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Stevan Spremo Talks About Boxing Up Bacteria for Space Travel

Season 1Nov 3, 2017

A conversation with Stevan Spremo, Space Flight Project Manager at NASA’s Ames Research Center in Silicon Valley.

Stevan Spremo

Transcript

Stevan Spremo

Host (Matthew Buffington): Welcome to the NISV podcast, this is episode 66. And sitting with me again I have Abby. Abby, tell us about our guest today.

Abby Tabor:Hey, Matt. Today we’re talking with Stevan Spremo. He works in the Chief Engineers office here at Ames, and basically his job is to guide other engineers through the development of hardware that’s going to go into space, and carry life sciences missions, for example. So Stevan started as an electrical engineering student at San Jose State [University], but he knew he wanted to work with medical technologies or life sciences, which is cool. And so he found the perfect role for that here. And he’s worked on cancer research that’s flown on the space shuttle, and studying how plants grow in space, and then coming right up in November, he has a new mission launching, it’s a small sat called EcAMSat, which he’ll explain. But this one’s very cool, it’s about studying how bacteria are resistant to our antibiotics, and whether that’s worse in space, and how it all works to make things better for astronaut health, and also for public health here on Earth.

Host:It’s always the thing I get a kick out of. Everybody thinks of NASA, they think of rockets, they think of telescopes looking out. But there’s a huge biology aspect. And not just the astrobiology of looking out into the stars and trying to find life…

Abby Tabor:No, but here.

Host:…but understanding biology. And it’s like this really neat overlap. Before we go too far into it, just a reminder, if you want to give comments, participate, give any kind of feedback, we have a phone number, it’s 650-604-1400. But if you want to do it the new hip way, we’re using the hashtag #NASASiliconValley, we’re on all the social media platforms that you can think of. So without any further delay, here is Stevan Spremo.

[Music]

Host:Welcome, Stevan. [Laughs] So we always start this off the same way. Tell us a little bit about yourself. How did you get to NASA? How did you get to Silicon Valley?

Stevan Spremo: Thanks for having me. I got to NASA – the story is when I was very young in third grade, my brother and I were writing to NASA to try to get like public outreach-type photos of the space shuttle or the planets and things like that. And we actually got responses, which was really cool. So that gained my interest when I was really young. And then through high school I had more interest as I learned chemistry and physics and went through all my classes. And I just really had this desire to come work for NASA. I really wanted to be an astronaut and went through college, and I got an internship in 1998 –

Host: Awesome. Yeah.

Stevan Spremo:– at NASA Ames here.

Host: Oh really. So were you already local so you already knew about Ames, or did you come from somewhere else?

Stevan Spremo:Yeah, I’m local from the Bay Area here in the Silicon Valley. And I went to San Jose State and was an electrical engineering student. And in 1998 I joined a group called Sensors 2000. I was doing life sciences, biological sensors. So I was an electrical engineer, and I had this focus that I wanted to combine electrical engineering with medical technology or life sciences experiments. And I thought there was a real future to that. And it was my future. And I started working on space experiments that included biological sensors, so –

Host: Okay. So it was like – so as a kid you were like, this is NASA. This is what I want to do. And then that kind of helped shape like what you studied.

Stevan Spremo:Yeah. I mean, I had this very specific focus of what I thought I needed to study. So like in college I had the electrical engineering courses, but I took classes like zoology and extra chemistry that biologists would take. So I really kind of started learning the language of scientists as well. And basically my career has been listening to requirements of scientists on what they need to study in space, and then I help design it.

Host: One of the things that Ames – bioscience studies, it’s like the experiments, the science experiments that actually go up into the International Space Station or went on the shuttle. So did you already know that that was a thing that Ames was already doing, or is it just happened to be that what you were interested in matched with what was already here?

Stevan Spremo:I had some knowledge that Ames was involved in life sciences. We had a friend of the family that had been working on experiments at NASA. And so I did have some knowledge.

Host: You had the inside track. You knew what people were already doing, a little bit.

Stevan Spremo:A little bit, yeah.

Host: And then when you did internship, did you just like go online? Did you know somebody? How did that work out for you?

Stevan Spremo:When I joined here, I did have someone who introduced me to the whole internship program. But it wasn’t like a direct in. So I interviewed with I think it was – I don’t have a count – but maybe like 15 managers.

Host: Really. That’s –

Stevan Spremo:And so what happened is I kind of had this very specific goal or dream of what I wanted to do. And they said, “Well, you really need to meet with this one specific manager.” And he was out on medical leave for a number of months. So what they kind of did was interview me and see if there were a number of different positions onsite that I would fit with. But then they kept saying, “You belong in the Sensors 2000 group.” And fortunately, they decided to take me on as an intern. And then they converted me to a full-time civil servant later. And then I got my position.

So I was a co-op student, which is a civil servant student, while I was going to school at San Jose State in electrical engineering.

Host: Yeah. Nowadays they’ve replaced it with what’s called the Pathways program. But before it was like they had different variations of co-op where people would be able to go to school and also work at the same time, or some mix of that, and then basically get into the civil service afterwards.

Stevan Spremo:Yeah. That’s – I started out as a civil servant from day one as a student. So all that time counted towards my –

Host: Oh that’s awesome.

Stevan Spremo:– retirement and all those things. So we were getting those benefits first – civil service.

Host: So when you come on while you’re working in this group, what is some of the stuff that you’re working on initially?

Stevan Spremo:So I worked on these electrochemical sensors. When I came to NASA, I was actually starting to run a wet chemistry lab, which was a little unusual being an electrical engineering student.

Host: I was going say, yeah.

Stevan Spremo:And so what I was building was electrochemical sensors to measure metabolic changes for cancer cells that are growing. So we look at pH changes in cancer cells. And the whole system that we were working on was an automated system that would sustain life for C6 neuroblastoma cells, brain cancer cells.

Host: Okay [laughs]. I was like thank you for clarifying [laughs].

Stevan Spremo:And so we were – the idea is to fly this up on the space shuttle on STS-93, which was a Columbia mission. In 1999 it launched. And we worked with the Army, Walter Reed Army Institute of Research. And the sensors were direct inline measurements with the cells. And so, as in microgravity environment changed, we were looking to see if cancer grew differently in space. And so the sensors I developed, I built like 150 sensors, and I think 16 of them flew to space –

Host: Oh wow.

Stevan Spremo:– and made these detections. So we had spares and extras in selecting the best of the group. But they were from scratch. We literally built them a hundred percent in the lab and then interfaced them with an electrical system that read it out, stored it, and then we retrieved the data when it came back from space.

Host: Crazy. Because like oftentimes when you think of like, cancer research, NASA isn’t the first thing that pops into your brain. So – but in the end, it’s like understanding how things grow with little to no gravity helps you to better understand how those things operate. And as you can understand them, you can better fight them or cure them or move them along.

Stevan Spremo:Right. Yeah. It was kind of an amazing opportunity to merge technologies and then benefit this cancer research, so –.

Host: And so even thinking about those sensors, I’m trying to think. So what does that exactly look like? Is it like a petri dish or some thing with the actual cells in it, and then you have your sensors that like kind of read it? What kind of changes are you looking at?

Stevan Spremo:So there were fluidic loops, so it was called Biona-C. And there’d be hollow fiber bioreactors that would grow the cells. And then there were media that was fed to the cells so it sustained life. And that would circulate through with a pump. And then from time to time we would take measurements or draws of fluid off from the sample where the bioreactor was, measure the H+ ion content or pH, and then read that out to a circuit card, and then store the data.

And then we’d see trends to see how things were growing in space versus on the ground. So we had the identical system on the ground as we did in space, looking for a difference in any kind of metabolic activity. Do the cells do something different in space or not? And so that was the experiment. And it was kind of a technology demonstration. The Army was also interested for their own aspects of research, too.

Host: So moving along from your work, then what did that eventually move into, what other things did you work on until you landed where you are now?

Stevan Spremo:Right. So the next experiment I worked on was a space station experiment. It was called European Modular Cultivation System.

Host: Okay, and [laughs]. You can – yes, tell me about that [laughs].

Stevan Spremo:So that EMCS was a – it’s a centrifuge system that’s up on space station right now. And we designed some cartridges that basically plug into that system. And we were growing plants – plant seedlings – in space, so

Host: Okay.

Stevan Spremo:– we were studying Arabidopsis thaliana seedlings, in which we were doing a phototropic response.

Host: Okay.

Stevan Spremo:And so basically, we would induce light and shine light on these while we were rotating it one-third gravity levels, which is the equivalent of Mars – would be the equivalent of Mars, one-sixth g which is equivalent of the Moon. And then microgravity levels, basically looking at how plants grow in space. There’re basically photoreceptors on plants that activate different responses. And because in microgravity the plants grow more in a tangled ball or confused state.

Host: Yeah.

Stevan Spremo:They don’t grow as well. So we were studying how do we, engineering-wise, alter that by having light affect these photoreceptors. And actually we can change how the roots actually grow –

Host: Okay.

Stevan Spremo:– and how the green, leafy portion or cotyledon portion would also grow. And with the altered state, we look at the RNA analysis for the genetic aspects, of what was going on, to study how to go to the Moon and Mars eventually. So that was another experiment I worked on. I specifically worked on the optics to make sure that the light was basically equally being distributed across –

Host: Yeah.

Stevan Spremo:– all the plants, and a number of other circuit-based designs that were supporting that. And then also making sure it’s biocompatible, because when you lock everything in a chamber there’re other gasses or volatiles that come off from the circuit boards or other things.

Host: All variations.

Stevan Spremo:And it can cause the biology to die.

Host: To – oh.

Stevan Spremo:And so we had to do other things to actually make sure that the air inside the chamber was basically clean enough that it wouldn’t extend the life of these systems.

Host: And I guess that makes sense because if you think of plants, which have evolved over millions of years with gravity pulling down on them, and then just seeing how once you remove that gravity, you change it to the Earth or Moon, different levels, I mean, it helps us to understand if we’re planning on eventually going to Mars and doing things that – to understand how those plants react. But that’s smart of thinking, like you’ve seen plants move to get closer to the sunlight. They kind of grow in those ways. So using that to manipulate it to change the way it grows is pretty neat.

Stevan Spremo:Yeah. So there’re two responses that we were studying: There’s gravitropic –

Host: Yeah.

Stevan Spremo:– and phototropic. So the light, I guess, from what the botanists are telling me, there’s a photoreceptor. And we were studying putting blue light and red light on the roots. And actually you can make them go away or toward those lights.

Host: Really. You can help control them.

Stevan Spremo:Yeah. So that – in addition to the white light that’s –

Host: Yeah.

Stevan Spremo:– kind of making the green, leafy portion, assimilating the sun. And also the roots actually do very specific things with light as well.

Host: No, I get a kick out of it because typically when people think of NASA, you think of rockets and astronauts. But at the same time, it’s like, yay, you’re in space, or you’re on the Moon, or you’re on Mars. At the end of the day, what are you going to do there?

Stevan Spremo:Yeah.

Host: So this is what these science experiments – there’re questions, there’re hypotheses to figure out, okay, what can we learn by being in these places that we can’t learn from Earth, and kind of working out those theories. So what are you working on now? What’s kind of like your day job?

Stevan Spremo:So I’m in the Chief Engineer’s office here –

Host: Okay.

Stevan Spremo:– at NASA Ames, which there’s a number of things I do in that role. I’ve been at NASA 18 years now, and I get called in if there’s maybe a problem on –

Host: Mm-hmm.

Stevan Spremo:– hardware development that can’t be figured out, or there was a mishap, something went wrong. I’m trying to figure out lessons learned, like why did we have something go wrong in the first place and identifying root cause. So also there’s a number of standards and procedures, like it’s almost like a prescription. Before you start a project, like how do you formulate it to, I don’t know, it’s not to guarantee success, but to increase chances of success.

Host: Yeah.

Stevan Spremo:So that’s kind of my role is to work with engineers and guide them and put some, I guess, milestones or gates to do checks.

Host: A checklist, of sorts.

Stevan Spremo:Yeah, it’s like the equivalent of a checklist to make sure that you’ve completed a number of tasks that would help in the reliability of a system.

Host: Well, you figure if something’s going to go wrong, you’d rather it go wrong here at Ames where you’re like – you’re working on experiments. Like [you’d] rather that it go wrong here while we’re practicing as opposed to while it’s in space. So it’s kind of, learn some of those lessons.

Stevan Spremo:Right. Absolutely. So we take things to the test chambers here – the vacuum chamber or the vibration shake table. And we simulate all the things that might go on in space to try to basically mitigate – or make sure that doesn’t happen in space, so an astronaut is not experiencing a piece of gear that is failing for any reason. So yes, that’s kind of what –

Host: [Laughs] Tested in advance. “That’s kind of what I do.”

Stevan Spremo:Yeah.

Host: Because I always think about it of not only being in space or surviving like the vacuum or harsh conditions, but it’s like you’ve also got to survive a rocket launch so that with the very intense moments where you don’t want your science experiment to fall apart [laughs] on its way up.

Stevan Spremo:Right. There’re basically 10 to 15 minutes that it’s a pretty harsh environment going up and through different stages of the mission of firing a rocket engine. There’re vibrations and there’re other environmental effects.

Host: Acoustics.

Stevan Spremo:Acoustics, and absolutely. And just the change to vacuum as well once you get in the vacuum of space and the thermal extremes, and so after you’re past that launch phase. But yeah, there’re a number of environments we test for, yes. So –

Host: So okay. So now I’ve heard of one of the things that you’re working on called EcAMSat. So that sounds like a fancy acronym.

Stevan Spremo:Yeah.

Host: Tell us a little bit about what that is.

Stevan Spremo:So it’s – EcAMSat stands for E-coli Antimicrobial Satellite.

Host: Okay.

Stevan Spremo:And so what we’re studying on this — it’s a CubeSat. So it’s a 6U spacecraft. 6U is like a standard – it’s basically roughly a shoebox size –

Host: Yeah.

Stevan Spremo:– like a large shoebox.

Host: Or a loaf of bread or something. It kind of –

Stevan Spremo:Yeah, this particular one is kind of like two loaves of bread –

Host: Okay.

Stevan Spremo:– in size as a good comparison. And we’re studying antibiotic resistance in space, which is –

Host: Okay.

Stevan Spremo:– a really big problem on the ground, as well as may impact future travel for astronauts in the future. And what we are finding out through a number of other experiments that have gone up – and this will help validate what we’re learning – is that E. coli or yeast or a number of bacteria are more virulent in space. They actually grow at a rapid pace.

Host: Oh really. They’re like stronger in space.

Stevan Spremo:They’re stronger.

Host: Oh wow.

Stevan Spremo:And the effects are – this was an unexpected –

Host: Yeah.

Stevan Spremo:– outcome. And so there’s a parallel between the ground and what we live – are experiencing on the Earth, antibiotic resistance, and trying to figure out what mechanism’s causing that in space.

Host: Okay.

Stevan Spremo: So EcAMSat is going into a microgravity environment, taking a 48-well plate microfluidics array. Basically each one of those –

Host: Okay, yeah.

Stevan Spremo:So it’s a fluidics card that has milliliters in scale –

Host: Okay.

Stevan Spremo:– of fluid going through it. So imagine – the best way I’ve been able to explain the volume of each one of these little cells is like an eraser head. So imagine 48 eraser head, like a pencil eraser, worth of volume on a card. And we flow through different antibiotic strains. So we grow up the cells. So we put it in hibernation before launch.

Host: Okay.

Stevan Spremo:And it sits on the pad. It’s in hibernation. We get up to space –

Host: It launches. It goes to the space station.

Stevan Spremo:And then when it gets up to space, it has a deployer, a dispenser.

Host: Okay.

Stevan Spremo:And then a door opens. There’s a container it rides up in. And after the primary satellite is gone and we can do no harm to it, the doors open and we eject this out with a spring pusher foot.

Host: Okay.

Stevan Spremo:And then it kind of has a tumbling effect.

Host: Yeah.

Stevan Spremo:And it has – it’s got a passive system to align with a magnetic field and knoll out this and stabilize.

Host: Figure out where it is

Stevan Spremo:And that takes about four days. After we’re stable and the microgravity environment is the best that it can be, the experiment starts. And for 150 hours we go through a number of events. And we feed the cells. They grow up to what we say stationary phase. And they’ve eaten all the sugars that we feed them –

Host: Okay.

Stevan Spremo:– is basically what happens. And then we have an optical detector that shines light through – red, green, blue. And the absorbance pattern is noticed on a photodetector below it.

Host: Okay.

Stevan Spremo:So we shine through the card and are able to look at how things are growing and see trends.

Host: Okay. So that’s how you know.

Stevan Spremo:And between the different color metric measurements, we can tell trends. And there’re things that happen in red, maybe not in blue.

Host: Okay.

Stevan Spremo:And so we calibrate that way. The other thing we do then is we administer antibiotic and stress the cells out.

Host: Okay.

Stevan Spremo:So different concentrations, there’s a response, okay? So you’re building the antibiotic resistance response. And in microgravity, there’s a wild type and a mutant we’re studying. And the principal investigator is looking at this – the scientist looking at this, Dr. A. C. Matin at Stanford [University], his hypothesis is that the two – the wild type and the mutant strains will stress at a different response rate.

Host: Okay.

Stevan Spremo:And then we can look at that and try to get the genetic marker to kind of explain what’s going on, why things are becoming more resistant.

Host: Okay.

Stevan Spremo:And then we basically look at the trend after that, administering something called alamarBlue.

Host: Okay.

Stevan Spremo:And alamarBlue is like a dye that it kind of changes color as the cells metabolize. So we can look at trends and curves of how the cells continue to live on and the way they intersect. And the graphs or the curve fit of the trends will tell us if, in comparison with the same exact experiment on the ground, if the space environment is responsible for doing something different –

Host: Okay.

Stevan Spremo:– like microgravity or radiation versus a 1 g environment on the ground.

Host: Yeah. It’s crazy because you think, obviously, as we look at like human exploration or having people in the Space Station, it behooves us to understand how [laughs] bacteria or E. coli, how things grow differently in microgravity. And having all of this, just seeing all the differences and understanding that better can then prevent or just – it’s not like only could it have benefits for us here on the Earth, but also help for that further exploration as well.

Stevan Spremo:Yeah, the idea here is this is decade – what we call decadal science.

Host: Okay.

Stevan Spremo:So it’s a decadal survey of what we need to do further space exploration.

Host: Oh, okay.

Stevan Spremo:So this supports astronaut health for long-duration exploration missions. So we have to understand this as kind of a keyway to the future.

Host: Yes.

Stevan Spremo:How would we administer antibiotics to an astronaut is really what the question is here. But there’s a secondary purpose on the ground is, are we going to discover something in space that could help antibiotic resistance issues on the ground. And that’s becoming a really large problem for terrestrial or Earth aspects of antibiotics.

Host: I like whenever they’re talking about the International Space Station, they always say, “Working off of the Earth, for the Earth,” because this all has benefits not only for going on the way to Mars – and this is – you’re talking about checklists before. This is one of those things you need to understand before doing that journey to Mars. But then the side effects can be finding out how to solve other problems here on Earth as well.

Stevan Spremo:Yeah, absolutely. In this case, we might need to have the astronauts having a dosing of like four, five, 10 times whatever the amount is. But also if we find a pathway of how antibiotic resistance, what the mechanism is, that’s something that might be a game-changer. One thing I didn’t mention is after the experiment’s done –

Host: Yeah.

Stevan Spremo:– we store all this data, and then we telemeter that back down to Earth.

Host: I was going to say. So the SmallSat hitches a ride on a rocket where somebody else has paid more money to go [laughs]. Once that goes off, it’s safe, you launch yours – start that science experiment. And yeah, is it just they – sends that data back to you guys here.

Stevan Spremo:Yeah. So we have a great program that basically we work with Santa Clara University, and they have a ground station. And we have two antennas on our system. And what happens is the electronics store the data as we’re going through the experiment – all this light measurement and –

Host: And you’re controlling all that from the ground. It’s up there? Or is it automated now?

Stevan Spremo:It’s actually automated.

Host: Oh wow.

Stevan Spremo:It will run on its own. And then we call it and ask for requests for the data through –

Host: [Laughs] Okay.

Stevan Spremo:– Santa Clara University. And it’s a great outreach thing because the students there are operating the satellite for us.

Host: Oh, that’s awesome.

Stevan Spremo:And they retrieve the data and deliver it to NASA. So it’s rough – it’s not a lot of data. It’s like a megabyte of data –

Host: Okay.

Stevan Spremo:– for the whole mission. It takes about two months through the orbits and everything that we do to get that small amount of data. We don’t have – this comparatively to the LADEE mission I worked on –

Host: Yeah.

Stevan Spremo:– we had major breakthrough on laser communications.

Host: And that went to the Moon.

Stevan Spremo:That went to the Moon. And that had 622 megabit-per-second download rate –

Host: Oh wow.

Stevan Spremo:– from the Moon per second.

Host: [Laughs]

Stevan Spremo:So it was like a DVD a second versus this.

Host: It’s smaller, but –

Stevan Spremo:It’s smaller, but you don’t need that much horsepower to this because the data file’s not very big.

Host: Okay.

Stevan Spremo:So it’s scalable. And what a CubeSat does is pretty interesting because it only operates off three to 10 watts of power.

Host: Yeah.

Stevan Spremo:If you think about your old incandescent lightbulb that you grew up with in probably your house or – this is a fraction of that –

Host: Yeah.

Stevan Spremo:– what it’s consuming, and doing all these tasks and reporting home and delivering a whole experiment that’s been automated. And I always find that aspect really amazing.

Host: Like being more compact, being easier, being like a small sat, using that CubeSat kind of like modules, it makes it cheaper. It’s easier to do. If something goes wrong, it’s like I’m sure replacing it isn’t the end of the world.

Stevan Spremo:The other thing that I didn’t mention earlier is the temperature requirements. We maintain –

Host: Oh yeah.

Stevan Spremo:– this – we have to simulate the body’s temperature. And so –

Host: And you’re doing that in space [laughs].

Stevan Spremo:Doing it in space. So 37 degrees Centigrade is normal human body temperature.

Host: Okay.

Stevan Spremo:So we’re simulating that. If we go above that, we’ll simulate a fever and ruin the experiment and kill off the E. coli.

Host: And how do you get that temperature while on the satellite that’s in space where it’s pretty cold, as I understand?

Stevan Spremo:Right. So we designed this so orbit after orbit, it’s capable of maintaining 37 degrees plus or minus a half degree Centigrade.

Host: That’s crazy.

Stevan Spremo:So we have looked at models, and we model this to – basically dynamically every orbit – maintain its temperature. We do models that are upwards of 650,000 calculations to look at all the situations that thermally it’s still stable. And so that’s really an engineering feat that this system –

Host: Oh, crazy.

Stevan Spremo:– is maintaining. And as a CubeSat, being low-cost, and we’re still achieving this requirement is kind of amazing. It’s taking a lab up to space and –

Host: Uh-huh. Like a mini-lab. Automated, too.

Stevan Spremo:Right. It’s automated. I think another thing I didn’t mention is that we take a little canister that’s got one atmosphere. It’s got lab air in it.

Host: Okay.

Stevan Spremo:So that’s something the cells also need to have to simulate the environment, so –

Host: And after the experiment’s run its course, you’ve got all your data. Then it just burns up in the atmosphere? Or how does that work?

Stevan Spremo:Yeah. Since it’s a low-Earth orbit system, we maintain orbits generally that are less than 25 years in life. And then just natural decay of atmospheric drag around the Earth, it will eventually pull down into the Earth’s atmosphere and literally vaporize.

Host: Vaporize.

Stevan Spremo:Yeah.

Host: So I can imagine somebody thinking, “You’re sending E. coli into space! What if this crashes on my house?” It’s like, “No, it’s never to get even close to that. It’ll get burned up and vaporized long before you even know it.”

Stevan Spremo:Yeah, so we get that reaction a lot.

Host: [Laughs]

Stevan Spremo:“What are you doing sending E. coli up to space? Is this a dangerous thing?” And –

Host: You probably have more in your bathroom [laughs] or on the doorknobs than you do in this –

Stevan Spremo:The particular strain we were sending up, it’s a common strain that actually people are treated for regularly.

Host: Okay, okay.

Stevan Spremo:So antibiotics and everything are regularly given to patients on Earth for this particular strain we’re studying.

Host: This is pretty – it’s a normal one. People can calm down. Anyway, it’s going to burn up in the atmosphere.

Stevan Spremo:Yeah, it’s going to – it’s never going to reach back down to the Earth, yeah.

Host: Anybody who’s got questions for Stevan, we are using Twitter, so we’re @NASAAmes, and we are using the hashtag #NASASiliconValley. So if anybody has questions, we’ll just push them on over to you [laughs] and he’ll respond.

Stevan Spremo:I’ll be glad to respond to anybody’s questions.

Host: Excellent. Well, thanks for coming over.

Stevan Spremo:All right. Thank you.

[END]