From theCUBE Studios in Palo Alto in Boston, bringing you data-driven insights from theCUBE and ETR. This is Breaking Analysis with Dave Vellante. Organizations have considerable room to improve their performance without making expensive changes to their talent, their structure, or their fundamental business model. You don't need a slew of consultants to tell you what to do. You already know what you need is to immediately ratchet up expectations, energy, urgency, and intensity. You have to fight in mediocrity every step of the way, amp it up, and the results will follow. This is the fundamental premise of a hard-hitting new book written by Frank Slutman, CEO of Snowflake, and published earlier this year. It's called Amp It Up, leading for hypergrowth by raising expectations, increasing urgency, and elevating intensity. Hello and welcome to this week's Wikibon Cube Insights, powered by ETR. At Snowflake Summit last month, I was asked to interview Frank on stage about his new book. I've read it several times, and if you haven't read it, you should. Even if you have read it, in this Breaking Analysis, we'll dig deeper into the book and share some clarifying insights and nuances directly from Slutman himself, from my one-on-one conversation with him. My first question to Slutman was, why do you write this book? Okay, it's kind of a common throwaway question. And how the heck did you find time to do it? It's fairly well-known that a few years ago, Slutman put up a post on LinkedIn with the title Amp It Up. It generated so much buzz and so many requests for Frank's time that he decided that the best way to efficiently scale and share his thoughts on how to create high-performing companies and organizations was to publish a book. Now, he wrote the book during the pandemic and I joked that they must not have Netflix in Montana where he resides. In a pretty funny moment, he said that writing the book was easier than promoting it. Take a listen.  They say, oh, I'm not doing another couple of tests. Now, the book gives a lot of interesting background information on Slutman's career and what he learned at various companies that he led and participated in. Now, I'm not gonna go into most of that today, which is why you should read the book yourself, but Slutman, he's become somewhat of a business hero to many people, myself included. Leaders like Frank, Scott McNeely, Jay Shree Ullal, and my old boss, Pat McGovern at IDG, have inspired me over the years and each has applied his or her own approach to building cultures and companies. Now, when Slutman first took over the reins at Snowflake, I published a breaking analysis talking about Snowflake and what we could expect from the company now that Slutman and CFO Mike Scarpelli were back together. In that post, buried toward the end, I referenced the playbook that Frank used at Data Domain and Service Now, two companies that I followed quite closely as an analyst and how it would be applied at Snowflake, that playbook, if you will. Frank reached out to me afterwards and said something to the effect of, I don't use playbooks, I'm a situational leader. Playbooks, you know, they work in football games, but in the military, they teach you situational leadership. Pretty interesting learning moment for me. So I asked Frank on the stage about this. Here's what he said. The older you get, the more experience that you have, the more you become a prisoner of your own background because you sort of think in terms of what you know, as opposed to getting outside of what you know and trying to sort of look at things like a five-year-old that has never seen this before and then how would you deal with this? And I really try to force myself into, I've never seen this before. And how do I think about it? Because at least the very different interpretations and be able to mind it just really avoid the rinse-and-repeat mentality and the abrupt people that have worked with me before, some of them got with me from company to company, they would have fallen prey to rinse-and-repeat. I would literally go like, it's not what we want. So think about that for a moment. I mean, imagine coming in to lead a new company and forcing yourself and your people to forget what they know that works and has worked in the past. Put that aside and assess the current situation with an open mind. Essentially start over. Now that doesn't mean you don't apply what has worked in the past. Slutman talked to me about bringing back Scarpelli and the synergistic relationship that they have and how they build cultures and the no BS and hard truth mentality they bring to companies. But he bristles when people ask him, what type of CEO are you? He says, do we have to put a label on it? It really depends on the situation. Now, one of the other really hard-hitting parts of the book was the way Frank deals with who to keep and who to let go. He uses the Volkswagen tagline of drivers want it. He says in his book, in companies, there are passengers and our drivers, we want drivers. He said, you have to figure out really quickly who the drivers are and basically throw the wrong people off the bus. Keep the right people, bring in new people that fit the culture and put them in the right seats on the bus. Now, these are not easy decisions to make, but as it pertains to getting rid of people, I'm reminded of the movie Moneyball. Art Howe, the manager of the Oakland A's, he refused to play Scott Hatterberg at First Base. So the GM, Billy Bean, played by Brad Pitt, says to Peter Brand, who was played by Jonah Hill, you have to fire Carlos Peña. Don't learn how to fire people. Billy Bean says, just keep it quick. Tell him he's been traded and that's it. So I asked Frank, okay, I get it. Like the movie, when you have the wrong person on the bus, you just have to make the decision, be straightforward and do it. But I asked him, what if you're on the fence? What if you're not completely sure if this person is a driver or a passenger? If he or she should be on the bus or not on the bus, how do you handle that? Listen to what he said. I have a very simple way to break ties and when there's doubt, there's no doubt, okay? When there's doubt, there's no doubt. Slutman's philosophy is you have to be emphatic and have high conviction. Back to the baseball analogy, if you're thinking about taking the pitcher out of the game, take them out. Confrontation is the single hardest thing in business according to Slutman, but you have to be intellectually honest and do what's best for the organization, period. Okay, so wow, that may sound harsh, but that's how Slutman approaches it, very Belicheckian, if you will. But how can you amp it up on a daily basis? What's the approach that Slutman takes? We got into this conversation with the discussion about MBOs management by objective. Slutman in his book says he's killed MBOs at every company he's led. And I asked him to explain why. His rationale was that individual MBOs invariably end up in a discussion about relief of the MBO if the person is not hitting his or her targets. And that detracts from the organizational alignment. He said, it's no flake, everyone gets paid the same way from the execs on down. It's a key way he creates focus and energy in an organization by creating alignment, urgency and putting more resources into the most important things. This is especially hard, Slutman says, as the organization gets bigger, but if you do approach it this way, everything gets easier. The cadence changes, the tempo accelerates and it works. Now, and to emphasize that point, he said the following, play the clip. Every meeting that you have, every email, every encounter in the hallway, whatever it is, it's an opportunity to end things on. That's why I use that title. But do you take that opportunity? And according to Slutman, if you don't take that opportunity, if you're not in the moment, amping it up, then you're thinking about your golf game or the tennis match that's coming on this weekend or being out on your boat. And to the point, this approach is not for everyone. You're either built for it or you're not. But if you can bring people into the organization that can handle this type of dynamic, it creates energy, it becomes fun, everything moves faster. The conversations are exciting, they're inspiring, and it becomes addictive. Now, let's talk about priorities. I said to Frank that for me anyway, his book was an uncomfortable read. And he was somewhat surprised by that, really, he said. I said, yeah, I mean, it was an easy read, but uncomfortable because over my career, I've managed thousands of people, not tens of thousands, but thousands, enough to have to take this stuff very seriously. And I found myself throughout the book, on the one hand, saying to myself, oh, I got that right, good job, Dave.  And the point is, to Frank's leadership philosophy, there's no one correct way to approach all situations. You have to figure it out for yourself. But the one thing in the book that I found the hardest was Slutman challenged the reader if you had to drop everything and focus on one thing, just one thing for the rest of the year, what would that one thing be? Think about that for a moment. Were you able to come up with that one thing? What would happen to all the other things on your priority list? Are they all necessary? If so, how would you delegate those? Do you have someone in your organization who could take those off your plate? What would happen if you only focused on that one thing? These are hard questions, but Slutman really forces you to think about them and do that mental exercise. Look at Frank's body language in this screenshot. Imagine going into a management meeting with Frank and being prepared to share all the things you're working on that you're so proud of and all the priorities you have for the coming year. Listen to Frank in this clip and tell me, it doesn't really make you think. You know, a lot of words and stuff and I've got a lot of questions for you here. And there's like 15 things, there are areas for you. I'm like, you're 15, you got none, right? It's like, you just can't decide what's important. So I'll tell you everything because I just can't figure out. The thing is, it's very hard to just say one thing but it's really the mental exercise that matters. Going through that mental exercise is really important according to Slutman. Let's have a conversation about what really matters at this point in time. Why does it need to happen? And does it take priority over other things? Slutman says you have to pull apart the hairball and drive extraordinary clarity. You could be wrong, he says, and he admits he's been wrong on many things before. He, like everyone, is fearful of being wrong. But if you don't have the conversation, according to Slutman, you're already defeated. And one of the most important things Slutman emphasizes in the book is execution. He said that's one of the reasons he wrote Amp It Up. In our discussion, he referenced Pat Gelsinger, his former boss who bought Data Domain when he was working for Joe Tucci at EMC. Listen to Frank describe the interaction with Gelsinger. One of my prior bosses at Gelsinger when they acquired Data Domain through EMC, that's not what he was telling me. And he called Andy Grove the same because he was the same time he was in command. And he said no strategy is better than his execution, which I find one of the most brilliant techniques. Now, before you go changing your strategy, says Slutman, you have to eliminate execution as a potential point of failure. All too often, he says, Silicon Valley wants to change strategy without really understanding whether the execution is right. All too often companies don't consider that maybe the product isn't that great. They will frequently, for example, make a change to sales leadership without questioning whether or not there's a product fit. According to Slutman, you have to drive hardcore intellectual honesty. And as uncomfortable as that may be, it's incredibly important and powerful. Okay, one of the other contrarian points in the book was whether or not to have a customer success department. Slutman says this became really fashionable in Silicon Valley with the SaaS craze. Everyone was following and pattern matching the lead of Salesforce.com. He says he's eliminated the customer service department at every company he's led, which had a customer success department. Listen to Frank Slutman in his own words, talk about the customer success department. I view the whole company as a customer success function, okay? I'm customer success, you know? I said in my presentation yesterday, we're a customer first organization. I'm leading the department. Now he went on to say that sales owns the commercial relationship with the customer. Engineering owns the technical relationship. And oh, by the way, he always puts support inside of the engineering department because engineering has to back up support. And rather than having a separate department for customer success, he focuses on making sure that the existing departments are functioning properly. Slutman also has always been a big, has always been big on net promoter score NPS. And Snowflakes is very high at 72. And according to Slutman, it's not just the product. It's the people that drive that type of loyalty.  He told a story about someone who came into his office to ask his opinion about a t-shirt. And he turned it around on her and said, oh, what do you think? And she said, well, it's okay. So Frank made the point by flipping the situation. Why are you coming to me with something that's just okay? If we're gonna do something, let's do it. Let's do it all out. Let's do it right and get excited about it, not just check the box and get something off your desk. Hip it up, all aspects of our business. Listen to Slutman talk about Steve Jobs and the relevance of demanding excellence and shunning mediocrity. He was incredibly tall of anything that didn't, or he didn't think it was great.  I'm not that aggressive in that way. I'm a little bit nicer about it, but I still, I don't want to give in to expediency and mediocrity, I just don't. I'm just gonna fight every step of the way. Now, that story was about a little thing, like some swag, but Slutman talked about some big things too. And one of the major ways Snowflake was making big sweeping changes to amp up its business was reorganizing its go to market around industries like financial services, media and healthcare. Here's some ETR data that shows Snowflake's net score or spending momentum for key industry segments over time. The red dotted line at 40% is an indicator of highly elevated spending momentum. And you can see for the key areas shown, Snowflake is well above that level. And we cut this data where responses were greater, the response numbers were greater than 15. So not huge ends, but large enough to have meaning. Most were in the 20s. Now it's relatively uncommon to see a company that's having the success of Snowflake make this kind of non-trivial change in the middle of steep as curve growth. Why did they make this move? Well, I think it's because Snowflake realizes that its data cloud is going to increasingly have industry diversity and unique value by industry, that ecosystems and data marketplaces are forming around industries. So the more industry affinity Snowflake can create, the stronger its moat will be. It also aligns with how the largest and most prominent global system integrators, global SIs go to market. This is important because as companies are transforming, they are radically changing their data architecture, how they think about data, how they approach data as a competitive advantage and they're looking at data as specifically a monetization opportunity. So having industry expertise and knowledge and aligning with those customer objectives is going to serve Snowflake and its ecosystem as well in my view. Slupan even said, he joined the board of Instacart not because he needed another board seat, but because he wanted to get out of his comfort zone and expose himself to other industries as a way to learn. So look, we're just barely scratching the surface of Slupan's book and I've pulled some highlights from our conversation there's so much more that I can share just even from our conversation and I will as the opportunity arises. But for now I'll just give you the kind of bumper stick and revamp it up. Raise your standards by taking every opportunity, every interaction to increase your intensity. Get your people aligned and moving in the same direction. If it's the wrong direction, figure it out and course correct quickly. Prioritize and sharpen your focus on things that will really make a difference. If you do these things and increase the urgency in your organization you'll naturally pick up the pace and accelerate your company. Do these things and you'll be able to transform better identify adjacent opportunities and go attack them and create a lasting and meaningful experience for your employees, customers and partners. Okay, that's it for today. Thanks for watching and thank you to Alex Meyerson who's on production and he manages the podcast for breaking analysis. Kristen Martin and Cheryl Knight helped get the word out on social and in our newsletters and Rob Hoef is our EIC over at Silicon Angle who does some wonderful and tremendous editing. Thank you all. Remember all these episodes are available as podcasts wherever you listen just search breaking analysis podcasts. I publish each week on wikibon.com and siliconangle.com and you can email me at david.volante at siliconangle.com or DM me at dvolante or comment on my LinkedIn posts and please do check out etr.ai for the best survey data in enterprise tech. This is Dave Vellante for theCUBE insights powered by ETR. Thanks for watching. Be well. We'll see you next time on breaking analysis.


Hey, this is Brett and this 2014 Chevy Camaro 2SS convertible is stock number 13034Z. I'm here at Summit Automotive in Fond du Lac, Wisconsin, your new and used sports car headquarters. This 2014 Chevy Camaro 2SS has the 6.2 liter V8 gas engine, puts out 400 horsepower with the automatic transmission. This car has been fully safety and inspected by our service shop as a fresh oil and filter change all the fluids have been checked and topped off and this car is 100% ready to go. Crystal red tint coat is the color and I shoot all my videos in 4K so if you have HD capabilities on your computer, tablet, smartphone or television I highly recommend turning them on right now because it is your best way to check out the quality, condition, options and cleanliness of the vehicle before seeing it in person. And if you like the video and how I do things here at Summit Automotive consider clicking the subscribe button to my YouTube channel in the lower right hand part of the screen and then click the bell notifications and you'll get updates every time I do the videos of the new and used inventory here at Summit Auto as well as a very large catalog of used vehicle walkarounds. This one has the 20 inch polished aluminum rims and it has Laufen S-Fit AS tires. These are 245, 45 ZR20s and they are brand new tires. We put those on in our safety inspection, you can still see the little wear knobs on there so brand new tires has Brembo brakes on it. Front fender is in excellent condition, I didn't see any major dents or dings on there. It does come with the HID headlamps, the LED running lights and the factory fog lights. Front bumper is in nice shape, I didn't see any major dents, dings or cracks on it. The hood is in very nice condition, I do really like the single heat extraction in the middle for this body style Camaro. The passenger side front fender looks really good as well, didn't see any dents or dings on there. And the passenger side rim, no major scuffs or scrapes, very nice condition. Crystal red tint coat has just a little bit of metal flake to it, really a cool looking color. And as you go down this side of this 2014 Chevy Camaro 2SS, take note of how clean the body is, how reflective and mirror like that paint is. Take these HD videos so if you are far away or even if you're close by and you just cannot make the trip down but you're still interested in purchasing the vehicle you can see the car, hear the car and of confidence in the vehicle that you're looking at before you even get here so when you do get here there's absolutely no surprises and you can make a smart and informed buying decision from wherever you're at. And if this video helps you make that decision let your salesmen know that you saw the video that it was helpful and that Brett sent you. Back rim is in excellent shape as well and the back tires are brand new too. Coming around to the back of the vehicle, this one does have the rear bumper parking sensors, the chrome tipped dual rear exhaust, rear bumper is in fantastic condition, I didn't see any dents, dings, scuffs or scrapes or cracks on there. Trunk area is very nice and clean. This is also where your RPO stickers are, or sticker is, RPO codes. Feel free to pause that to make sure that this one has all the options that you're looking for. At your soft top down you have to have this shield right there, I actually have a video showing you how to do that. Make sure that your soft top goes down. And you do get the LED tail lamps on the back there. Coming around to the driver's side, rear quarter looks really good. You can see major dents or dings on there. And for full disclosure this back room is in excellent shape. The only thing I did see on the entire car is just one little scratch right there. Other than that, looks really good all the way around. Driver's side door, didn't see any dents or dings on there. It does come with the heated power mirrors.  Both of these front seats are heated. The SS stitched into the headrest, side curtain airbags, didn't see any rips or tears on the seats. Factory floor mats, Boston premium audio, power windows, power locks, power mirrors. You also get this silver metallic finish to the inside of the doors. That wraps all the way around the dash. It's pretty cool. Back seats are in fantastic condition as well. You get the latch child safety system for any child car seats you may have. And I didn't see any rips or tears back there. So we'll hop inside, check out the miles. The radio will take the soft top down. You can see that this one has 13,882 miles. You get a nice information center there. Instrument cluster is very nice and clean. Didn't want to point out that this one does have the heads up display in the windshield. So that's a pretty cool system right there. Has the leather wrapped steering wheel, cruise controls and paddle shifters. Bluetooth and audio controls on the right. Steering wheel is an excellent chip and insinirips, tears or scuffs on it. Smells with the Chevy MyLink system, AM, FM and Sirius XM radio capabilities. You do get heated seat buttons there. Down here you get an oil temp, oil pressure, volts and trans temp gauge. Stability control and the six speed automatic transmission. Passenger side, floor mat and seat are in excellent condition. No rips or tears in there. Smells very clean inside this car. I don't think it's ever been smoked in. The headliner is in fantastic condition as well, or the inside of the soft top if you will. You do get home link buttons right there for your garage door security systems and lighting systems and on-star and SOS buttons in the rear view mirror. So we'll put this soft top down, so you gotta release it like that. And then you can just press that and you can see that that works really nicely. And it'll stop once it's complete. It'll tell you in the center console here that folding top motion is complete as well. Alright, we'll start it up and take a look under the hood. Starts right up, no check engine lights or anything like that. There is your backup camera that is working nicely as well. I would personally like to thank you for checking out the video today. Hopefully from this HD video you have been able to verify the quality, condition, options and cleanliness of this car all the way around inside and out. There are those HID headlamps, LED running lights, and the factory fog lights. I think for a 2014 this car is in fantastic condition. Under the hood we have the 6.2 liter V8 engine, 400 horsepower, engine bay is very clean, runs extremely smooth, has a cold air, air aid intake. And once again this car has been fully safety and inspected by our service shop as a fresh oil and filter change, all the fluids have been checked and topped off. And this car is 100% ready to go. There is your emission sticker. You can see the shock is doing a nice job holding that hood up. And this car is 100% ready to go and I would highly recommend this 2014 Chevy Camaro 2SS in crystal red tint coat. Actually I'm going to do a final walk around with the top down here because it does look so good. But to see more pictures of this car or one of our other 450 new and used cars, trucks, SUVs, sports cars, Camaros, Corvettes, Challengers, Chargers, Mustangs, GT500s, ZL1s, you name it we got it. You can go to that website which is right there at www.summonauto.com. All pictures and descriptions of every single vehicle from two locations all at www.summonauto.com. And if you'd like to check out more HD videos you can go to www.youtube.com. Remember to like, subscribe, and share on this video and all the videos that you see there. Click the bell notifications, you'll get updates on the videos I do each and every day here at Summit Auto. In fact in a second you will see a link to subscribe to my YouTube channel on the upper left. A link to more Camaro videos like this one on the upper right, a link to this vehicle on our website on the lower left, and a link to one of our latest YouTube videos in the lower right. Check us out and we're super excited to help you with this Ultra Clean 2014 Chevy Camaro 2SS Convertible and Crystal Red Tint Coat. Thanks again for checking out the video. Remember to like, subscribe, and share on my YouTube channel. I really appreciate it. Thanks again.


Hi and welcome to the session. I am Asha and I am going to help you solve the following problem which says, without actually calculating the cubes find the value of each of the following. The first one is minus 12-hole cube plus 7-hole cube plus 5-hole cube. And the second one is 28-hole cube plus minus 15-hole cube plus minus 13-hole cube.  And if x plus y plus z is equal to 0, that is this term is equal to 0, then we have x cube plus y cube plus z cube plus 3xyz is equal to 0 since the product of any number with 0 is 0, which further implies that x cube plus y cube plus z cube is equal to 3xyz. So this is the key idea. We are going to use in this problem to find the value of the given two problems. Let us now start with the solution and the first one is minus 12-hole cube plus 7-hole cube plus 5-hole cube. Now this is in the form of x cube plus y cube plus z cube where x is equal to minus 12, y is equal to 7 and z is equal to 5 and the sum of these three numbers that is x plus y plus z is equal to minus 12 plus 7 plus 5 which is equal to minus 12 plus 12 is further equal to 0. So this implies that x plus y plus z is equal to 0 and hence minus 12-hole cube plus 7-hole cube plus 5-hole cube must be equal to 3 into x into y into z where x is minus 12, y is 7 and z is 5 and on multiplying 3 with minus 12 7 and 5 tips minus 1, 2, 6, 0 and hence the value of the first part is minus 12, 6, 0. So this completes the first part and now proceeding on to the second part where we have to find the value of 28-hole cube plus minus 15-hole cube plus minus 13-hole cube. Again this is in the form of x cube plus y cube plus z cube where x is equal to 28, y is equal to minus 15 and z is equal to minus 13 and the sum of x plus y plus z is equal to 28 plus minus 15 plus minus 13 and the sum of minus 15 and minus 13 is minus 28 and the sum of a number with its additive inverse is 0 so we have x plus y plus z is equal to 0 this implies x cube plus y cube plus z cube is equal to 3 x y z 28-hole cube plus minus 15-hole cube plus minus 13-hole cube is equal to 3 into 28 into minus 15 into minus 13 and on the crime we get 1, 6, 3, 8, 0 and that's the product of the second part as 1, 6, 3, 8, 0 so this completes the second part and hence the solution so hope you enjoyed this session take care and have a good day


Jean-Philippe Smet, better known by his stage name Johnny Halladay, is a French singer and actor.He has been called the French Elvis. He was married for 15 years to popular Bulgarian French singer Sylvie Vartan and the two were considered a golden couple for 20 years. Halladay has completed 181 tours, had 18 platinum albums, and has sold more than 80 million records worldwide, making him one of the world's best-selling artists of all time. Halladay remains largely unknown outside of the francophone world, thus earning the nickname the biggest rock star you've never heard of in English-speaking countries. Nevertheless, at an international level he is still considered as the only non-anglophone rock musician having a large international reputation. He was made chevalier, knight, of the Legion Donor in 1997. Early Life Halladay's father, Leon Smet, 1908-1989, was Belgian, his mother, Heugette Clerc, 1922-2007, was French. Born in Paris, Halladay took his stage name from his aunt Desta's husband and dance partner Lee Halladay, a pseudonym for Lee Ketchum, an expatriate Oklahoman, performer and emcee at the Café de Paris where Johnny began his singing career. Johnny was raised by Desta and Lee from a very young age, with Lee being his first manager. Johnny was always very loyal to Lee and secured him a permanent job as a producer and on-dar man with his label, Phillips Records until Lee's retirement and relocation back to Oklahoma. Career Influenced by Elvis Presley in the 1950s rock and roll revolution, Halladay became famous in the 1960s for singing rock and roll in French. His debut single, Les Lafayas was released on the Vogue label in March 1960. His first album, Hello Johnny, was released in 1960. In 1961 his cover of Let's Twist again sold over one million copies, and was awarded a gold disc. It topped almost every European chart, although the track did not appear in the UK singles chart. He appeared on the American The Ed Sullivan Show with American singing star Connie Francis in a show that was taped at the Moulin Rouge nightclub in Paris. He also staged many appearances in the Paris Olympia under the management of the late Bruno Coquatrix. For their first concert, the Jimi Hendrix experience opened for Johnny Halladay in Evru on October 13, 1966. Black and white footage, also from October 1966, exists of Halladay partying with Hendrix, his manager Chaz Chandler and others. There also is a widely published monochrome photograph of Halladay walking somewhere in Paris in late May 1966 with Bob Dylan. At the end of the 1960s, Halladay made a string of albums with Mick Jones and Tommy Brown as musical directors, and big Jim Sullivan, Bobby Graham and Jimmy Page as session musicians. These are Jeanne Aum, Riviere, Hoverton Litt, aka J.E.Sue's N.E. Dan's La Rue, and Vi. On J.E.Sue's N.E. Dan's La Rue, Halladay also hired both Peter Frampton and the Small Faces. Amongst their contributions are the songs Amen, Bang Bang, Reclamation, News Report, and Regard Pourmoy, What You Will, which are variations of Small Faces and Humble Pie, Frampton's band, songs tracks and they all play on the album. Often forgotten is Halladay's non-LP single and EP track KJETMA from the same sessions. By 1969 alone, his sales of records exceeded 12 million. One of Halladay's later concerts, 100% Johnny, Live à la Tour Eiffel in 2000, attracted an audience of 500,009.5 million television viewers, the show was broadcast live on French TV. In December 2005, Halladay had his third number one single in France, Monday plus Bonoel, after two ensemble and Marie, dedicated to his adopted daughter Jade. Shortly before announcing his retirement from touring, he released a blues-flavored album, L.E.Cour d'Unhame, on November 12, 2007. The album hit number one in both France and French-speaking Belgium. In addition to the lead single Always, L.E.Cour d'Unhame features T.M.A.R.S. I.M.A.L., a duet with blues-man Taj Mahal and I Am the Blues, an English language song written for Halladay by U2's lead singer Bonoel. His next album, C.A.N.E. Finirajama, released in 2008, another number one on the French album chart, and its lead single, C.A.N. Finirajama, also reached number one. In 2008 he recorded a series of acoustic songs with French musician Drexel Joné. Halladay's most recent album, also a number one hit in France, is Tour 66, Stade de France 2009 – a live set recorded at Stade de France during his farewell tour with appearances by Drexel Joné on the guitar. French movie producers Michel and Laurent Petin introduced Johnny Halladay to Hong Kong film director Johnny Tu. Halladay was cast as the main role of Tu's vengeance after meeting with the director in early 2008. The shooting began in November 2008 and concluded in February 2009. Personal Life Halladay was introduced to the great Chanteuse Edith Piaf in the late 50s and became one of her biggest fans up until her death in 1963. He attended every concert she performed in and was said to have been friends with her throughout his career. In a recent article he claimed that Edith tried to seduce him during a dinner with her in 1960, and resulted in him fleeing from her in despair, but a 1962 photograph of Edith, her husband Theo Serapo, and Halladay shows that she earned his trust back. Halladay performed Edith's 1950 hit Himal Amour in 1995. In November 2005, Halladay started a procedure to obtain Belgian citizenship but his request was turned down in 2006 because he failed to fulfill the residence requirements. In late 2006, Halladay announced that he would move his permanent residency to Gestod, Switzerland to escape the high tax rate imposed by the French government. Swiss law allows wealthy individuals to claim residency if they live six months and one day in the country and then pay a fixed tax based on expenses, such as rent or assets in Switzerland, rather than a percentage of their income. Halladay has said that he would move his residency back to France if it changes its tax laws. He maintains a second home in Los Angeles, where he has been spending increasing amounts of time in recent years, enjoying his relative anonymity in the US. In January 2014, Halladay revealed that his current residence was in fact in the United States after an investigation by Swiss journalist showed that Halladay did not spend enough time in Gestod, Switzerland to qualify as a resident. One of his favorite leisure activities is riding his Harley Davidson on long trips through the California desert, staying in small motels along the way. Halladay married Bulgarian-French singer Sylvie Vartan on April 12, 1965. Their son David was born on August 14, 1966. Halladay and Vartan divorced in 1980. He was then briefly married to Bae Beth-8 Yen, the marriage lasted just two months and two days, ending in February 1982. A four-year relationship with French actress Natalie Bae followed, their daughter Loris Met was born on November 15, 1983. He was married to his third wife Adeline Blonde-Eau for two years, divorcing her in 1992. He has been married to Laetitia Buddha since March 25, 1996. The couple have adopted two girls from Vietnam, Jade Odette Desiree, born August 3, 2004, formerly Bui Thi Hoa, in November 2004, and Joy, Mai Hoang, born July 27, 2008, in December 2008. He also has three grandchildren through his son David, their names are Emma, Leona and Cameron. Halladay supported Nicolas Sarkozy in the 2007 French presidential election. The Masked Avengers referenced this in their prank call to American vice presidential nominee Sarah Palin when an Avenger impersonating Sarkozy named Halladay as his special American adviser. In July 2009, Halladay was diagnosed with colon cancer, and underwent surgery. On November 26, 2009, Halladay underwent surgery in Paris to repair a herniated disc. He suffered complications and was admitted to Cedars Sinai Medical Center in Los Angeles. Doctors announced that they had put Halladay into a medically induced coma so they could repair lesions that had formed as a result of the surgery, and to relieve his pain. On December 17, Halladay and his wife started legal proceedings against Dr. Stéphane Délageux, who had performed the original surgery. The conflict was resolved in February 2012 following Délageux's vindication by medical investigators.


Hello friends and welcome back to another Pokemon how-to guide my name is Lee also known as Osiris and in today's guide We're gonna be looking at how to get hidden abilities in Pokemon sword and shield before we get into it If you do enjoy this sort of content, please remember to drop a like on the video Do subscribe to the channel for more Pokemon content and leave your comments down below Let me know what you think of the guide in general if you found it helpful And if you have your own methods with how to get hidden ability Pokemon that you found effective so far in this game So hidden abilities are something that are available on the majority of Pokemon and for the most part They are really useful abilities to obtain because normally they are a little bit better than what you're gonna get Generically from just catching a Pokemon in the wild and for the competitive players It does open up a lot of different opportunities to use these Pokemon and utilize them in battle So very important to get but a little tricky because there isn't a really defined way of getting them within sword and shield At the minute, so there is only one way to obtain them like I say within max raid dens and max raid battles So it is a bit difficult and there is a little bit of a grinding process to doing this But you can do it with one wishing piece and that is all you need So as long as you've got one wishing piece You can find a den that that Pokemon that you're after is located in and once you do have that den Located you can just reset over and over again on that one den until you do find that Pokemon with its hidden ability I'm gonna share some of my own opinions with how I've had a lot of success Finding Pokemon with hidden abilities in this guide And I think that's one of the reasons why I wanted to make this because I've had quite a bit of success in my own methods Doing this although they are quite similar to a lot of other methods that players do tend to use But I found that there is some success with what I have done and obtaining hidden abilities so far So that was the reason really why I wanted to share these with you in this guide now the first thing that we want to do I think the the example that I'm going to use in this guide is to try and get Zen mod darmanitan So what we really want to be doing is heading up to a hammerlock hills here And we want to head down to a specific den and to find the dens that you want to catch your Pokemon in There is obviously in every single den there is a bunch of different Pokemon So determining what Pokemon are in there It is sometimes pretty hard one of the really good resources that you can use at the minute is serabi.net It does list every single den with the Pokemon that you can attain and then more importantly It does list those Pokemon with being able to find their hidden abilities or not in these specific dens So we're going to go to this specific den here Which is hopefully if you follow this guide and you're wanting this specific Pokemon and you can use this method So what we're going to do as I say we'll save in front of this den because one of the methods that I found Most successful is having a solid purple beam Now I know a lot of players will argue that you don't necessarily need the purple beam And this is probably true But I found a lot more success having a purple beam than not having a purple beam with when obtaining Hidden ability pokemon So what you want to do is when you found the den like I say you can check serabi for the exact pokemon that you're looking for In the den location you find the den like we are here We're going for that zen mod darmanitan You want to save in front of it and then you want to drop a wishing piece And you want to set your text to slow as well. Remember this is just the date manipulation Glitch that we're going to be using here to get this purple beam to start with so it's like a few step process So I want to save the game here and then we see the the beam is not red So you just want to Shut the game down and then come back into it And this isn't really too hard because it's not a slow process It's not the quickest process the game loading up does take a little bit longer than probably what we'd all like it to But I mean it's not the slowest process in the world And you maybe want to be taking a couple of minutes to really get that solid purple beam I'll be interesting here from you guys about if you've had success with The solid purple beams and hidden abilities or just the regular red beams that have been rumored to Be pressing too quickly there. So let's drop it in and a bit trigger happy there But yeah, if you've had a lot of success finding hidden abilities with just the regular beam Then I would love to hear from you because I've not really had very much success at all I don't think I found a single hidden ability pokemon with just the single red beam And they've all been the solid purple beam So I don't know if there's been information out there literally scoured reddit All of the different pages across the internet different youtube videos and things like that to have a definitive answer on it And I can't find one So I'm just summarizing my kind of findings here and hopefully if you do use this method You'll have as much success as I have because I have got bunch of hidden ability pokemon already in the game And it hasn't really been too hard for me to find so as you can see what we're going to be doing now Is just resetting the game over and over until we do get that purple beam by dropping that one wishing piece Into this then so what I am going to do cut the video now And we'll come back when I have found it just to save a little bit of time rather than going through this over and over again So as you can see friends, we finally got the purple beam I haven't been doing it for too long. Maybe this is about the 10th try of resetting So it's not taking too long now. We've got the purple beam. That's the first step in this process Obviously, I'm just talking you through this now We want to save in front of the den because the next step is where we use the date manipulation technique So we want to click into the den Remember we are looking for a galarian doorman attack here So we want to select invite others and once the timer starts you hit that home menu This is a bit more chilled out. So we're not relying on getting ahead of the save date So you want to come down to date and time make sure that the Synchronized clock by the internet is off come down to your date and time Just knock it forward by one day come back out and into the game again quit out of this screen here So we want to quit out the red den And then we will see the weather change and that means yep We're into a new day. We've tricked the game and finally we get it. So this is good for the guide So it's taking a little bit a little bit quicker than what I thought would might do So we do have the doorman attack here So we want to check whether it's got its ability or not Now one of the ways we want to do this before going into this raid is just to make sure that we do save the game And the other step that you want to make sure that you are doing is going into your system settings here in the game So options and you want to go down to auto saves and make sure that your auto saves are turned off Would you like to save these settings? Yes So because one of the issues is when you come back into the the wild area from the raid It will automatically save Not allowing you to go back and then go into this platform once you have checked for its hidden ability So what we want to do is click into the raids. I would recommend inviting others But for this we're going to use the NPCs See how quickly we can get it. It would have been a lot quicker if we invite others to this But we unfortunately aren't going to be doing that in this one. Hopefully it doesn't take too long The big thing here is though to identify if it is zen mode I think don manatans one of the easier ones to identify Especially in a raid battle then maybe some others that have the abilities that don't activate within a battle But once we get don manatans down to 50 health It should change its form and that will mean that we are able to tell whether or not it has that hidden ability If it doesn't transform at 50% or below then we know we've not got the right one What we can do then is we can reset the game and then start doing the cycle through the max raid again Until we get another don manatana and then we'll try it again with that one Now we will just probably skip ahead a little bit until we get it down to 50% Just so we see whether or not it is the hidden ability don manatana or not So as you can see this should take the don manatana down to about 50% now We are using the max guys and now here is the moment of truth for this specific pokemon So this is a good method to use to see if this is the hidden ability don manatana or not And it is getting desperate This putting the shield up is that ability going to activate it doesn't look like it is going to and No, I don't think this is the hidden ability don manatana. Unfortunately So in a way this is quite a good example for the guide where we can see that this isn't the hidden ability don manatana So we are just wasting our time at this point. So what we want to do is just come out come to the whole menu close the game And then we'll go back into it The purple beam should still be intact and we should still be able to cycle through doing the date manipulation So what we want to do is change again and you get the don manatana back as you can see we're back here Yep, we know it's not the hidden ability don manatana. Unfortunately So what we have to do is just go past this one do the date manipulation again We'll invite others go down to date and time go to the date change afford one day come back out into the game And then quit again and hopefully it doesn't take too long to find another don manatana We'll come back I'll keep cycling through this and as soon as we do find another don manatana Come back and we'll see if that one is the hidden ability or not So as you can see we finally come across another don manatana So what we want to do is again just save the game Before we go into this one so we can check whether or not it is going to be that hidden ability don manatana Not it is important to save right here Make sure that you do have those auto saves turned off as well So when you come back out the den you can reset it if it isn't the hidden ability or not So we're into the match now against this specific don manatana as you can see we have got it nearly down to 50% we're going to be just about there and soul rock actually doing some work for once Not really something you see too often It does break the barrier will this take it down to 50% So just above we're going to have to use one more attack without sassian and that should knock it down this next turn But the talk will actually coming in handy here as well our NPCs putting in some work taking it down to 50% And we are going to see the shield activate now here We should see the hidden ability activate if it is and we do so this is we know now This is probably an easier one to get the grind is pretty hard to get here But we now know that this is the hidden ability don manatana We have the game saved prior to coming into this max raid battle So we know if we do fail to catch it we can reset We know quite confidently that this has the hidden ability that we want So this is a good way to show you how to get the hidden ability of this pokemon And hopefully it doesn't take too long for us to catch this one And i'm so happy because this is a pokemon that i've wanted for a long time And i'm sure it's one that you if you haven't already got it You would like to be able to access and get a hold of within the game So let's get forward a little bit we'll get to the catch point here So we are using a dust ball make sure that you are using this Ball because it is probably the one that gives you the best catch rate We aren't hosting the raid unfortunately that would be a better way to do Once you do know that it has that hidden ability If you host the raid you're pretty much guaranteed to get the catch But here we are into roll three and we do get it So there we go we've got our hidden ability don manatana and that is great And this is just an example to show you how you can get hidden ability pokemon Within the sword and shield titles now i have said that searching for that purple beam Does generally give me better odds sometimes it's not always going to work Because some pokemon like i said are only available with solid purple beam But some pokemon aren't available with that solid purple beam So you have to just go with the regular beam maybe that's a little bit quicker And then you just use the day manipulation to get the pokemon in general But as you can see on your screen right in front of you now that it has got that zen mode So we have got the hidden ability Finally it does take a little bit of time to get there But there is a method to do it to make sure that you're not spending too much resources Too many wishing pieces and too much of your time to do It's just a case of getting to the right den that you can find in cerebe The link is down in the description below It will tell you whether that hidden ability is available on that pokemon in that specific den Once you have that pokemon all you need to do is cycle through With these methods in this guide and you should be able to get that hidden ability pokemon that you want So hope you've enjoyed the guide my friends this does wrap it up here We'll be back with another guide very soon Have a great one thanks for tuning in Do drop a like subscribe to the channel And i'll see you all for the next one Until then take care and bye bye


This year, Red Hat made architecture changes to OpenShift container platform and released OpenShift version 4. You may have used OpenShift 4 by now or at least heard of it. In this series of videos to follow, I'll take you a little deeper into OpenShift 4.x architecture to showcase the awesomeness of this platform. OpenShift 4.x allows you to quickly set up a cluster on the infrastructure of your choice. These are different cloud providers such as AWS, Azure, Google Cloud, et cetera, and your own infrastructure such as infrastructure with virtualization like VMware or OpenStack. Or you can install even on bare metal. OpenShift also comes with a developer friendly version. This can be installed on your workstation and it is called code-ready containers. You are seeing the landing page from where we can download and install at cloud.redhat.com slash OpenShift slash install. Let us see one of the cloud providers. OpenShift can be installed in two different ways. One with full stack automation commonly called installer provision infrastructure or simply IBI or you can use pre-existing infrastructure commonly called user provision infrastructure or UBI. With IBI, the installer manages the infrastructure as well. It spins up the machines and then installs OpenShift. It also manages the operating system in addition to the OpenShift platform. IBI uses only Red Hat CoreOS as the operating system. If you choose UBI, you as an administrator will set up the infrastructure and start the installer to use that infrastructure to set up a cluster. With UBI, you have a choice of using Red Hat CoreOS or Red Hat Enterprise Linux 7 as the operating system for the worker nodes. For masters, you will still use Red Hat CoreOS. If you use Red Hat CoreOS as the operating system, then the OS and its upgrades are all managed by the OpenShift container platform. For the purpose of the first install, let us use IBI. So I'm going to click on this IBI button. We can download the OpenShift installer from this page. We can also download OpenShift command line tools, CLI, which is OC from this page as well. The pull secret that you see here will be needed to download images from Red Hat's registry. We'll come back and copy this when we start the installer. I already downloaded the installer, so let us try to install now. I can simply install the cluster by running the install command like OpenShift install create cluster and by providing a directory where the installation artifacts will be stored. This installation directory will store all the cluster configuration information. As an example, if we were trying to destroy the cluster later, we would need those artifacts. So this directory are pretty important. However, as the documentation in the landing page shows us, we'll customize our deployment a little bit. In order to make some customizations, we'll create an install config, which is an installation configuration that can be edited to change a few things. So I'll run OpenShift install create install config instead. I'm using a directory with name first where all the install artifacts will be stored. So ask me a few questions. I'll choose an SSH publicly to use. AWS is the platform on which we are going to install, and I'm choosing a region and a domain name for my cluster. I give my cluster a name. I'll just call it first. Now it is asking for the pull secret to be used to pull the images. So let us copy the pull secret, paste that here, and the installation configuration is generated. Let's look at the install config file. So the default install config that it generated shows that it's going to create three workers, three masters for the control plane, the network sider to use, and the IP address range for the services. The rest of the information includes pull secret and my SSH key and things like that. I made some minor changes to this install config. To include a specific size for my worker nodes, I'm going to use M4 extra large. So as you can understand, we can make changes like these, which include the sizes of the machines, the network sider, the service network, and things like that. Now that we have edited the configuration, we'll use that configuration to install the cluster. So I'm running OpenShift install command to create a cluster, pointing to the directory where this configuration exists. The cluster installation has just started, and it will run for about 15 to 20 minutes, and then the cluster will be ready. While the installer is installing the cluster, let's look at how the installation actually happens. The cluster installer, which is running on my workstation, creates a bootstrap node. This is the temporary node on the infrastructure where I'm installing the cluster. This bootstrap node sets up a small Kubernetes cluster, installs three master nodes. The master nodes form the control plane, and once they are up and ready, they take over the control from the bootstrap node. The master node will have the complete machine configurations for the workers that are yet to come. So once the masters take over the cluster, then the workers would come up based on the configuration stored on the masters. In case of installed provision infrastructure, all the masters and the workers would use Red Hat CoreOS as the operating system. Once the control plane on the master comes up, the bootstrap node will be destroyed. The cluster installation is now complete. As I explained just now, first it brought up the bootstrap node, and once the control plane got set up, the bootstrap node was destroyed, and then the workers came up. The installer also creates an admin user, the credentials to log into the cluster. We also have two different URLs. One is the URL to access the API server from OpenShift CLI. The other is the master URL to access the web console. In this video, we have learned to set up a new OpenShift 4.x cluster with minor customizations using installer provision infrastructure method. In the next video, we'll explore this cluster that we just created and look at the structure of the cluster and all the components that the installer created for us.


There are no more hands. No, yeah, there's Cornelia. And there is a question in the chat. First time I see it. Okay. Thank you for the great presentation. Please scan the mixing, deregulate the sequence in the modeling. Yana. I'm not entirely sure I understand the question, but perhaps Monday or Adiaha can rephrase their question while Olivia is giving her talk, and then we can discuss it later. Yeah, maybe it's better. So here is Olivia. And she is a PhD student at the University of Washington, and she has a great method to measure conjugation, and I've seen her talk at Plasmids, and I was like, wow, we need to have Olivia at the workshop, and finally there she is. So please, Olivia. Thanks, thank you so much. Can you hear me okay? Okay, yes. All right, so hello everyone. I really want to applaud Alice and the organizers for the conference and everything you put together. Thanks, Alice, for bringing us all together and doing all the hard work of organizing, which is I know from limited personal experience that is challenging. So, but yeah, today I'm really excited to share with all of you. Kind of Yana had a great talk and talked a lot about why I also am like her, and I care about transfer rates and estimating them accurately and why accurate estimation can be important and help us not get misled about the data we're collecting. So with that said, I work in the lab with Benjamin Kerr at the University of Washington. I'm a six-year PhD student also graduating, maybe moving on to a postdoc because it's kind of similar to Yana in position, which is kind of fun. So I want to thank everybody that was on this project first and foremost, it was really close collaboration with my advisor, Ben Kerr, also with Ava's Topps Lab at the University of Idaho and Ivana Bozik's lab at the University of Washington. And so I also am going to give a little zoom out about why I'm going to make a plug for the estimating conjugation rates are important. And I kind of come at this from a similar but slightly different angle. And I like to use this example of COVID-19 pandemic because it's a familiar topic to everybody and kind of gives us some intuitions about modeling and why parameters are important in them. And so during the pandemic, we're just really, there was an extraordinary effort early on for us to try and understand how fast the disease was going to spread in the population. And so to do this, most of the models were extensions of some standard epidemiological models and Alice gave us some interesting intro to all of that. But this SI model just is simple we are tracking two populations, a susceptible pool, people with not that don't have COVID and then the infected individuals. And the model determines how fast susceptible individuals are going to turn into infected individuals and that's dependent on this transmission rate that we call beta. And this, of course, we all know depends on actually making human contact with somebody that is infected. So you have to have some human contact for transmission to occur. And so we need to add this to our model and we can take a slice out of our chemistry textbooks to do this and we're gonna use the law of mass action. And this just allows us to use the density of the two populations, multiply those together with the transmission rate parameter. And that will kind of tell us how many of those infected individuals will be created over time. If you're not familiar with transmission rate, you're probably more familiar with R0, which is like this basic reproduction number that we kind of focus on when we think about pandemics or viral spread. And you can clearly see by this equation that I'm showing you here that essentially beta is really important for R0. And why I bring that up is because early in the pandemic we all remember these forecasting models people were using about how fast COVID was not spread and they were sometimes good and sometimes not very good. And a lot of it has to do with the parameters we use underlying the models to give us good, accurate or potentially inaccurate predictions about the biology. And so what I think is really fascinating is that the way we model conjugation and we think about plasmid spread is like really similar, like almost the same. So when we care about plasmids in our communities that we've heard about in the gut or in the hospital settings or in wastewater as we've heard a lot this week, we wanna know how fast these plasmids are spreading through our populations over time. And so similar with the COVID example, we're tracking two things. We're tracking our plasmid-free types and we're tracking our plasmid-containing types. And the way they turn into plasmid-containing type is the transfer rate parameter that we are all interested in knowing, estimating correctly. And this is also density dependent. So for conjugation, which is just one mode of how horizontal gene transfer happens but conjugation is cell to cell contact dependent. So we add that law of mass action parameter into our models and then we can model this and how fast plasmid-containing populations are gonna come up over time. But transfer rates are not trivial to estimate for many reasons but one of them is that there isn't just one transfer rate. You can't say this plasmid has this transfer rate or you can't say because they're gonna be very dependent on the environment and the cells that they're around. So to kind of illustrate this and make kind of an analogy, we know that there's transmission rates across species for viruses just like how we probably, the COVID pandemic came from maybe a species like a bat and that transmission rate is quite different from human-human transfer of the virus. And so this is similar in our microbial communities but much more relevant because we think about these species being in close contact with one another all the time. And so here the transfer rate of the between the blue species is probably quite different than the donor, the plasmid is transferring between the red and the blue species. So how do we estimate conjugation rate? Yana gave a good example for the intro of this and I'm just gonna go through this really, really briefly but I think about it in three phases and I'm gonna kind of walk through the mating ass in very specific ways. And this will hopefully help us understand how our new method is quite different framework to how to think about how we estimate conjugation rates and makes it so that you estimate kind of measuring different things in the laboratory. So again, we mix our donor and recipients together in a tube just like normal and as we call this a mating assay and we're just gonna track our populations over time and they're going to essentially grow because there's gonna just be division of your donors and your division of your recipients over time. But at some point at this critical time point you're gonna have a conjugation event happen and you're gonna create this first trans-conjugation cell which is what I'm showing you right here. And this is going to happen at some critical density and gonna matter for what the donor recipient density is matters and then what that actual transmission rate between the transfer rate between the two cells is. And here I'm representing the trans-conjugation with a blue cell because that's the recipient but it got the red plasmid and then I just fill in the cell with purple just so you can easily track them in the presentation. All right, and then as we let the tube go we're adding two additional dynamics in our tube that are important. The trans-conjugations are now growing because that population, that third population has been created and importantly that third population now can donate its plasmid to the recipient like Yana was saying earlier. And so we have these five kind of key events that can happen in the tube as it incubates through time. And these dynamics that are occurring in the mating experiment were modeled using a set of differential equations by Bruce Levin and colleagues where each population type is a variable in the model. Yana kind of described this but the three populations have exponential growth and we're showing the growth rate parameter with this brown side parameter. And it's very importantly the same across all three populations in the original model. And then we also have trans-conjugates are being created due to plasmid transfer events into recipients and from donors. And those are happening with this transfer rate and gray again same for both types of events. Okay, so later on we got the Simonson method because they found an analytical solution to these sets of differential equations like Yana said and I will refer to this conjugation rate as the SIM method.  So we have an initial population size, a final population size and then importantly your three populations you need to get an estimate of their density at some critical point in time as well as the max growth rate, just like Yana said. Okay, but our method updates some of these assumptions. The first two are similar to Yana's. So I'm gonna go through them really quickly. We know that things grow at different rates especially if they're different species like if I grow my Klebsiella in the lab and my Echoli in the lab my Klebsiella grows a bit faster at least for me, quite a bit faster. So we care about that. So we update the growth rate parameters and we allow there to be population specific growth rates and I'm showing you that with the red, blue and purple side parameters. We do the same things with the conjugation rates especially because Yana showed some good data that within species conjugation rates can be quite different between cross-species conjugation rates. So we have these population specific transfer parameters in our model. And this is the diversion with other previous estimates is that we use a stochastic branching process framework to derive our metric rather than a deterministic approach which is what other methods use. And we incorporate the fact that conjugation is a random process in your populations and this is the case with mutation, right? So let's make an analogy to that because it's a bit easier to understand. So we're gonna track a population growing through time. We have a wild type cell and each cell when it divides there's a probability that a mutation is going to occur. And for this population, the first mutant arose here and I'm highlighting that with purple halo and then this also goes through clonal expansion and those mutants divide through time. But the important aspect is stochasticity. So if we ran a few populations in parallel for a short amount of time, what we're gonna see is that some mutations some populations do not acquire mutations within that timeframe and other populations do. And this heterogeneity across different population is kind of highlighting the random aspect of mutation and what's kind of the classic key observation or key intuition that the Luria-Dellpark approach has and what they actually use to estimate mutation rate. What's really cool is conjugation rate is conjugation events are similar. They're also transforming the genetic state of the cell because you're taking a recipient and you're adding a new DNA element. So it's a type of genetic transformation. But instead of tracking just the blue population we also need to track this exponentially growing donor population through time. And that's kind of like the difference between mutation and conjugation. There's these two population types that are particularly important. But when you can also look at mating assays, those pretend they're all like nine mating assays in the lab and you can see that some of them if you run them for a shorter period of time will not have a conjugation event occur. So no trans conjugates are present. And then you have some populations that do have trans conjugates. And this is really similar to the mutation process. So using this stochastic branching process framework and the updated model, we derived a new estimate for the conjugation rate which we call the LDM. And it stands for Luria-Dellpark method because we were really inspired by their approach to estimating mutation rate. And the important distinction with the LDM is you do not need an estimate for the trans conjugate density. This has a lot of advantages when you're doing lab work and I'm really happy to talk about, go into that in detail, but instead I'm gonna kind of highlight what you do actually need to measure for the LDM. So you need an initial estimate of the donor recipient densities and a final estimate of the donor recipient densities, but there is no trans conjugate density estimate. Instead we have the P naught right here. And P naught is actually the probability of having zero trans conjugates. The easiest way to think about it is that what's the probability of getting one of these gray squares? So the probability of no population that has no trans conjugates. And the maximum likelihood estimate of that is actually just the proportion of populations within this array that don't have trans conjugates.  You're gonna allow them to grow and incubate for a short amount of time and allow transfer to occur. But transfer importantly, because you're only running it for a short amount of time will only occur in some of the populations and not in others, which I'm showing you here, one population with trans conjugates and one without. And then you add trans conjugates selecting medium to your plate essentially dilutes everything. So you can kind of like, you're like stopping conjugation because you're like diluting everything down and so making things less dense and you're adding things that are going to inhibit the growth of the donors and recipients. And so anything that has trans conjugates with this new medium added will grow up to a turbid culture. And then after this incubation you're gonna be able to identify the populations that had trans conjugates and then the populations that didn't. And what's great about that is you just look at your plate by eye and you're like, count the number of wells that are not turbid and you just make a fraction and you say, okay, here there was 31 populations that were not turbid out of 96 and that gives you your P-naught estimate which can be used in the equation I showed you before. Okay, so that's great. We have a new theoretical framework, new analytical estimate and then a laboratory assay. So how accurate is malaria-delbrach method compared to other approaches? That's a good question to ask whenever you make a method, right? So I'm gonna show you some stochastic simulations very briefly. And then my most exciting thing to show you if you were at Closet's Roundtable is that our new experimental data at the end. So I'm really looking forward to sharing that with all of you. So the simulation, so briefly we just run, here I'm showing on the Latin deterministic simulation. So you just set some initial donor and recipient densities, you pick some conjugation rate and growth parameters and you run a deterministic simulation. This is what other people assume in their methods is that things happen at this like average deterministic, just means like your average trajectory that you would get. But we made a stochastic framework because that is similar to how we actually, what we do in our theory, you allow things to be random. So what you do when you run it, one of the stochastic simulations is that you're gonna get a trans-conjugate trajectory that deviates from the average. And that could be because the first trans-conjugate showed up later in time than the average expectation. If you ran another simulation, it might show up earlier in time as I'm showing here. And so what we do is we use the simulated populations to calculate the conjugation rate using both the LVM and the SAM equations. In the paper, there's actually other metrics that we use and that can be found in the supplement if you're interested in comparison with other metrics that are around. But I will reiterate here something really important which is that again, the difference between the two equations mainly is that you need a trans-conjugate density to have. You need to have a trans-conjugate density that's not zero when you do Simonson. And for ours, you need to have proportions of your populations that do and don't have trans-conjugates. And so the timeframe at which you can assay these to do these metrics are different. The LVM can be assayed where you have a proportional population without which is gonna be around this timeframe. And then Simonson needs to happen after you already have trans-conjugates showing up. And that's gonna be an important thing to keep in mind when you interpret the simulation results. So for the sake of time, I'm just gonna show you one simulation scenario that is particularly relevant to me because I'm interested in microbial communities. And so here I'm showing you where I set the cross species conjugation rate to be much lower than the within species conjugation rate. And what we're trying to estimate is gamma D, which happens to be the cross species conjugation rate in your assay. So the one that's happening between donors and recipients, not the rate that's happening between your trans-conjugates and your recipients. So we set that, we set a conjugation rate, which I'm showing you here. And then we're gonna use our simulated populations to look at our two metrics. What you can see with the LVM is that it's accurate over time, which is great. And then what you see with Simonson is that you get increasing inaccuracy as incubation time increases. The intuition for this is that, remember those three phases I told you about in the mating assay where you only have donor and recipient growth, you have that conjugation event occur which creates that third population type. And then there's the phase where trans-conjugates are present and you're having trans-conjugate growth and trans-conjugate conjugation occurring. The longer you incubate, the longer the trans-conjugate event types are gonna contribute to the accumulation of that population. And that's essentially why you're getting over, you're getting inflation and overestimation of the donor conjugation rate through time. Okay, so let's move on to our laboratory experiment. So I'm gonna show you a cross-species case study where I set up mating assays in the lab between Klebsiella and E. coli and with an in-gef conjugated plasma. And here on the x-axis, I'm gonna show you what I got for the LVM method side by side with the Simonson metric in the lab and then our estimates on the y. And this is what we got. And so you can see there's a huge discrepancy between the LVM estimate and the Simonson method where the Simonson estimate is a few orders of magnitude. It's quite shocking, above what the LVM got. And so there's some inaccuracy seeping into here. But one thing that could explain the overestimation, I kind of set you up for it with the simulation, is to think about that, what is the within-species conjugation rate? So if the trans-conjugate conjugation rate or the within-species conjugation rate, so with an E. coli is much higher than the cross-species conjugation rate, our simulation results suggest that that could potentially be making the Simonson method overestimated. And so what I did is I took the LVM metric and measured the within-species conjugation rate to see if it's higher. And in fact, it is by a lot. It's about six orders of magnitude higher with any coli than I got when I did my cross-species conjugation rate estimate with ClpCl2 coli. And so notably, this supported my hypothesis that Simonson was overestimating the cross-species conjugation rate due to potentially a higher within-species conjugation rate. But I did one more test to kind of get some more supporting evidence for my hypothesis. And my test came from the intuition that I started to get from the simulations, which is that what happens with incubation time is you get more and more overestimated. So I ran, this was a traditional Simonson assay. And the traditional Simonson assay includes a 24-hour incubation time. And so what I did is I ran a truncated Simonson, so I shortened the incubation time a lot to see if that would result in the estimate of Simonson coming down and getting closer to what the LVM gave me. And in fact, we did see that with our truncated assay that the Simonson estimate came down. And that was our second piece of experimental evidence that supported our hypothesis that the Sim estimate was inaccurate due to the asymmetry between cross-species and within-species conjugation rates. So I was pretty excited about this data. So just to wrap up in conclusion, so we developed this new LVM method and we think it really offers some new possibilities to be able to accurately measure conjugation rate between many types of plasmids, species, and in fact, many different environmental conditions that we might be interested in. Particularly what Yana was talking about where we're kind of need to understand how temperature and different biotic or abiotic factors actually affect these rates. And we think our method offers the possibility to look at some of those things and accurately. So here I showed you, we derived a new estimate. We checked accuracy with and precision with our stochastic simulations. And then we applied our laboratory protocol to a cross-species case study alongside what has been kind of the longest running gold standard approach Simonson for some decades now. And I want to just make a plug, an important plug that I think the most important thing that I learned with this project is check the assumptions of your model when you're running, when you're trying to estimate things and check the assumptions in the laboratory too. Because there's things that can bring bias into your estimate because of how you're running things in the lab, are you running them correctly? Are you getting things into a constant growth rate, exponential growth? Are you using a well-mixed system? And are you executing it in a line with how the model actually is telling you that it should be ran? I'm really happy to go into all the assumptions that I think should be checked because I have definitely seen that if you violate any of these assumptions in the laboratory when you execute it, it will give you quite different values for your transfer rate, which could be really misleading, especially when you're trying to compare things in the lab. Does this have higher conjugation rate than that? Well, it's not a systematic deviation and potentially if you're violating an assumption, it's not going to just systematically elevate or lower. It's going to maybe change the rank order of your conjugation rates. It might be giving you the wrong answer. So just kind of a cautionary note and something that I've noticed of being like, wow, if you do one thing, even a little bit different, it can give you quite different estimates, which is like, okay, we've got to be really careful. And another analogy I like to make that I feel like kind of communicates how important the laboratory execution is, is like the rigor we apply to RTQPCR, right? When you do RTQPCR, you know that you need to check the efficiency of your primaries. You need to have more than one reference gene. You need to do a concentration gradient of your DNA. And so when we're looking at how to execute those protocols, we have very stringent criteria that we follow and I think measuring conjugation rate needs to move sort of in that direction so that we can be really confident in the estimates we're actually getting in the lab. And with that, I really want to thank everybody on the team being my sounding boards with all of my questions and problems I ran into along the way. Thanks to my funding sources and thank you all for hanging on and listening and thanks to organizers for a great week. And I'm really happy to discuss anything that I talked about today. Yay, this was great.


I wish to acknowledge and show my respect to our elders past visit and emerging and thank you for inviting myself to perform the work in the country. I'd also like to extend a special welcome to the Marines who have travelled to Larakia country to be with us today and wish you all the best while you are here and also a safe passage in your journey when it's time to go home. So it's just important today as it has been in the past that we endeavor to safeguard and preserve our way of life, our land and our democracy. We have fought shoulder to shoulder and we have contributed blood and treasure in every conflict since we've started with our Australian partners. It was extremely well said. What you do here today is really important. This is about preventing conflict. That said, if deterrence fails, we will be ready to respond to fight and win and we can only do it together. So I'm honored to be here. I thank you very much for your efforts. Thank you. They applaud the most praise and have some fun. It was all really good. How about you? Afghanistan 2019-2020. Glad you're back safe. Well done. A lot of challenges now. They're moving out of the desert and they're out here in the Pacific. Yeah. So we all got to be ready. Did you bring him here or not? We've got an artillery battery left. Thanks for what you're doing. Be careful, all right? Make sure you get home to mom, dad, and the kids. But enjoy yourself. Thanks for letting me come out and spend some time with you. Thank you. All right. Hey, hello. How are you doing? It's all dressed up for me. You got it? Not too much. All right. Well, free challenging theater. Having Marines and Australians all to prevent a war. Not to happen. All right.  You're working with the best partners we have. All to protect our way of life. So thanks for what you've done. See you around there. All right. Do it yourself.