and macula are non-human primates some non-human primates um and and human primates so uh we they used somatic uh cell nuclear transfer to do this and then what we did was to create and characterize six different founder animals um and the important thing is that they carry this human p23h redoxin mutation and so any therapy that we find that is efficacious and safe uh can then be easily translated to the clinic because it's the same mutation that you'll be treating in in the in the human patients so a lot of what we do um in terms of the electrophysiological assessment relies on the erg and i know all of you are familiar with the erg here's one of our uh more uh mature uh pigs with a jet electrode on his um on his cornea this is the lkc gansfeld and we we literally shoved his place gently his head into the the the gansfeld and what you know is that the erg is a gross potential and so it it it reflects all of the processing of the retina both peripheral and central and that's going to be important uh in terms of some of the uh in some of the challenges that we face in gene therapy so the a wave as you are all probably familiar is the is the the change in the polarization of the photoreceptors after a flash of light it's a doubt and it's just a and it's followed by the b wave which is the post receptoral component primarily from the depolarizing bipolar cells and if you see these little tiny wiggles on here those are called oscillatory potentials and they reflect a lot of the circuitry here in the inner in the inter plexiform layer so we can uh assess photoreceptor function and bipolar cell function and you can and as i said if you have congenital stationary night blindness you'll see a normal uh amplitude a wave but no b wave in rp what happens is that the a wave decreases and the b wave also decreases because it's dependent on the polarization of the of the photoreceptors and we can change the lighting condition so we can dark adapt the animal and and use very dim flashes to assess rod photoreceptor function or we can put an adapting background which saturates the rod response and then and present a bright light light flashes and assess cone function so we can get both rod and cone functions separately uh in using the erg then the other thing of course you know is that the erg is a non-invasive gross potential so we can use this on a weekly or monthly basis to track what's happening in terms of the degeneration process or in terms of functional recovery after therapy so i'm going to take you through the characterization of the of the six founder pigs that we that we did uh to figure out who to use to for for the rest of our experiments and what we found electrophysiologically is that if we looked at the scotopic b wave and we started at three months we found two different classes of founders we found the ones that were severely affected and had no rod function at from three months all the way through 18 to 24 months there were some that were moderately affected so they had rod function at three months and then that declined um and then in terms of their cone function uh the the severely affected had some change in the in the cone b wave uh at even at three months and then that is pretty solidly maintained all the way out to 18 to 24 months so this is very similar in in terms of the the human function except that the humans have rod function so they're more similar to the moderately affected but when we were trying to figure out which animal to use as our founder for the rest of our experiments we had one other factor that we needed to take into account and that is the size of the animal so these are mini pigs but mini pigs are still pigs and so once you get out to about uh you know 22 months these are substantial animals they weigh in at about four 500 pounds and so we decided that from the point of view of animal husbandry uh that that a severely affected animal should give us a phenotype that we could work with in a time frame where the pigs only got to be about 150 or 200 pounds at termination and so a lot of this electrophysiological work was done by Juan Fernandez de Castro uh who was a postdoc in the lab and is now one into private practice in in Florida so here are some beautiful histological preparations from Brian Jones who helped us with looking at these founders this is an adult domestic pig so here are the photoreceptors out here in the outer nuclear layer and the and their outer segments and the rest of the lamination pattern is beautiful and normal in this adult in the two moderately affected animals at at 18 and 22 months you can see that there's still some of photoreceptors that are around these are primarily cones and and here's our one of our severely affected animals at 12 months and you can see that all they have left are these cone photoreceptor nuclei and then the inner nuclear layer and then the ganglion cell layer so uh but this is so when when we first started with these guys they wouldn't let us work with them before they were before three months of age and so we didn't know whether they had any rod function at birth and so that was something that we needed to do in terms of once we got the f1 or the offspring from whichever founder we we selected so we selected this one called 53 one and as i said we knew that he had no uh no no rod function at three months of age but we knew that his cone function was similar to wild type and we knew that there was a slow and progressive decline of cone function so because of these limitations in terms of husbandry we selected him as the founder and so then we wanted to know uh for in terms of what the model could do for us whether the this this founder transmitted the trans gene in Mendelian fashion so as an autosomal dominant mutation whether the f1s have the same phenotype as the founder and then we wanted to be able to use these guys so that we could look and see whether rods were present at birth and whether they functioned and then declined and then we wanted to know whether the cones developed normally during those first three months before that they declined and so i'll go through those data now so here are the morphological data first and so here are the wild type animals on the left and the p23h transgenic pigs on the on the right this is at e105 so you can see that they have a full complement of photoreceptors right prior to birth the gestation time in a pig is about 113 days and if you count the number of photoreceptor nuclei you can see that they're identical at p0 there is a small change in the in the outer nuclear layer that's shown here in the summary and here's p0 and p3 there's a there's a further i'm sorry this is p3 there's a further decline and then as you go past between p14 and p16-60 there's a real decline in rod photoreceptors that at p90 and all the way out at p120 you see that there's a single layer of nuclei and and they have the morphological appearance of of cone nuclei and and they and we have i'll show you we have cone function out at p120 and then these data were generated by Wayne Wong and Patrick Scott in our department there's a central to peripheral decline in rod photoreceptors that's shown here summarized on this slide in these butterfly plots so we go from p0 to p3 all the way to p120 and this this image of that's a schematic of the of the fundus shows where the most effect is which is here in temporal ventral retina so if you take a look at what's plotted here is that there's more of a decline in temporal retina early here and here temporal retina is lower than the nasal retina and then the ventral retina is more affected than the dorsal retina until you get out to about p120 which is why this dark shading is here in this area in terms of the in terms of the ultra structure these are much higher power images than i've been showing you before and you can see that at p0 the cones have very nice structure they have inner segments and outer segments and there are even some rod photoreceptors that have inner and outer segments sprinkled in here at even higher power at p0 you can see ribbon synapses in both the wild type and the p23h and you can see that the cone pedicles are normal in both of these animals at p3 and as we go through from p14 to p60 the cone outer segments retract and so do their pedicles so at greater than p90 the cone pedicles have retracted as well and even though they are they've retracted we still have some signaling through the cones in the retina and i'll show you that right now so over here are representative traces of ERG responses at p3 in gray is the wild type and in black are the transgenic offspring so we have these animals born at the University of Louisville and so we can begin to to assess them as early as p3 we find that before p3 it's a little difficult to anesthetize them and then bring them back but they're pretty hardy by p3 and at p60 this is so at the top here is the rod response so as i showed you in the in the founder animal there's there's no rod function here but unfortunately we have no rod function even though we have full complement of rods nuclei at this point but but at p3 and all the way close to p60 the cone function is is pretty good and pretty robust and the and the the summary data is shown here on the right so this is the scotopic or the rod driven responses we don't see any in the in the offspring of of 53 one just like in 53 one which is plotted here in the triangles whereas the wild type pig has a has a normal development of rod function and then that plateaus after about p60 in terms of the photopic response p3 and p14 and p30 are almost exactly the same between the wild type and the transgenic so this is nice because the cone system seems to develop normally and in many of the very aggressive forms in the rodents the the the degeneration of rods and cones occurs at the same time as the normal development so you have these issues of are you looking at a change in developmental sequence or are you looking at a change in photoreceptor degeneration and so we in terms of our cones we can say that the cones develop normally and a lot of this erg data was done by a graduate student in my lab who's finished and heard whose name is jennifer noel so to summarize what we know about the offspring is that they have no evidence of rod function from birth onward but their cone function is normal and then it declines so in terms of cone function they're very similar to what happens in the human patients in terms of rod function they are they are not as good a model so and the other thing is that they all what i didn't tell you is that they inherit in the Mendelian fashion so about 50 percent of the animals that are born in every one litter are transgenic or animal wild type and so they all have the same phenotype as the founder so they're in in terms of the genetics they're a good model for for rp so this slide shows what happens in terms of the redoxin expression and now the transgenic is over here on on the left and the wild type is here on the right and this is a anisostein to remind me to tell you that this is from our one of our anatomical studies and so it's upside down so the photoreceptor inner and outer segments are shown here with and and then in the transgenic the redoxin is actually expressed all the way through the cell bodies and into their terminals and so this misolocalization is probably part of the reason why these rod photoreceptors are dying in terms of the expression pattern the wild type now is shown up here between p 30 and p 120 and we can see redoxin expression in the transgenic at p 30 and just a smathering at p 60 so even out to p 60 there's some redoxin that's still being expressed and so the hope is that if we can do something in terms of eliminating the mutant protein that we might be able to rescue some function and i'll show you some data about that later in the talk okay so let me stop here for a moment and summarize so what we see in the in this p 23 h transgenic pig model is what is the way we interpret this as three stages of retinal degeneration in terms of retinal structure and function the first stage is is the is the is a loss of rod driven function okay that's that's happening here p 0 to p 14 and then we have a mid stage p 30 to p 60 where the the world we've lost the rod function completely and the cone function starts to decline and then we have this idea of end stage disease which is larger than p 90 and so depending on the stage the the the therapy is going to have to be tailored to the stage of disease and i'll talk about those therapies in a second but what i want to do is also tell you one other way that we assess visual function in these animals so as i said we when we use the erg it's got the advantage that we can use it successively over time and we can actually assess weekly if we want to but as i showed you in that earlier slide it gives you the the view of what the entire retina is doing and i also showed you that the cone function has a central to peripheral gradient and so at any one point in time with the erg we are looking at how good the peripheral retina is whereas the central retina has probably lost function but what we'd really like to know is what happens it in particular locations in the retina and so you can use the multifocal erg but i'm not a big fan of it um but what my lab also can do is actually take portions of the retina and place them on a multi electrode array and put them into a dish and then use visual stimulation to activate the retina in vitro and we can record from the retinal ganglion cells and ask questions about visual processing using different light flashes or using different kinds of visual stimuli and we can do this either here in the central retina or we can take a we can dissect a piece of retina out from peripheral retina and and if this is where we place our treatment then we can ask whether treatment has affected this very particular localization in the retina versus in an intra eye control or in the control fellow eye which may not be which we may not have treated and what we end up doing is recording the action potentials of these ganglion cells on these electrodes and the nice part of this approach is that we have 60 electrodes we can usually get about 90 different ganglion cells on our on each recording and so therefore we get a nice survey of what the ganglion cells are doing in a particular area versus in another area and so we did that with these p23h animals and here's the here's a here are schematic not schematic these are what we call post stimulus time peristimulus time histograms and at the top each of these little ticks is an action potential and so then we can look at the average over many trials we usually do 10 to 20 trials and we use different brightnesses of of light stimuli and what you can see is that you get this nice peak firing and then sometimes a maintained firing for the two seconds that the light is on and that sustained component usually increases with the brightness of the light and sometimes we see a decline in the peak but this has to do with light adaptation when we use the same visual stimuli in transgenic animals when when we're in rod isolated conditions we see no response so the rods are not driving the ganglion cells at this low level but you can see that once the the we reach cone function that you can see these beautiful responses among the remaining cones and so here's the summary of this kind of an experiment where we're plotting the light intensity on this on the x-axis versus the percent of visually responsive ganglion cells wild type again is in gray and transgenic is in black so you can see at under rod only conditions we have no ganglion cell ganglion cells being driven in the transgenic animal but as soon as we get up to someplace where the cones can become sensitive we start to pick up on the visual responses and the cones then saturate in a hundred percent of the cells of the cones that are there have have you know have really good responses and this is out at p120 so this plots the percent of visually responsive cells as a function of age and that's stable in the wild type animal but in the transgenic animal you can see that uh that there's a decline in in cone function all the way out to p90 but the cones that remain have very good and robust responses so we we're losing drive to the cones but the cones that have responses are being maintained in a in a in a very stable fashion and this actually also is similar to what happens in in humans until they start to lose cone function all right so this takes us back to that schematic that i showed you earlier about the three stages of transgenic photoreceptor degeneration when we're here the question that we want to address is can we restore rod function and prevent cone degeneration by getting rid of the mutant uh rhodopsin and then in the mid stage the question is can we prevent further cone dysfunction so can we do something to support the cones in some way shape or form so that they they are arrested from the time of treatment onward and then in end stage disease there are a lot of treatments that other people are trying and i'm not going to talk about this but they include prosthetic devices that will drive the circuit optogenetics that will replace the the drive of the photoreceptors and stem cell therapies and and other and other transplantation strategies so what i'd like to do now is just to talk about what what these two what what you might want to do with gene therapy in in these two stages of the disease so as i said what we'd like to know is whether we can restore rod function by manipulating and in our case the p23h rhodopsin gene expression and then here we want to know whether we can just delay cone dysfunction and degeneration and the idea here is to in in our hands is to provide some kind of trophic support or some neuro protection strategy so just for everybody in the audience who might not know about gene therapy this is a brief summary of how we deliver the virus and how the the viral delivery of the genes and and the protein expression so we use adeno associated virus because it's usually well tolerated and we use these and and we we pull pull out the guts of the of the viral genome and replace it with whatever genes we want to use in terms of our in terms of our functional strategy so we can manipulate the AAV vector coat and this is what some of my collaborators do and and in manipulating that coat to the protein then it will recognize certain receptors on the surface of some of some cells but not on others so this helps to create a selectivity of targeting of the virus to particular cells and in our case what we want is is to either target broad photoreceptors or cone photoreceptors so the coat may change depending on which of the cell classes we want to target and then the delivery method so there are two ways of doing this you can use a sub retinal injection and that's good if you want to target photoreceptors because now they're very proximal to the injection volume or there's the idea of using intravitrile which is from us from a therapy approach in humans is probably more palatable because it's more easily tolerated you don't create a retinal detachment that has to settle down and then the promoter is also used to enhance the selectivity of of the of the gene is being delivered so for example we want to target the broad photoreceptors the redoxin promoter is a really good thing to do that and the down here is summarizing either sub retinal or intravitrile injections so the red is a really great place to try approaches gene therapy approaches and part of it is because of the transparency of the ocular media this allows this allows us to see really well when we're doing both intravitrile and sub retinal surgeries like a lot of you do in in patients the limited access of the sub retinal space and even the eye eliminates systemic side effects so inflammatory in general systemic inflammatory responses there's a certain amount of immune privilege that occurs in the intraocular environment so that's also helps with any inflammatory responses that might occur in some gene therapies you know for example those that are targeted at liver because it's a bilateral disease we always have an untreated or a control treated eye as the as the control for the treated eye and so that's a nice intra-animal control and then we can track changes in noninvasively with oct so here's another fundus image off of our oct equipment and here is a one of the one of the one of the animals oct where the photoreceptors now here or at the bottom and the ganglion cell layer is here at the top so we use oct as well as the erg is a noninvasive way of tracking what's happening in terms of degeneration and also therapeutic all right so what I'd like to do now is to talk about how we're tailoring gene therapy to the mutation so the what many of you may have read about is is the is the gene therapy for the null mutation in labor's congenital amaurosis and this is probably the poster child for gene ocular gene therapy and in in labors that you that you lack a particular the expression of a gene so what you have is a null mutation the mutation creates no protein and so it's a relatively simple disease to treat can you replace the absence of this mutant protein with a normal protein and in doing that can you rescue function and there's a and I think the jury is still out on whether that is working perfectly in these in these patients with labors there's there are some reports of a very good application and of the of gene therapy and there are some reports where the the therapy is not doing as as good a job and that so as I said the jury is still out on that but the idea is can the the idea initially that we had was can we use gene augmentation to try to ameliorate this dominant mutation so the idea is can you express more wild type rhodopsin and therefore reduce the ratio of the wild type reduce the mutant to wild type ratio and if you do that can that have a therapeutic effect we started to do that with Al Luen and Bill Houseworth at the University of Florida but the first thing that we needed to do was a control experiment to ask whether the the viral vector that we were using was going to give a specific infection of the photoreceptors the rod photoreceptors themselves so what we used was a green fluorescent protein marker that was driven by the rhodopsin promoter and here's an image a fluorescence image of the fundus that shows the infection area of in this particular animal so the optic nerve head is down here and so you can see that the area that we get of infection that we get is pretty broad it's not the whole eye but it's it's a it's several disc diameters in area and if you look at the sections of the animals so the blue is a dappy stain to show the nuclei of the of the of the various cells in the layers of the retina and so you can see in the transgenic we get some we get very nice infection with with the AAV and if you look there's a little row that where there where there's no fluorescence in both this animal and this animal and that's represents where the cones are so the rods are nice are are nicely affected probably not every rod but many of the rods are infected in this in this application so we then switched vectors not not not the external coat vector but now we used at the same AAV driving radopsin in this gene augmentation and then this is the experimental design so we inject we do these sub-retinal injections at p3 when we know that we have rods around and then we start to do fundus exams erg and oct at four weeks and carry it out as long as possible so this is the the the transgenic uh and wild type data untreated data that I showed you before and then we use controls that include the gfp so sometimes we mix a little gfp in with the in with the therapeutic vector to try to show where the um where the infected area is and as I said we did this work with Al Luan and a postdoc in my lab erin rising and one of our fellows uh niloforpeery to do these experiments so um unfortunately we didn't see rod rot radopsin augmentation didn't work in our hands in in this model so there's no difference between baseline and and the treated and untreated animals and either four weeks uh eight weeks or 12 weeks uh in terms of the rod isolated response so these are these are representative ergs and the shaded area is is the uh is the standard error of the mean of the responses but we did see an impact on the combs so these are the summary data from individual animals and the dashed line is is a untreated transgenic control this animal just got the gfp whereas our transgenic animal got the aav row plus a little gfp and then the wild type is plotted in black so what you can see is that at uh and this is time across here so at most of the ages past two weeks which is what we expect we expect to see that the trans gene will start to be expressed between two and four weeks and so you can see that it's helping to keep the cone function more robust uh in the in closer to the wild type than the untreated treated animal and that also occurred in transgenic animal number three but in this animal which did not receive treatment the the transgenic the the the two eyes were exactly the same and these are the individual cone responses from those animals that we've summarized here and here so red ops and augmentation is limited in its efficacy it is safe but it's but it's limited in its efficacy it doesn't help with rod photo receptor function and it probably delays the the decline of cone function but probably isn't isn't going to be something that we want to pursue in the future so the next strategy that i'd like to tell you about what that's also been done in collaboration with albulin is an idea of knocking down the rhodopsin expression and and reducing it now what we'd really like to do is be able to knock down only the p23h mutant rhodopsin and leave the wild type copies in intact but the shRNA approach isn't that specific and so what we are doing is instead knocking down and then using a replacement so we have an shRNA that's specific for rhodopsin and then a second a second expression cassette that will express wild type rhodopsin at the same time and these experiments were really exciting so shown here are 18 the some the average of of the b-wave response in 18 wild type animals at p3 in 22 transgenic animals at p3 and in this particular animal 145-12 at p3 at the time when this animal received the shRNA plus the row of replacement 12 weeks later the untreated wild type looks again like this this is the same 18 animals the untreated transgenics as i showed you before have no rod function but in this particular animal both and both eyes received the treatment we saw a very significant b-wave so first thing i thought was we had misgenotype this animal and that the first p3 recordings weren't any good and so we re-genotype this animal three times we re-genotype the animal at 12 weeks which is shown here we took the animal out to 16 weeks we saw the same kind of b-wave response and then at termination we also took tissue and re-genotype this animal and this animal was a transgenic animal every time so why don't i have a whole litter to show you this is the most unfortunate litter of animals that we ever had and they and all of its litter mates developed a respiratory infection shortly after they had the treatment and they all died with the exception of this guy so i only have this animal to show you for right now but last week we did a whole litter of 14 with this this shRNA row and so hopefully in four weeks we'll be seeing something that's significant and i'll have more to say this is the OCT from that animal so here's an untreated transgenic here you can see that we're losing a lot of the definition in the outer retina shown down here at the bottom here's your untreated wild type animal which shows all this beautiful definition for what happens in the in the photoreceptors in the in the outer outer part of the retina and here's this 145-12 which shows some thinning of the ONL which is what we'd expect because treatment is at p3 and the gene's probably not ramping up until about four three to four weeks of age so there's going to be some degeneration in the middle but at 12 weeks this animal had a had a significant outer nuclear layer that was not present in any of the untreated animals that we've seen and these and these these experiments have been ongoing with uh buban sahu govinda pangini and genel adenir adeniron uh in who who who were just doing these experiments to replicate last week so we're really excited about this idea of the efficacy of shRNA uh knocked down with replacement and then the safety is shown here on this slide so as as as expected from everything else i've told you there is no uh the the cone function in in all these animals treated and untreated transgenics is normal at p3 and it remains normal uh at p12 so the shRNA isn't with the replacement isn't doing any harm to the to the retinas so that's also one very important aspect of any gene therapy or any therapy trial you have to both show efficacy and safety okay so the last thing that i'll tell you about is a neuro protective strategy so what was found by um joseph the hall and his colleagues uh uh was that there is a factor a secreted factor that the rods produce that appears to keep the cones healthier and happier and it's called rod-derived viability factor and john flannery and lea bern who was a postdoc in john's lab but now is a assistant professor at the university of pittsburgh they had shown in a rodent model that rdcvf could actually delay uh cone cone driven uh cone driven declines and so we tried this particular rdcvf in the in the p23h transgenic animals and as expected uh we don't see a change in rod driven function these are animals out at 12 weeks post injection uh the red is the rdcvf treated i and the black is uh is the bss treated i and in terms of cone function we are seeing perhaps a little bit of a of us of a rescue of cone decline uh in the transgenic versus the bss treated animals but these are experiments that are now still ongoing uh and we're doing a lot of more analyses to convince ourselves of whether this is uh how the prevalence of this so not all animals respond equally and that's going to be the case with with patients as well and so we're so the statistics and the analyses are pretty involved when you start to come down to trying to compare one individual animal to a large number of transgenic animals that are in our database and so if anything what we're seeing again is a is a subtle effect on cone function and as i said our analyses here are a new way one last thing that i want to point out about this is that these we use intravitorial injections and it's been a completely different viral vector for for these treatments so uh i need to before i i close and take questions i need to acknowledge Hank Kaplan uh because it was Hank's idea to create this model and i wouldn't be talking to you today and i wouldn't have this translational component of research in my lab if he had not supported this idea and funded this idea in many ways throughout the last seven to eight years um Doug Emory and Doug Dean are both people that helped with the characterization of the of the animals that i didn't tell you that i told you that i didn't acknowledge previously and then we've had some very good support from both the NIH and several of the of the foundations that are associated with the University of Louisville as well as the foundation for fighting blindness and research to prevent blindness so thanks for your attention and i'm happy to take any questions


Good afternoon. Mr. Brennan. Present. Mr. Rickerman. Here. Mr. McDowell. Yes. Mr. Duvall. Proven. Mr. Vine. Here. Mr. Davis. Here. Mayor Benjamin. Here. Everyone please stand and join me for the Pledge of Allegiance. This is the flag of the United States of America and the Jewish Republic which is dancing. One nation under God, indivisible, with liberty and justice for all. And it's great to see the tabeta blue flying with us today. Thank you, Mayor St. Magic. The Red McDowell, would you mind giving us a word, please? That is for our head, creator and merciful father, for your goodness and mercy, for all that you've done for us, for the opportunity and the privilege of waking up this morning. And even though it's rainy and cloudy, we know that the sunshine of your grace shines upon us. But we know that there are various issues and situations within this city. But we also are very aware that your love and your mercy covers each person within the confines of this city. Bless and keep us. We ask it in your name. Amen. Amen. All right. Are there any amendments to the agenda, Madam Clerk, Madam City Manager? If none, we'll entertain a motion to adopt the agenda. So moved. Is there a second? Second. Any discussion? We'll move the previous question. Clerk, call her all. Mr. Vernon. Yes. Mr. Rickerman. Aye. Mr. McDowell. Yes. Mr. Duvall. Aye. Mr. Vine. Aye. Mr. Davis. Aye. Mayor Benjamin. Aye. Thank you. As the Mayor and Council, we ask that you approve the September 7, 2021 City Council meeting minutes and the September 8, 2021 emergency meeting minutes. Is there a motion? So moved. Is there a second? Second. Any discussion? Seeing none, we'll move the previous question. Clerk, call her all. Mr. Vernon. Yes. Mr. Rickerman. Aye. Mr. McDowell. Yes. Mr. Duvall. Aye. Mr. Vine. Aye. Mr. Davis. Aye. For your presentation, these are always special to them. Mayor Benjamin and Council when we are recognizing one of our long-time employees, Ms. Vicki Shafley, human resources specialist upon her retirement. Ms. Pamela Benjamin, Chief of Staff, will come forward for the presentation. So thank you. Are some of the most important words we can say? And so I want to thank the Mayor, Council, Ms. Wilson, our city manager for giving me the opportunity to say thank you to Vicki Shockley for over 30 years of service. Vicki was hired by Ron Cunningham. I don't know if anybody remembers that name, but he was our HR director back in 1989.