It's a pleasure to introduce Professor Taikjib Ha, who comes to us from Johns Hopkins today. He's been at University of Urbana-Champaign for 15 years from 2000 to 2015. Before that, he was at Berkeley, where he did his PhD, with the leaders in the field of bringing physics to biology, working with Raymond Genleau's, Steven Chu, Simon Weiss, etc. And before that, he was at Seoul National University, where he did his undergraduate degree in physics. So he's a physicist, a Bonafide physicist, who's come over to biology and has brought with him a variety of concepts, approaches from physics to biology, and now has been able to address some of those mysterious things like helicases and other enzymes that act on genomes, complexes that function within cells, building ever-most sophisticated tools to actually look at functioning molecules in the milieu within which they function. For doing this, pioneering this frontier along the way, he has won every possible award that you can imagine, starting from Seoul scholars, Sloan, early days, to the Ho-Am Prize, which is considered the equivalent of an early Nobel Prize that is given to Korean American scientists, to this year, he was elected to the National Academy of Sciences. We noticed his work early when he started talking about DNA structures and their plasticity, right from the days of early papers, about 200 papers ago, 197th paper, that talked about Stephen Chu, by the way, who's the energy secretary also, has done enormous amount of work in single molecule studies, bringing physics against biology. So how DNA bends, we had studied in school that DNA has a persistence length of about 500 nanometers, and here was someone who's claiming that DNA is far more plastic than that, far more mobile, far more move-up-changeable than that. And since then, we've been following his work in every paper. This year, I believe he's had three or four papers in science, cell, et cetera, already. And I'm looking forward to his stories today, and hopefully he'll continue and collaborate with small molecules that we've been developing in the lab. With that, let me not continue further and hand over the stage to TJ Ha, as he likes to be called. Thank you for coming. Thank you very much for the kind introduction and invitation. Does it work? So I'm between jobs, so it's nice to be invited to get some free food and housing. I'm a physicist by training, I forgot most of my physics and haven't learned enough biology to call myself a biologist. So to paraphrase a bridge in spears who's saying, I'm not a girl, not yet a woman. I'm not a physicist, but not yet a biologist. So it's really fun to be here, lovely, beautiful campus and wonderful colleagues and students. I've learned a lot, thank you very much. Many years ago, Watson Creek published this paper proposing the structure of double helix. The paper ended with a paragraph saying that, well, it has not escaped a notice that the specific pairing suggests a possible copying mechanism for genetic material transfer. And they actually worried that this was not explicit enough, and that maybe someone else may take the credit for the discovery by making the claim more explicitly. So a month later, they published another paper proposing this base pairing, and then the rest became history. That paper ended with a paragraph, a lot to be learned, even if this is correct. For example, what makes the pair of chains unwind and separate when DNA forms a stable double helix? It took more than 20 years until people discovered a class of enzymes called helicases. Helicases for DNA molecules, DNA unminding enzymes, unzipping proteins. They can be also considered as motor proteins because they use ATP energy to power themselves on the DNA tract. In fact, if you give them a single-celled DNA, they will move on the single-celled DNA in a directional manner, 3 to 5 prime or 5 to 3 prime of the DNA. So you can call it a translocase. They function in many different processes inside a cell. They function in essentially every possible imaginable DNA RNA metabolic processes. They unwind DNA or RNA. They can remove proteins bound to nucleic acids. They can cause branch migration, highly resumption. They can do chromatin remodeling, RNA protein complex remodeling, and so on. And when they are not well, if you have mutations in some of these enzymes, you can get serious human genetic diseases. Some helicases form a ring of hexamers, and others are non-hexameric. There is some discussion whether they function as a monomer or dimer that I touched upon today. You know, there are DNA helicases and RNA helicases. And some 5 to 3 prime helicases, meaning that if you want to see unwinding of a DNA, you need to present the enzyme with a DNA with a 5 prime tail so that it can bind to the tail first and then translocate in the 5 to 3 prime direction to unwind the DNA. And vice versa for 3 to 5 prime helicase. Some people are so in love with these enzymes, so they pay additional money to the state to have a license plate like this, it unwinds. So you can actually, if you're not interested in the topic, you can just sit and unwind for the rest of the day here. So this is actually from Missouri, my long-time collaborator, Tim Newman, who has received his PhD in biochemistry here with time record. I am not, I'm too cheap for doing this. So you know, interesting questions are, you know, how do they function and how are their functions regulated? And for example, you know, if you switch, you know, how do you turn on and off the helicase functions when you need them and when you don't need them anymore, because if these enzymes float around inside a cell and unwind every DNA, RNA they see, it can be digested. You need to tightly regulate their functions and how do you achieve this? Okay, this is an important number because we do one molecule experiment, a single molecule experiment. So the main tool that we've been using is called a single molecule fret, where if you want to measure conformational changes of an enzyme, you put green and red dye molecules on the known site so that you can distinguish between closed and open conformations. In the closed conformation, when you excite the green molecule using your laser, energy is transferred to the red and you get red photons instead of green. In the open conformation, you get green photons instead. If you want to measure how quickly the butterfly flaps its wings in an ensemble, you must synchronize a reaction by convincing them to start from one conformation, one sitting on the leaf and then you can frighten them into taking off the leaf and perform relaxation measurements. But as you can imagine, in many cases what you measure is not the wing flapping frequency, but how long it takes for the butterfly to take off the leaf. Ideally, you want to measure this in real time, looking at a single enzyme as a function of time and measure the anti-correlated changes in red and green intensities so that you can deduce the flapping frequency of the wings and also amplitude of the motion. This can be done in many, many different labs in the world these days and can be used for studying amazing variety of different systems. You can also measure intermolecular changes using a single molecule threat. For example, if you have a helicase moving on a single-send DNA from 3 to 5 prime N, putting a green dye on the protein and a red dye on the destination end of the DNA, you can imagine seeing as a function of time, greens it is going down and red is going up due to increase in threat. This is not actual data, it's a powerful animation that I made, but our data is actually almost as pretty, so this is actual data. As a function of time, you see red signal is going up, green is going down, and the surprise here was that once it goes to the end of the track, it somehow goes back to the beginning and repeats the process many, many times. We did a lot of experiments of this kind to get at the physical mechanism of this process and also what it might mean biologically. We wrote up a manuscript and we were able to convince some people that they can publish this paper several years ago. And then I paid a lot of money to a local artist in Urbana-Champaign to draw a cover illustration. This is one example where this old man is sitting on the DNA and then reeling in the DNA loop and then kicking up a protein's bound to the DNA. And unfortunately they didn't take it and instead they picked a picture of a diabetic mouse, so it was very disappointing. It's okay, sometimes you win, sometimes you lose. Here's another picture that they didn't take. My student, Gia Park, is sitting on the DNA and then she's the one who did the work and then reeling in the DNA and then kicking up the asteroid's bound to the DNA. So I spent so much money on these cartoons, I have to show them somewhere, so that's why. Another very popular single-marking method is called optical tweezers and it's basically chopsticks made of light. So if you shine infrared laser in a tightly-focused spot, then a small particle can be sucked into the middle of the spot and then if you move the laser beam around, you can manipulate the position of the bead. If DNA is attached to the bead and then there's an enzyme sitting on the surface pulling on the DNA, you can play a tug-of-war and you can apply very tiny forces, about a peak on the level of forces and then measure the response mechanically using forces and also by measuring the DNA length as a function of time, it can go down to a single base-pill resolution as Stephen Block has shown. The idea of using stretching to extract information is not new and this is an old idea. In medieval times, people are stretching other people to extract information and asking questions such as, what is this? So because my lab was doing just fluorescence measurements for many, many years, I was really envious of people doing optical tweezers and getting this wonderful data and I am a man of many envies so I had this force envy and this I wanted to combine the optical tweezers with single molecule fluorescence and the idea is to measure conformational changes using fluorescence or FRED as a function of applied force and actually there have been many other attempts in this direction and some are listed here so I'm just telling you about our own version of this direction. So if you think about the optical tweezers, you are performing mechanical measurements but with your eyes closed like that but if you do purely fluorescence measurements, you have arms tied in the back but looking at the process with your eyes passively and the idea here is that by combining the two, you can sample the best of both worlds. So Michel Wang was kind enough to host my former post-Saxon Hong for a week, so he spent a week in Cornell University more than 10 years ago to learn about optical tweezers and then he came back and built an instrument combining single molecule FRED and optical trap and he was able to show that you can measure DNA junctions dynamics using fluorescence but as a function of force now he is on the faculty of Seoul National University and another student then continued on the same instrument Rubozo who is now a post-Saxon Hong at Harvard what he did was to work with team Romans group to pull on the single cell DNA wrapped around a protein called SSB and then when you begin with this state of a fully wrapped DNA where two dies are close to each other you get a high threat, may need red signal and then as you increase the force you will gradually peel the DNA of the protein surface giving rise to gradual decrease in threat that you can measure as a functional increasing force. So this is the actual data that he acquired so as a functional force you can increase a force showing gray from low to high value gradually you can return to the low value and then repeat it five times and these are the raw data of red and green intensities of the DNA die and they are changing in an anti-collect matter and the blue is the fred efficiency that is dropping gradually as you increase the force really showing that DNA is getting peeled off gradually little by little as you increase the force showing that we can do these kind of measurements and so on. Actually data was so beautiful that when my students showed me the data for the first time I almost cried but I didn't instead I told them go back and take some more data as every good advisor should do. And then we became more ambitious so Jan Shemla is a colleague a former colleague in Albanian pain and so he came from Carlos Busmante's lab so he's an expert in high-resolution optical tweezers. So we combined together single molecular fluorescence and optical tweezers of dual laser traps you can actually have much higher resolution and sensitivity and here the idea is that if you trap two beads and then you can shine protein in the middle of the DNA you can measure fluorescence from that protein but at the same time measuring the mechanical response with down to single base resolution of tweezers. Here we had to go back and forth between different lasers because if you have the trapping laser on at the same time as a fluorescence laser excitation then you get 10 times faster photobleaching so we have to actually do this like 60,000 times a second to avoid that issue. So it's actually pretty complicated instrument that that Matt Comstock built. Matt is now physics professor in Michigan State University so I'll show you some data that he'll acquire using this instrument later. Okay so coming back to the helicases how does a motor move on the track processively? One possibility is to use the inchworm mechanism. You have two units of a motor protein three different colors and then in the first step you expand the inchworm but in the process if you weaken the binding if you have a weak binding to the front then upon expansion the front head will go forward but next time you compress the inchworm but if you modulate the binding affinity so that now the front head is bound strongly then upon compression the rear head will move forward and you can repeat this process multiple times to move in the track linearly and for non-hexameric helicases the rigorous lab has shown really good data using structural biology that this is how the helicases can move on the single-send DNA track one base at a time. So here's an example of helicase from Hepatase virus NS3 and and this mechanism requires two non-hexameric subunits and these are provided by REC-K4s as you are familiar based due to my my caucus work on REC-K so you have two like REC-K like domains here and the ATB binds in between and the ATB binding then causes the two domains to approach each other compression and weight ATB associated inchworm expands and this can repeat many many times. So here's a structure of the hep C helicase bound to a piece of DNA and if you look at the structure of the protein without any ATP then highly conserved Australian residue is shown here and blue in contact with the phosphate backbone of the DNA three nucleotides apart in this confirmation but if you look at a similar helicase bound to an RNA in the presence of ATP analog then there's compression, inchworm compresses and then there's two Australian residues now two nucleotides apart so that gives the basis for one nucleotide movement along the DNA per ATP hydrolysis. So here's an animation that illustrates how this could happen so two REC-K like domains here and here can move on the DNA backbone one base at a time for ATP hydrolysis. But then how do you couple that translocation into DNA unwinding? One possibility is a following you unwind one base pair every time you translocate on the DNA backbone by one nucleotide. There will be number of base pairs on one versus number of nucleotide translocate one at a time and this was proposed by structure biologists you know this example for example by Wei Young's group where they solve the structures of UBRD helicase in many different forms and then they were able to show that there is a one base pair unwinding for each cycle of ATP hydrolysis. So this model will say that you have continuous process of one at a time indefinitely until the enzyme force of the DNA but we have some interesting single molecule data that we actually use to propose that for at least for NS3 helicase that unwinding is not happening every time you translocate one nucleotide we propose that you wait until three nucleotides have been translocated and then you unwind three base pairs all of a sudden and it continues this way more like a spring loaded mechanism. So in this animation what we are trying to depict is the idea that as a helicase remains move on the DNA backbone the third domain stays behind due to phenylalanine residue stacking against the DNA and residue and then after three base pairs of translocation you unwind three base pairs in a burst. Another data of similar kind was actually was obtained by my former postdoc Wang Lu-kui and in this case we are looking at what's called RIP44 it's an exoribor nuclease that chops the RNA from the end one nucleotide at a time but you can also dice it as RNA that has secondary structure as you flex portion without using an ATP so it uses the hydraulic energy here to cut the RNA one at a time and we also found that in this case our unwinding of the duplex does not happen one base pair at a time although we know that motion itself is prepared one nucleotide at a time and we estimated that it's about four base pairs unwound every after four steps of one nucleotide digestion step so that you can essentially compress the protein with four steps and then it can snap open multiple base pairs at a time and the idea of this type has been actually proposed for other systems too for RNA polymerase initiation there is a mechanism called scrunching where you can build up energy in the system until you make the transition from initiation to elongation phase and escape the promoter and 529 DNA packaging motors have been shown to have multiple layers of actually a stepping not just single size step that repeats indefinitely so so that was my introduction to for the main part of the talk so we are studying super family one helicases named rap UVRD and PCRA at their function in replication we start mismatch repair and basic decision repair and PCRA actually functions in basic replication but they are all bacterial origin they are all stripped to 5 prime DNA helicases again they require 3 prime tail for unwinding to be seen in vitro and they are actually very similar in terms of structures and in fact I have a structure here but I don't even remember which one it is because it doesn't actually matter because they're always similar there are two major confirmations that differ in in the rotation of this domain colors differently here around the vertical axis and this will become important later in the talk so we have interesting questions you know what is a functional form is it a monomer or dimer that is required to unwind the DNA and when you have two forms of the helicase close and open which one is active for DNA winding and also how is unwinding regulated so there's one model that you know Tim normal and I propose based on mostly based on his data but also based on some of our data I share with you shortly that UVRD helicase functions in this manner as a monomer you can move on the DNA three to five prime directionally but it cannot unwind the DNA at least in a detectable manner as as a monomer but when another one comes by then magic happens and you can see unwinding and the other model is you based here that you need just one monomer to unwind the DNA processively and this process can continue you know on and on so my student Kyung Seok Lee built a set up combining optical trap optical tweezers with terp for total internal reflection fluorescence microscopy so the idea is that you tether one end of the long singles and DNA on a surface five prime and and then you stretch the DNA using an optical trap and when the protein lens on it moves from three prime to five prime direction then you will see fluorescence signal moving in the laboratory frame in in your video microscopy and this is how we prepare the DNA so we start with double-send DNA tether to a surface we use an external clays to digest one strand sometimes all the way down sometimes we know not not quite so you can have some meaning double-send the DNA portion and then we can use the optical trap to hold one end three prime and then then stress the DNA vertically in this direction and then then start the experiment so that's the beat at this is a surface attachment point and I will show you a movie and this is a fluorescent junk so you should ignore technical difficulty sorry so what what you would have seen is actually spot appearing in the middle of the DNA and then going down gradually and then in addition the spot becomes brighter and brighter okay and and you can actually plot this in the form of chymogram so you can take the section of a movie apply it as a function of time floating by somewhere here and then it comes down toward the surface for the five prime and and it gets brighter because not because it's collecting cosmic dust but because it's getting closer to the surface you know we have this evidence field excitation or that decays exponential with it as a function distance from the surface in fact you can use this measurement to determine the depths of the laser penetration into the solution and and you can then determine the speed and so on very accurately so we on some DNA molecules we are we are lucky you can see many many evrd helicases binding and translocating to the surface one after the other and I call this my meteor shower because it's so beautiful and you can measure the slope and then the speed that we get is it's almost identical to what Tim Norman measured using his work phase essays in some cases we see that evrd comes down and then actually stops here there's another one coming down but this one is another coming down stopping at the same location here and this is not because the enzyme hit the surface because you are still quite a bit far away from the tethering point right here so idea we have here is that monomer comes down and then it encounters a duplex DNA because of incomplete digestion and just get a stole right there one a support we have for this model is shown here what you can do is to perform mechanical measurements of DNA stretching to estimate how long the doubles and DNA is remaining here and then we can also measure that distance of the store position to that tethering point and you can plot them one at relative to the other and we get a straight line with the slope of one supporting our idea that the enzyme as a monomer stores over it encounters a doubles and DNA because it cannot mind the DNA beyond the junction more direct way of looking at this is the following we can put DNA oligo in a defined location using a particular sequence and you put two dice there just to make it a bit brighter and then here is the DNA location where duplex in a exist and then you feel the binds here and then comes down and it stores in the same exact location and there's another example here this is where we mark the DNA for the duplex junction you really comes down is storage at the same location in fact if you measure that this separation between this and that is essentially zero with plus minus 20 nanometers close to our precision of the measurement so supporting the idea that when you really encounters a double-sand DNA it stops there it doesn't online beyond the junction at least within a resolution of about 20 base pairs if you do experiments in higher protein concentration you do see unwinding and here's an example so you really comes down and stores at duplex junction and then later on then it starts to move again but when it starts to move here fluorescence suddenly becomes brighter so it's saying that there's another protein that just came to the spot at the same moment and so the idea we have is that it's coming down at this rate and then it stops here and then another one comes and then as two proteins then it can unwind the DNA but at a slow rate it's known from teams measurement that unwinding is about three or four times slower than translocation in addition you can measure the force on the bead and then show that at the very moment that force starts to decrease now because now generating additional single-sand DNA so so the kind of data we have here and then other kinds of that is just that UVRD as a monomer is not able to unwind DNA at least not very well you need to have one more than one protein to achieve so the second interesting question was the following you know why do you have two different confirmations open and close for PCIe UVRD rapid cases in in the open confirmation this was called to be domain is so open and the close confirmation is swivels around the vertical axis by 130 degrees to obtain this confirmation and it's a really large movement some pairs of residues on the protein can change the distance by more than 50 angstroms and because this was crystallized with duplex with a 3 prime tail a natural substrate for the enzyme it was natural to propose that this closed form is a functional form for DNA unwinding just want to show you you know all of the structure that have been solved over the years PCIe was the first ever helicase whose structure was determined Apple form in the open form and red was solved in by Tim Norman and Waxman bound to singles and DNA both open and closed and PCIe bound to partial duplex set in the closed very young structure also you know UVRD closed and UVRD as by as Apple is is open so basically all three helicases have two different forms nearly identical and because of the these these structures it was believed that the closed form was the active form for unwinding which is I think very reasonable but but we have some different ideas for example when team moments lab deleted a to be domain then protein still function so that was in contrast to the proposal here where this to be domain in contact with the duplex is essential for unwinding the DNA actively and in addition team group showed that this mutant without the to be domain on my DNA as a monomer not very well but still pretty good you can on my 30 base with DNA as a monomer and third we have some single molecule data where where the rapidly case can move on the three-prime tail but it encounters a duplex and snaps back to the beginning and repeat the process many many times but if you if you label the protein in such a way that high-fret between the two days reports on protein domain closing what we see as a functional time is that as a protein approaches the duplex fret goes up gradually meaning that to be domain closes gradually and eventually then it goes back so we thought that maybe closing of the to be domain is a signal that it has reached the junction that needs to go back as a regulatory mechanism so this is a cartoon that we made is a DNA with three three-prime tail and butterflies approach approaching the junction as you approach the junction it slows down the flapping and then stops at the closed form and the stops there and then it goes back so that was the idea so that's why we thought closed form is inactive for unwinding so so that was our proposal so five years ago I met Mark Dillingham who is a structural biologist in UK at a meeting and we had a small argument that I told him that I think is the open form that is active on winding and Mark told me that well TJ I like you I like your work but I think you're wrong here so he said is it must be the closed form so if I thought well you know we probably need to have actual data right to address this point so I emailed my student Zinan well you know why don't you cross link two systems two systems on the helicase so that you can stabilize a closed confirmation and show that that form is inactive and and that should prove him wrong you cannot prove me right but you can prove him wrong so so that's what he did so he put two systems here on 2B and 1B domains shown in the red residues so that in the closed form there are seven enzymes apart so you can cross link them using bi-functional cross linkers but in the open form there are 42 enzymes apart you cannot cross link so actually if you do it this way then you cannot take the open form so so we called this version rep X for X for cross link then we made a control construct where we put the systems in this manner so that they are next to each other in the open form but 28 enzymes apart in the closed form so that should stabilize a protein in the open form we call this rep oh I miss this spoke I would say rep wide so about that so question was which one is active on winding and I think I'm gonna skip this slide but I was really impressed because my physics student was able to come up with essays to to show that a cross linking is actually intramolecular but not intermolecular and and then he did unsung 80 pH essays and it turned out that 80 pH 80 pH rate didn't really change upon cross-linking so I was very disappointed but I told him there maybe you need to give them some more difficult task of unlining the DNA and then you'll see a difference so the essay here is a flat base essay ensemble fret you have a label DNA initially high fret but if the enzyme on minus the DNA you get a reduction in fret so an ensemble fret measurement as a functional time rep wire type on my DNA very slowly even at high concentrations but rep X on my DNA much faster so I was actually surprised because I had to pay some money with against mark 50 cents and but but I knew that my student was not fabricating the data because he was doing me directly opposite to what I was expecting to see I was right so and then he did some more controls so if you have two systems but do not use cross-linking then you don't get this fast unwinding of rep X and if you do the same with rep Y cross-linked into the open form that is the same as the wild time rep X is much faster so it's not just cross-linking or topological enclosure of the DNA that causes fast unwinding but really the closed form stabilization so the next question is that why is it because you form dimers better with cross-linking or maybe you have now mono-modic helicase that is very active so with this single molecule fret experiments we immobilize the enzyme using a histate histin tag and antibody on a surface and then we added DNA shown here a label in such way that you begin with relatively a low or medium fret and and so you get similar intensities in green and red and when the protein binds DNA binds to the protein on the spot you see a sudden increase in fluorescence and nothing happens until DNA dissociates and then another DNA bind dissociate and so on especially when you have rapidly case bound to the surface and it captures the DNA it doesn't on mine it just you know horizontally forward and then release it releases the DNA but and same for rep Y but if you do rep X you see something different so you see initial binding and then fret actually increases and then fret dropped to zero and then you lose total fluorescence signal and you see three events that happen in succession on the same helicase molecule so what's going on here is that when helicase unwinds the DNA then on one strength becomes coiled up bringing the that die closer to the green on average and then that gives you an increase in fret when you unwind the DNA fully then red strength is gone so we you get zero fret here and then finally when you release the green strength then you go back to the base level fluorescence and you can compete as many times it turns out that 80% of the binding events actually result in full unwinding it's showing that rep X monomer is now active as a monomer and it can unwind you know these duplexes are quite well with high high yield so then we decide to test how how good a helicase is by using optical tweezers experiments so we work with a student in Jan Schemner's group in Urbana so there we have two dual-trap two tweezers and there's a bead with a DNA six kilobase per long DNA with a three-prime tail of various lengths and then second bead is coated with antibodies against the histidine tag so you can grab wrap X on the bead surface and then then once you form a link between the two beads and if you have ATP then helicase will unwind the DNA and then the DNA between the two will become shorter and shorter so distance between the two beads will decrease as a function of time so this is the actual experiment you can in the microfluid chamber you have a capture channel with with ATP gamma s to establish a link and then you move the tethered DNA to the ATP chamber and then when when you move into the ATP chamber shown by this boundary then the length of the DNA starts to decrease and monotonically with some pauses and basically what we find is that every binding event you see result in full unwinding of this long DNA I mean at the bottom we have to stop the reaction because two beads are collided each other where you cannot do measurements anymore but you know we believe that if DNA is much longer than it probably have no problem in unwinding even longer DNA so it's highly processive let me likely buy the lengths of the DNA and this is true for different tail lengths and also different force and with and without SSB so that was really impressive because you know you know because it can be unwind DNA as a monomer for such a long distance and this is comparing wrap X with wrap Y and wrap you know fraction of our but unwinding events if you establish a tether only wrap X show this incredible ability to unwind DNA over long distances you can also perform the measurement where you don't have the force feedback so the force is free to increase and then you can see that enzyme can unwind 30-40 piconewians of forcing force before tether breaks and you can also measure the average velocity normalized velocity as a function of force it doesn't decrease with increasing force up to the moment the DNA undergoes its own transition so we now call this wrap X a super helicase because this is super at least to our mind so the conclusion here is that I think I lost my bet with Mark and so the lesson here is a never bet against a structural biologist now if the closed form is active in unwinding then why did nature create the open form this question can be looked at using what's called the hairpin essay so we we have hairpin in the middle of a DNA and double trap configuration and the protein comes and unwinds the DNA then as you unwind the DNA hairpin the distance between the two base will increase as a function of time and then you can read using the optical trap but because now we have this instrument that can also measure fluorescence in the middle of the DNA you can then use that instrument to measure copying on the above the protein monomer dimer or protein confirmations so this is the experiment where we are measuring our number base based on one as a function of time for the hairpin and you can see that DNA is being getting on run by a single UVR D enzyme labeled with two dies so that high fat corresponds to the closed form and low fat open form and you can see the unwinds at least on the tension you can unwind DNA as a monomer to a limited degree and then it can but instead of continuing fully comes back down and unwinds and receives online receives and it repeats it many many times basically sitting there idling you know without really going very far eventually it dissociates if you measure of a fret from the same molecule simultaneously these are the raw data of green and red intensities and this is a fat efficiency that you calculate and we can begin to assign different fresh stage low fresh stage for the open form and high fat state for the closed form and and then then we can actually color different segments of the trajectories when Fred is high you color this segment in red and for all of the high-fat segments and likewise for the low-fat segments then if you look at the optical trap trajectory when Fred is high DNA is being unbound when you can see them here here and here in here and then when Fred is low generally DNA is getting received so the situation is actually more nuanced that you have the closed form high-fat form is used to unwind the DNA but then after unwinding some number of base pairs then enzyme somehow switches back and then causes re-zipping of the DNA but in that process it takes the open form you can do this from many many molecules and plot a two-dimensional histogram Fred versus velocity so when Fred is high velocity is positive you're unwind the DNA and Fred is low it's negative you are re-zipping the DNA so closed form for unwinding open form for re-zipping so we have this model called a strand reversal model where helicage is moving 3 prime to 5 prime direction to unwind the duplex DNA for after unwinding some number of base pairs it undergoes some kind of transition to strand switch to the 5 prime strand and then it are translocated on the 3 prime to 5 prime direction on this strand and then it causes re-zipping of the DNA upon its awake and they switch back and then repeat the process many many times so that's the idea behind this model and the idea is that when it's unwinding it takes the closed form contacting the duplex DNA with the 2B domain and then when it needs to switch to reverse the direction it maintains a contact with the duplex using the 2B domain but it undergoes a conformational change into the open form so that now you can engage the other DNA tail and translocate on that tail and causing DNA re-zipping so that is the strand reversal strand switching model that we proposed so the idea that we have right now is that in DIBO strand switching and re-zipping after unwinding just a few base pairs can keep the enzyme in the off state because you want to regulate the function again you don't want these enzymes to go berserk and unwind every DNA that they see right so you want to keep them in the off state but in the right place so if it's idling there unwinds a little bit and come back and so on comes back and and then likely in DIBO a partner protein can come into stabilize the closed form and then switching it on and making it a really super active helicase in RepEx we actually make it a super helicase by this allowing the open form required for strand switching so you cannot strand switch anymore now it has to keep going in the same direction we also did the same experiment with the PCRA and likewise Fred and then optical trap measurement showed that PCRA X in the closed form becomes a super helicase and for PCRA we have a known protein that is known to activate this helicase function so what we did was we purified this protein called RepD and then measured the PCRA proteins confirmation using single molecule Fred and without this activating protein we see single molecule Fred histogram shown here in black mainly open form but if you add RepD protein that is known to activate helicase activity then you stabilize selectively the high-fat concoct confirmation so this suggests that perhaps RepD makes the PCRA enzyme a really good helicase by stabilizing the closed confirmation and by preventing strands and switching reaction so we have a monomeric super helicase that's nice because you don't have to assemble multi-meric enzymes and there's no nuclear activities unlike in red big CD so we can envision several useful directions in biotechnological applications one idea is to use this for single market DNA sequencing in nanopores why because nanopores can read the DNA sequence by changing its current through a pool the helicase can provide DNA one base at a time at a measured pace because our helicase is very processive and robust against force you can read thousands of base pairs in one in one DNA second application is called isothermal DNA amplification in PCR you have to go up and down in temperature because you have to melt a double-sand DNA you just made but if you have an enzyme that can unwind the DNA to the melting for you then you don't have to do that you can do everything in one temperature and that can be useful if you want to have essays for detecting pathogenic DNA in Africa or if you want to have a quick test for DNA content in in few situations where you don't need you don't want to wait for an hour before PCR is complete so here's an example you're working with Jens a good luck in University of Washington he's a single model key sequencing platform and so we are using our super helicases to feed the DNA into the nano pool and then read the current as a function of time you see many different levels for different sequences and you can call different levels and then and there is an algorithm to align the current data to the known sequence of this phase DNA then we can actually see pretty good correspondence between our measured current levels in red and then predicted current level so you know it's all the voice shows that super helicase can be used for you know getting information from DNA at the single molecule level over you know many many thousands of base pairs here's another example of doing isothermal DNA amplification so we add this PCR X which is actually from a thermophilic organism so it functions at high temperatures and then you add DNA polymerase and primers and so on with a 1k base 1k or 5 kilobase DNA templates and we can amplify 1 kilobase DNA 5 kilobase DNA at a constant temperature there's no thermocycling if you use wild type then you get a smear here so it's actually beginning to work you can amplify DNA you know even very long DNA and I think there are several interesting applications here let me stop Sinan Arsalan was my main hero for the super helicase story we have a really long standing collaboration with Jan Schoenler's lab metcom stock is the one who built this fancy instrument and measured EBRD commercial changes during unwinding we've been working with Tim Norman's lab for many many years and Chris Thomas was a collaborator on the web D protein thank you very much yeah so on the way back the speed also depends on the 80 concentration so it's powered by ATP not just sleeping so that's a very good question you so published a really beautiful structure this year with the same same screw on the racu helicase in the closed form and there's a very striking change between closed and open forms right so so I think it'll be really interesting to also cross link the protein in the closed form and see if it becomes a super helicase right and my prediction is that it can be done but it'll take a lot of effort because you have to use a different approach racu has a lot of assistance like 30 system so you need five register times to we have to use other other approaches but I think the mechanism like this would be probably I wouldn't call it general but it'll be found to apply in other classes not all but okay so that is is so I think that this is okay so that is a complicated question to answer so wrap why is it in the open form but if you have high high enough concentration that why does unwind DNA not not very well but so that this means that closed form per se is not essential you know essential for unwind thing but it's more that open form is needed for strength switching and we don't see any difference between wrap why and wrap in terms of you know long distance on minding yes so yeah yeah so okay the first question was actually it doesn't slow down we may be reporting to store stores as we see at defined locations so when the enzyme keeps moving there's no slowing down continues to move in the same with the same speed now you you had you have sharp eyes to notice that when you had two proteins appearing to unwind the DNA there's no prior history of following this DNA by the second protein it just seems to appear on the same spot and so we have we have really limited number of molecules that we I think we have maybe less than 10 events like this because if you go to higher concentration to increase this event frequency then then back from that becomes an issue so we don't have a lot of data but we do have some examples where we see another protein following from approaching it by so we see both types


Hey everyone, welcome back to Nintendo Prime. We have, woo, some big stories today. Three big ones in particular, with sort of a fourth topic thrown in. I'm really excited about today's Prime news. I hope you are as well. I got the suit on today because it's business as usual and we got something we really need to discuss here.  You know, I would really appreciate if you would just hit that like and subscribe. We have a brand new giveaway happening at 80K as well. I've talked about it in prior videos. So if you wanna find out what it is, I guess go watch those. I don't really wanna take too much of your time. So instead, let's hop right into our first story. And you guys have probably seen the news, but there's so much of it, I wanna sort of encapsulate it here in this one topic. And that is because Nintendo has dropped Bayonetta 3 release date and brand new trailer today, along with a bunch of information. So the trailer, as you're seeing right now, looks absolutely incredible. It shows an additional playable character. So that's really cool, a new playable female character. The trailer actually gave me a little bit of an odd feeling. I'll get to that in a moment because the trailer is a release date trailer. So the release date is October 28th. So this is actually pairing up really nicely with Sparks of Hope and then the billion third party games dropping on Switch in October. So yeah, October's a really packed month. But beyond all of that, the trailer gave me this weird vibe because Bayonetta's always been, well, very sexually confident as a character in terms of her look, in terms of her presentation, and in terms of her personality. And we saw a little bit less of that in this trailer because Bayonetta always stayed completely clothed no matter what the attacks are. And if you guys know from previous Bayonetta games, her hair is basically what her clothing is. That she uses her hairs for her attacks. So what she's doing attacks, her clothing would basically vanish, leaving her almost nude, not necessarily completely nude.  But you didn't see that in this trailer. Well, that is because they are adding a naive angel mode, which allows you to basically turn on and off the risque nature of Bayonetta. So essentially, hey, if you play it and it makes you feel uncomfortable, you can turn it off. I guess that's kind of cool. Options are always great. I think a majority of people are just gonna play Bayonetta the way it was intended to be played, but that's because we've already seen this for two straight games. But hey, it is cool that they give you the ability to turn it off.  Now, that being said, there's a lot of other things happening here as well. They announced the Trinity Masquerade Edition, which includes a 200 page art book and three swappable game covers so you can take out the cover inside your Switch cartridge and put in other ones. I think that's really neat and interesting. They didn't announce pricing on it. Technically, it was only officially unveiled in Japan and the UK, however you can't pre-order it yet, but in clarification in talking to Nintendo, they have told me through email that they are going to be releasing it in the United States and that pre-order information will be available at a later date. I do have some concerns, of course, of how Nintendo is going to handle these pre-orders. We've seen with special editions in the past, such as Xenobay Chronicles III special edition that, hey, they would only make it available through the My Nintendo Store, the website would crash, and then even when they implemented the Q system, bringing it back, there's many of you that still do not have it, and yet there's hundreds of available verified pre-orders for purchase on eBay from scalpers. I don't like when things are only available through a single provider. I've never liked that. I don't like the valves doing it with Steam Deck. I don't like the Nintendo that it was Xenobay Chronicles III, and I obviously do not like that this is probably what's going to happen with Bandit III as well. Maybe I'm wrong and maybe they'll let me actually walk into a local Walmart or GameStop or something and actually lay down a pre-order, but for right now, I'm just presuming that's what it's going to be. Pricing's not available for that version of the game yet. Now, they do have the actual physical game available to pre-order right now. You can pre-order it at Best Buy or Amazon. I will have affiliate link codes down in the description. Obviously, you get it from Best Buy, but you get a higher percentage, but hey, you know what? I appreciate you guys even using our links at all if you would like to pre-order Bandit III. I know I'm slapping down a pre-order today myself, so I'm really excited for Bandit III. Oh, but the Bandit News isn't actually done there because what was really surprising in this drop wasn't just that they did it on social media and all that. Nintendo actually just did this yesterday with that new Kirby game. It's that they also dropped that they will be releasing a physical version of the original Bandit. So Bandit I, which does not have a physical version on Switch, is now going to have a physical version. It's only gonna cost $29.99 and they're releasing it on September 30th. That's great. I do wanna note though, they did say this will be a limited run of the physical version. So essentially, I think you'll be able to get it September through the holidays, and then that's it. I know that Nintendo plans to keep printing Bandit I into 2023, so if you really want that physical copy of Bandit I, you're gonna wanna get it now. If I could find pre-order links, I will include those down in the description as well because again, this is gonna be a limited run. Nintendo's warning us now, this game physically is not gonna be sticking around Bandit I. So if you would like to get that for Switch as well, I guess look forward to September 30th. Yesterday we talked about Bandit Namco hack. Essentially, this group had hacked Bandit Namco and is trying to claim ransom for them, ransoming private information for millions of dollars and all that. We don't know the exact details because at the time Bandit Namco hadn't even confirmed the hack was real. And some people actually yesterday were in the comment section being like, oh, this is just speculation. Well, speculation or not, Bandit Namco has come out today and confirmed the hack is real. They have confirmed that Bandit Namco has been hacked and they are currently assessing the scope of the damages from the hack and that is it and that they will have a future update on this down the line. So next week or so, there'll be something officially coming out from Bandit Namco, explaining the scope of the hack, what was compromised and what Bandit Namco's actions are going to be. I presume they're not gonna be paying the ransom. A lot of times these hack ransoms are not actually paid and the information just gets released. This is what's happened with a few other things. We have like Nintendo private emails during the Giga leak getting leaked out there. So companies don't like to pay millions of dollars in ransom for somebody who broke the law to hack their company. In fact, they just like to pursue legal action and try to get those people in jail. Nintendo actually successfully did that with the Giga leak. We'll see if Bandit Namco can be successful. This hacking group has actually hacked other companies as well and has yet to be caught. So they are wanted and have already broken the law several times. By the way, there is a leak out there supposedly from the hack that's suggesting future games and content coming from Bandit Namco. I just wanna note that if you see anything about that yesterday or today, that none of that's true. That's all been debunked. Nothing from the Bandit Namco hack at the time of recording this video and likely at the time of publication has actually been released. The hacking group has come up themselves to state they have not released any of the information from the hack at this time. So if you're seeing that floating out there, that was just people trying to take advantage of the fact that it's known now that Bandit Namco was hacked. So let's get our 15 minutes of fame and just make up a bunch of games and stuff that's supposedly in the hack. And yeah, there might be information that comes out from this hack and we'll cover it as it becomes relevant. At this time, nothing from the Bandit Namco hack has actually been released publicly. So if you're seeing game lists and stuff like that, just know it's not real. So don't really have to pay it any nevermind. So the Sega Genesis Mini 2 or the Master System Mini 2, whatever you would like to call it, has now had a public listing at Amazon with a release date of October 27th. Now this system was previously only confirmed for Japan and now that it's appearing on Amazon US, it makes it feel like a reveal of it coming to North America is imminent. Now there is a game list. We don't know the entire game list yet. It's being updated. I'll link you to an article down below from Nintendo Life that goes over the currently known games in it. Right now I think there's about 20 something games known to be part of this. We know that Sega in the past has obviously released systems. This is their second of the mini classics. So it'll be interesting to see what's on here in comparison to the first one. But yeah, the actual first Sega Genesis Mini Classic did really, really well and was received really well and had some of the best emulation from these sort of miniature Sega Genesis consoles that have existed forever, had really good emulation on the last one. So there's a lot of excitement around the second one as well. So yeah, we'll see what happens. But I just wanted to let you guys know that it is available. You can actually go pre-order on Amazon right now. I think for $109 or something like that. I don't know if that price point is actually official. Don't know that the release date of October 27th is official, but that's there and it's a thing and it's on Amazon. And I expected an announcement probably by the end of this week. But you know what I am not expecting an announcement of? A Nintendo Direct. So we talked yesterday about a story from Pierce Schneider last week where he said he had heard that there had been talks of a first party Nintendo Direct for this month. However, and he noted this, however, yeah, he wasn't really sure if that information was going to be true, AKA these aren't like the most solid sources he's ever talked to. So what's interesting is obviously we just had Bayonetta 3 with a trailer drop and release date today. That was something people thought that we needed her to direct for. We also had the Kirby game yesterday. And also people sort of dismissed the Kirby game as mattering for direct because Nintendo always randomly reveals these smaller Kirby games. So it really wasn't that shocking that that ended up being revealed in the way that it was. But Bayonetta 3 being unveiled in the way it was, the whole idea that it does look like we're gonna get an announcement for Mario Kart 8 DLC soon. It kind of feels like they might just social media drop DLC wave two. It's making people really doubt that there's going to be a Nintendo Direct this month. Now, I don't know if there will be a Nintendo Direct this month. I've never known. I predicted, I speculated that there would be, but I didn't have any actual information. And obviously reported on Pierce Schneider yesterday. But again, he wasn't even confident in the information he received either. So will there be a Nintendo Direct in July? The answer is still, I don't know. But I'm always about hyping responsibly and setting expectations. And at this point, I would suspect there will not be a first party Nintendo Direct this month. Maybe an indie world, I don't know. But setting the expectations, I think we should start to not expect that. And instead, expect maybe more of these social media drops. And this might be Nintendo's plan all along for the summer is that they're gonna do that third party direct and then they're gonna take the first party stuff and just give you these social media drops. Now, that doesn't mean I don't expect any more directs the rest of this year. We can still get a general Nintendo Direct in September. And I absolutely expect a Splatoon 3 Direct next month. Remember, we got a Xenobank Chronicles 3 Direct last month. So I fully expect a Splatoon 3 Direct next month. So I'm not saying we're not gonna get directs. I'm just saying that maybe the whole idea of Nintendo replacing the June Direct was actually just third party direct and Twitter drops. And that was Nintendo's plan this entire time. Now, obviously we don't know if Nintendo's gonna participate in E3 2023 at this point. We should know by the end of the year because the ESA announced that they would actually have the exhibitor list and all that stuff announced by the end of this year. And Nintendo is on that list. I'm gonna be doing a booth like they have at every E3 that's ever existed. You can start to say it's pretty safe that Nintendo would be planning to do an E3 show next year. And maybe even the return of Nintendo Treehouse, which would be Nintendo Treehouse Live, which I think would be incredible. I almost think Treehouse Live is better than the E3 Directs. But anyways, that's next year. That's worry about this year. And what's coming right now, Nintendo Slate is filling up quick.  But it feels like if they could drop Bayonetta on Twitter, they could drop that as well. So I guess we'll just have to wait and see what happens. Anyways, folks, I am with Andrew Roblojans from the Center Prime. I'm making you guys for tutoring him. And we'll catch each and every one of you in that next video.


For a lot of my time in education, I would hand write notes and I would write a lot. So when I eventually got deep into my clinical years in fourth year of medical school, I made a huge change to my note-taking style. And basically what I did was I stopped taking notes. When I stopped taking notes and I just used past paper questions to distill the most important information, I found myself really only saving or writing down the very few things that I thought were most, most important, most high yield and most likely to show up or be tested on for our exams. Looking back, I just wish that I had trusted this idea that not writing notes was a viable option.  And if you think that there's something that can be improved upon or something that you could be doing better, then do it. Go for that change. These types of changes could make you more efficient with your studying and it can give you more time to do things outside of school that you really enjoy, that you love and you want to get done.


We are at Pueblo Motorsports Park in wonderful Pueblo, Colorado. We are here to shake down a 991 Turbo S, which we recently tuned for Porsche of Colorado Springs. They'll be racing at this year's Pikes Peak International Hill Climb. So Pikes Peak is the second oldest motorsports race in America. It actually started in 1916 as a way for the town to draw on tourists. In over its 93 years, has seen all sorts of vehicles, cars, motorcycles, and even trucks. The race is almost 12 and a half miles long, and climbs from around 9,000 footers start to just over 14,000 feet of the summer. I've been tuning for Pikes Peak for the past seven years, and this year we're excited to work with the 2014 991 Turbo S. Today we are meeting with Dave from Porsche of Colorado Springs. He is the one that is spearheading this whole project. We've been working closely with him to get the car prepared for Pikes Peak. We are also meeting David Donner. He's a longtime motorsports veteran of 27 years, and aside from his countless victories in multitude of racing, he's a four-time winner of the Pikes Peak Hill Climb. So this is what I'll be running in the Pikes Peak Hill Climb. It's a 991 Turbo S. We're running in the production class, which is called Time Attack 2 on Pikes Peak. To do that, we have to remove the interior, all the interior items for, you know, fire safety, and it takes weight out. We'll be putting a roll cage in it, otherwise it's a stock vehicle. Ceramic brakes come with this car, the Pirelli tires, and obviously, the important part, do some tuning. So the first time out, I was thinking I'd just go out and maybe do a couple of reconnaissance laps and just feel everything out, you know, just generally how the car is feeling. I haven't driven it before, so... Yeah, to start we'll just get you on track, just feel the car out a little bit, and then from there we'll get the access put on the car, and then we'll start to gather data. Sounds like a plan. All righty. All right. Good luck. Thank you. The most important thing in Time Attack 2 production class is the driver and the tuning. As we're only able to modify the exhaust system, the brakes, and the suspension with Porsche factory parts, we have to rely heavily on the tuning and the driver to accomplish a solid race day. My hope out of testing for today is that we can verify the calibration that's on the car. We've already built the calibration in Southern California, and we just need to make sure that it's sufficient for this elevation and we're not going to run any issues related to the altitude or temperatures. Start of Pikes Peak is approximately 9,000 feet of the start line, so it's important for us to get some testing at a higher altitude. Where we're testing today is approximately 5,000 feet, so while it's still not the same elevation as the start line, it gives us a good idea of what we're going to encounter at Pikes Peak. How does it feel? Wow. Yeah? Yeah. Do I need to let it idle? Yeah, just let it run for a minute and cool down. First driving impressions. It's incredible. The amount of torque is amazing. Yeah. Absolutely phenomenal. It just keeps pulling, pulling, pulling, pulling. Especially out of the really tight turns and stuff. Okay. You can feel the front sort of biting. Okay. Which is kind of an interesting sensation. I haven't run all-wheel drive like this with this much power. Handling-wise, there are a couple of things that are a little different. It kind of caught me off-surprise. Unloading into a corner of the tail kind of wanted to come around. Okay. And that just might, it tried to swap ends on me. Okay.  I'm not used to that. How do the temperatures look? Everything was sort of standard. Okay. All right. Fantastic. Well, we'll get some computers and stuff in the car and then we'll start to push it and see how it does. So now we are getting the access port in the car and we're going to start to data log the parameters that we need. Measuring ignition timing, torque delivery. The other nice thing is it allows us the ability to chart his driving and see, you know, consistencies, things like that. So right now I'm just setting up the access port to do those things and we'll get them back out on track. The biggest challenges we face are generally altitude and then just air density is really hard. There's not a lot of oxygen towards the top. So things start to overheat. There's not a great way to cool them. Typically the cars at towards the top of the mountain will lose a decent amount of power. So the turbocharged cars tend to thrive well there due to the turbochargers, whereas a lot of the naturally aspirated cars towards the top will start to run out of steam just because they've lost so much power. Okay. So how did the car feel? You know, it was strong. Yeah. It's very strong. There's no hesitation. Good torque, no, you know. Everything looked pretty good on my end in terms of, you know, boost control, torque control, throttle angle and all that stuff is doing what you're requesting. Temps look good on my end as well. I mean, both engine and coolant temp or engine and oil temperatures are around 200 degrees. So, you know, totally manageable, totally good temp. So we're going to make a few tweaks and we're going to see if we can get the car to go any faster. Well, the mapping that's in it, the car, I don't even, I can't read it because the boost gauge maxes out. So it's strong. It's really strong. The thing is, is what I've learned is that you don't, you don't need to shift as much because the car's got so much torque that you really only have to shift a couple of times. But so it's got so much torque you can go into a corner and one gear and exit in the same gear and then shift up. All right, Dave, so how did the car feel? The car felt great this last run. I mean, all the sport mode stayed on, the traction control stayed off and the car was neutral. I'm still learning a few things about the all-wheel steering but the power is exceptional. I mean, it's pretty crazy. Yeah, tune looks good. I think we're going to leave the calibration where it's at because of, you know, obviously the elevation change and even the start line is going to be much higher than what we're at now. I think trying to push it any harder isn't going to be... Not going to prove anything. Yeah, it's not going to do anything for us right now. So the car's running really well.  We'll tear through the data. There's a lot of data and, you know, we'll keep testing and once we get closer towards, you know, running to the top of the mountain, we'll start to push the calibration probably a little bit harder and see if the car can go a bit faster. All right, well, we're all done. We'll see you guys in a few weeks. All right, thanks, Mitch. Yep, thank you guys. Thanks, Mitch.


Now, this is a fascinating story. I'm here to interview Ralph Czaplinski, who is the country manager for Zero Motorcycles. Zero Motorcycles is the world leader in pure electric motorcycles, and these have no compromises, these machines. We have had them exhibit at our show in California, and I remember we were always getting a new anecdote. We were absolutely fascinating because we were told then that one of these motorcycles, one customer in Texas was charging it from the local river. He'd got green all the way, so it's not only a zero-emission motorbike, but he was actually charging it with a turbine in the local river, which is amazing. I think it was Texas or somewhere, but anyway. Haven't heard about it yet. No. So, Ralph, could you lead us through? What are these models now, and how are things going with the company? Can you tell us the story, please? Sure. Where should I start? Right in the beginning, in 2006 when the Zero Motorcycle was founded. Show us what you're doing, show us the machines. It's the machines, it's all about, I guess. Okay, yeah. I brought you the latest models. It's a model year 2018. That's the two poles of our model range. We begin with the smallest bike. It's a 7.2 kilowatt hour battery powered bike, so it's the smallest, as I've said. Nevertheless, very torquey. We have an enormous torque of more than 100 newton-metre and 46 horsepower. The range of this motorcycle with this smaller battery is about 100 kilometers. It depends on your riding style, but that's what it can do. Right. Where does a Honda come from? This is very much an American company, isn't it? Forget about that one. It just has to be the website. This is 100% American company, isn't it? It is 100% made in California and Santa Cruz. This sticker is just from my Berlin dealer who was so kind to let me this bike because I... Right, so he didn't take the publicity, but...  Actually, I think that Yamaha, Honda, and various others are working on pure electric motorbikes, but the Zero Motorcycle is ahead. We think that it's entirely possible that in a market of hundreds of thousands of motorcycles a year, this type of product could well create a billion-dollar business. It could well be well in excess of 100,000 a year, which would be multi-billion dollars, but it easily become a billion-dollar business. So I think you have our admiration. Can you lead us to the Bug-Eyed one, or that's this one, the other one? There are two different ones here. Yeah, that's a completely different motorcycle, a completely different area of application that's our high-performance street bike. It's called Zero SR. It's at 14.4, the 14.4 stands for the kilowatt-hour battery, and that's double the capacity of the smaller one. So this takes me easily 200 kilometers with a single charge. I can tell you of a world record. We had three weeks ago here in Berlin, where one of our Berlin customers rode 1,113 kilometers in 24 hours with exactly this bike. He did it by just quick charging with our charge tank and with a standard onboard charging. So you see, it is possible to do already touring with our bikes. This is our high-performance bike. As I've said, it has 146 Newton-meter torque, 70 horsepower. It accelerates from zero to 103.3 seconds. And this shows that electro-mobility is not only boring, and no max control from zero, isn't it? I mean, amazing compared with anything else. And well, of course, this is our most expensive bike, but it's funny, but it's our best-selling bike. Now, how long have you been selling the bikes in Germany? It's spread over the early days, is it? We started the European business in 2010. Oh, right. And I joined the company in 12, and since then I'm accompanying the sales in the markets, Germany, Austria, Switzerland, and some Eastern European countries. And yeah, I put every year 30% on top, which is not a rocket science. Thinking of the basis where we come from, but at least it shows a tendency. So we're talking about that is on-road off-road, and this is an on-road touring bike. We have something in between. There's only two of our 800s in total, and there are dual-sport bikes, which can do both, at off-road, on-road, and in between. And so you've been selling for a while, although these are relatively new models, you haven't been selling these for all that time, and do you have a feel for where your market's leading you? What's the best-selling vehicle likely to be? It will be the high-performance bikes. We are known for premium, our customers are okay with premium prices, so our bikes best-selling are the higher-priced ones, with the most performance, most power, and the highest battery capacity, as well as the charge tank, which normally sits here, but this is not equipped with it. This is an expert, it's an accessory, and our top model equipped with a charge tank is a lot of best-selling models. Excellent, and the Germans love their cars black, don't they? Would they like this in entirely black, or do you do differences at all? We only have one color per model. It makes it easier for the customer to decide. And the white, we don't have that much experience. The first container is now coming over to Europe, but the first reaction from customers is quite positive. So, very much looking forward. And this is a very special white, as you can see. Yeah, it's a silver. They will like it. Silvery white. And I love the way it's a quality product. The whole thing is a huge quality, doesn't it? Absolutely magnificent. We get some of these hobbyist bikes that are very big and ugly, some other electric bike manufacturers have made mistakes, like putting the weight in the wrong place and having sometimes huge hub motors that alter the whole ride. But this is obviously built by people who ride motorbikes. Yeah, and they did it for a long time. Take our top engineer, the CTO, A. Pascamasi, who was a long-time right-hand of Erich Buhl, Wisconsin. He brought some really nice features from Buhl, like the mass centralization, where we have the really heavy motorcycle components in the middle of the motorcycle, as deep as possible, to get the center weight down. And it makes great handling of this bike. Yeah, and you've got an aluminium frame for light weighting of the frame.  And this weighs only more than 80 kilos. So it's quite heavy. And I think in the States, they're selling police bikes where they wanted battery swapping. And that was provided. Is that right? But you don't have the police bikes here? We do sell them in Europe. Ah, interesting. The first orders are coming in from German police stations. They are to be delivered soon. And there's not only police, there's special forces, there's military. Yeah, and in many of those applications, the fact you can come up silently and no one knows you've arrived can be a distinct advantage. There are so many technical advantages with our bikes, they're quiet. They are easy to maintain or there's nothing to maintain. You don't have so much down times. And the cost of operation, which is an obvious advantage of our bikes, is liked by the authorities as well. So the resale price ought to hold up well as long as the battery has a reasonable life. It says five years, that's your guarantee. And the warranty of the battery lasts five years, it should last longer. But you have a warranty for that. It does, absolutely. That puts people's minds at rest. Absolutely, they are always a little afraid of this monster battery. How much is it if it breaks? Well, of course, it's a large amount of value. But we can't tell you what it will cost after five years. No, no, sure. The next five years you're on the safe side. And the probability that it will be much cheaper in five years' time is very high. So we can't tell you. But still, as you've said, it won't be broken after five years.  So if you... We like the car, really. The car batteries must be similar to that. If you multiply this by the number of charging cycles times the average range of a single charge, you end up in more than 400,000 km until the battery gets weaker. So that's more than a motorcycle lifetime. And what does your warranty constitute? What are you warranting for? Accident of damage? If the battery goes completely dead, you will replace it even at four and a half years. We guarantee in the next five years, if you purchase it today, we guarantee the next five years at least 80% of capacity. From the original battery capacity. Absolutely. That's great. And two years on the rest of the motorcycle were just industry started. And you sell charges? Absolutely. What's the speed of the charges? Are there different options? Give me one second. Good. Key for the bike and I'll show you. I hope I have the cable with me. But I usually don't go with all the cable. That's all you need to charge it in everyday life. That's a normal household socket. That's, as you can see, like a computer cable. We put it in here like this. And normally this is about two meters long. Put it in your household socket and with the onboard charger placed underneath here it charges from 0 to 95% in about nine hours. That's quite long. Either you work or you sleep. But we can do it much quicker. Therefore we use this space here. We get in a charge tank with a type 2 charging socket on it which will be here like a tank cap. And you can go to public charging station and charge it completely full in two hours. Less than two hours. And that makes this bike capable of doing tours. Our customers do nowadays already go to 500 km a day. You need to ride or plan your ride a little bit more You need to plan it. Not just go and look for a gas station. In e-mobility it says charge when you can and not when you have to. Yeah, yeah, yeah. Which is like your mobile phone, isn't it? You top it up. And we don't have a memory effect that you can charge it at every state of touch. It doesn't have a battery at all. Is there competition for anyone in Europe wanting to buy an electric motorbike? For your star product here? Do you see competition? Not yet. I wish to. I'm not afraid of competition at all. I can't wait to get the big players on board because they would attract and they would attract many more of their customers on electric motorcycles. Because so many people know the existence of electric motorcycles in general or the zero in particular at all. People thought that it would limp along and they got amazed. But I think Yamaha has some off-road bikes but they're very light electric bikes. I haven't seen them in Europe. I don't know. Yamaha to launch two models already for two years now. They announced it already for Eichmarshow 2015 I think or 2016. I haven't appeared yet. And BMW had a scooter an electric scooter but that doesn't in any way compete with you. Sort of maxi scooter and not a proper motorcycle so we have complete different target groups. I like the cosmetics. I like the design. I think it looks gorgeous. Is there a reason why that one has the two lens light and this has a more conventional light? Is there a functional reason? Just styling. Just design and styling. No functional reason behind it. Well good. Thank you very much. I think this is superb. You're most impressive. You're a billion dollar company in the waiting. Thank you very much. We should follow you every year. Bye for now.


Hey, what's up guys? This is Chris Pike. My friends call me Big C. Back in action today. I got some more heartbeat moments for you And in this one, I want to show you yarn hub. This is a super Super high quality channel. I came across the other day and they feature all sorts of different war videos for the most part But here's the thing they do it a little differently than the average one now You've probably seen lots of war videos with like old footage or with Animations and with you know Adobe Illustrator and After Effects, etc This one's done in unreal engine unreal engine is amazing for video games But they're using it for renders for war videos on YouTube and this is a totally unique idea I'd never thought of it before but it works and it works well So here we go in this one. We're gonna be going to yarn hub And this is about captain Vladislav Reginis and I apologize if I said that wrong because I probably did Anyways, he commanded a defense operation in a small Polish town called Vizna Vizna Vizna and anyways He had a hunt. He had 720 Polish soldiers and he faced off against a gigantic German force 42,000 men 450 tanks 108 150 millimeter Howard sirs and 58 lighter guns and anyways the battle was it's just yeah You got to watch the video. It's amazing. Let's get into it with the first moment. All right here We go. So I am on heartbeat if you want to follow along with me Just go to h.ki or hki as George says and then just click on my name and I under my moments You're gonna see a whole bunch of moments here. I've I did about 40 today, but these ones are really cool here So let's get in here. Let's see what Vladislav Reginis and his Polish defense does so let's kick it off right at the beginning Here we go. I'm gonna click on the first moment The 7th of September 1939 just outside of the town of Vizna in Poland Yeah, so that's a bit of an understatement. Let's let's just be blunt here So anyways, there you go. It kicks off September 7th 1939. Let's kick Let's move forward a little bit here and let's get into some of the action You know Keep in mind this is done inside unreal engine. I believe this is in five 5.4 is the latest release and this is just Absolutely amazing like I just can't believe how good this is. Anyways, let's keep going see what happens here Suddenly an explosion shakes the earth Soldiers in the trenches are rushing for the bunkers as the air fills with fire and trapped Look out aircraft then war over head dropping bombs one of the men rushes for the phone and gives a message They've all been dreading So those I believe are stuka dry dive bombers But please in the comments if I'm incorrect in that, please correct me. I'm not a hundred percent sure on what those bombers are The dry brushes set a light and fires spread across the trenches in both positions the air becomes foul smoke and dust Look at that though. Look at that render of that picture black and white with the correct Like with the fold up at the top left there and a little bit of creasing like this is just just ultra high quality Big kudos to these guys. So anyways, they take their they're taking their beatings here And now let's get forward a few seconds until the battle really begins. Well the calm before the storm Let's just leave it at that Defining explosions to the most disturbing silence. Yeah The men at Visna lay low not daring to expose an inch of their bodies outside something else breaks the quiet What's your boys?  So this is one of the reasons why I use the heartbeat tool I mean, there's a product placement here that lasts about 40 sec 50 seconds And while I get it, you know what these channels need to make their money great on them for this type of you Recap type reaction type video doesn't make sense to show it too much So let's get forward until the tank battle here at two minutes 38 seconds Yeah Visadig most At the front the men Probably thinking of that scene from saving Private Ryan where they put the C4 Sticky bomb stuff to blow the bridge. Well, they did it here, but with dynamite it appears Now I don't know much about tanks, but I don't think they're the best at floating and swimming So anyways all hell breaks loose here, but let's move forward a little bit here until one of my favorite scenes in the whole thing In fact, I like it so much. I'm gonna click on a heart here Which means I'm ganking it a favorite and I've entitled this what kind of gun is that marmot Okay One more shot of the massive rifles washes across the battlefield and the huge bullets slams into the enemy armor So this part is you know, I did some research on this and apparently there were rifles or I guess it's kind of like Special sniper rifles that the Polish used that could shoot bullets Into tanks like normally, you know a tank is pretty highly armored and they're not you're not gonna be able to snipe your way into it But this was a special I can't remember the name of the gun But anyways, this is for real so kudos to them for their accuracy and I mean look at this as the explosion comes in Everything's blowing apart Absolute amazing. Let's let's watch a few more seconds and then we're gonna skip forward to the tanks going over the river They fire again and again with some shots bouncing and some punching through The tankers are thrown into disarray is the transmission of their injured brothers makes them keenly aware The horse by the way is the official Polish tank in this in this video the danger they're in They fire back with their cannons aiming at bunkers at random as they retreat on the fire escaping the Polish ambush So they go back over the bridge or back the way where they came from of course They're gonna come back and do some fighting But it's interesting to see that such a small, you know group of men can fight off such a massively overpowering force Let's get forward a little bit here and we're gonna go looking at the anti-tank rifles howitzers That's what I've enabled this moment Explosions rip through the formation sending the German infantry scrambling for cover Which doesn't exist while the tanks are shaken and disoriented by the incessant detonations a Few are brutally taken out of action with a direct impact while the rest struggled to fight under the heavy Riflemen continue firing through damaging more enemy tanks The bunkers continue to fire at the exposed Germans as the entire battle becomes a duel of artillery But only one side has bunkers the German position is completely untenable Tanks and men are just trying to survive through the storm of shells So there you have it as much as the Germans had the overwhelming force the Polish had bunkers And it's gonna be hard to bust them out. So let's see how that goes In this case, I'm gonna skip forward a little bit here to bombs away here come the Germans So yeah, the Germans are coming back and they're bringing some friends Men stuck outside die for cover as bombs rock the earth around them The relentless bombing resuming in anger Eventually it subsides and Reaganist knows that can only mean one thing The Germans fire at the defensive positions Advancing from cover to cover through the trees and grass the poles return fire from the bunkers and trenches cutting down Germans left and right, but they have the numbers they reach one of the trenches and drop in taking cover from the bunkers as they fight off the Polish soldiers So this is hand-to-hand combat at it's just nastiest, I mean one of the ways you can bust a bunker of course is through you know dive bombers or Bombers or any type of bomber really and then all you can throw a Potato mashers or grenades into the into the front slit there and that's what they're doing This is kind of like what they did in the Pacific Ewo Jima in particular, but here we go Let's skip forward a little bit here and take a look at the 60 Panzer tanks that are coming I call this one 60 Pang Panzer tanks raw roll It's 60 panzers from the 10th Panzer division They unleash their guns and the shells slammed the first bunker where the poles open fire with all they have Yeah, all arms do little and the few anti-tank rifles can't handle the sheer amount of vehicles as Shall after shall slams into the fortified emplacements Yeah, I heard stories of people going deaf and all kinds of stuff just from the impacts But anyways, let's get going a little more here the men in the first bunker received the brunt of the enemy attack No good panzer shells Hummeling the bunkers as men fight for their lives Across the main road in the opposite town Captain Schmidt is facing the same impossible odds Gunfire rings down everywhere and tank shells slam into the heavy concrete Right, so this moment here on the right side. You'll see here. It's called direct it You'll see why more and more shall slam into the first bunker Finally a Shell strikes right Look at this Let's look at how amazing this sinner. This looks like a cinematic. This is this isn't a video game. This is a YouTube videos It's absolutely unbelievable In the blink of an eye Yeah within moments the German infantry breaks down the door and pushes in So the German infantry I've got the first bunker and then the next one I was called a fixed bayonets Yeah, you know that it's on when when you get the command to stick your bayonet on your rifle. Yeah, it's it's an hour never here We go Yeah, the Germans here a war cry and men charge out of the destroyed bunker Schmidt at the very front Bray Germans try to flee some continuing to fire as the mad poles run through the hail of bullets Schmitz men furiously attack the enemies the Germans try to fight back with the cohesion of their lines been lost and the poles This reminds me of that scene from we were soldiers they weren't expecting this were they the absolute just Unbelievable bravery of these people. It's just it's unbelievable They run back to the safety of the bunker and make it almost completely unscathed it doesn't last for long Right, so let's skip forward a few seconds here the next moment that I use the heartbeat tool for is called Schmidt goes Sisu and if you haven't seen Sisu It's a strange movie, but it's got a pretty tough dude in there And he's kind of like from one of these countries Finland or Iceland or something I can't remember and he was all sorts of scary. Here we go. Let's go to it Schmidt go Sisu Schmidt's bunker is surrounded Schmidt opens fire with his rifle joining one of the remaining machine guns in the defense German after German falls The panzer takes notice the tank takes aim Yep, right out of saving private Ryan.  Yeah, this is exactly what this reminds me of Let's see what happens Good idea Explodes wrecking the machine gun and firing shrapnel across the room Schmidt the machine gunner and three other men are here Germans can quickly surround the complex banging on the doors Schmidt realizes.  Let me tell you let's skip forward now to one of the final moments at 14 52 Where Reginis or Reginis gives his speech and then we'll just wrap this up Yeah Keep in mind this is actually in polish. That's how high quality this video is. This is unbelievable He opens the door a line of Germans stand outside, but don't open fire Reginis holds the door open as his men walk out one by one sharing a look of utmost respect earned The Germans throw them to the ground But Reginis doesn't look the last man exits leaving Reginis on his own And he closes the door Yeah, this is rough moments later The men hear a grenade go off inside So there you have it guys, this is an absolutely unbelievably high quality video This is the kind of stuff that the heartbeat tool was made for I hope you know in the future that people see this and they go check out their channel This is incredible stuff. Thanks for watching


The luminosity of a supernova depends on the mass of the star. If we knew the mass, we'd have ourselves another standard candle. But for most explosions across the cosmos, we don't have that information. But there is one scenario where we do. It's called a Type 1A supernova. It is based on a particular binary star setup, and it is recognizable via light profiles and spectral analysis. Here's how it works. 1. A massive red giant star has a small stellar companion. 2. Mass flows from the giant to the dwarf through the L1 Lagrange point. You'll remember Lagrange points from our discussion on Jupiter in this segment on our solar system. 3. The mass of the dwarf star increases, and 4. Once the mass of the smaller star reaches a critical level, its ability to hold off collapsing under the force of gravity comes to an end. The result is a total collapse inside a few seconds. This creates a supernova explosion that rips the smaller star into pieces.


A classic potato salad has the perfect balance of potatoes, eggs and a little crunch. Which of these two classic recipes would you prefer? First up, potato salad with red potatoes. Waxy red potatoes are a great choice because they hold their shape so well when cooked. This recipe has a slight tang due to the addition of mustard and the crunch comes from fresh celery. Our second contender is potato salad with yellow potatoes. This version is creamier. Potatoes hold their shape when boiled but yellow potatoes have a softer mouth feel compared to their waxy red counterparts. In this recipe, the crunch comes from dill pickles, so if you love dill, then this is the recipe for you. Either way, remember to keep your potato salads cold until ready to serve. Enjoy!


There is very little room left to deny the fact that ancient cultures throughout the world were all connected in the ancient past. We see depictions of flying machines in Mexico that are very similar to the ones of the Viamana. We see in India for example, and there is of course also the handbag of the gods. The handbags are literally depicted by every culture on every part of the earth, but how can this be? This obviously points to the fact that the world was once connected in ancient times pretty much like it is today. And you never know, perhaps in a few thousand years people will look back at this day and age and make similar comparisons regarding mobile phones or something like that. Seems plausible, does it not? But are we destined to forget the day and now as being unimportant at some point in the future to the very point we actually forget the timeline of our civilization all over again just like we have done for thousands of years. Wait, do you hear this? How is it possible the ancient Olmecs, Aztecs, Egyptians, and cultures of those in New Zealand shared depictions of their gods which are almost identical? Is this just random cultural spread? Or is it possible there is more to it than we are willing to accept? The Leventa Steel 19 offers a curious representation of the feathered serpent, a humanoid figure sitting in some kind of vehicle or chair as if manipulating some sort of device. At first this may not sound extraordinary or weird, however it becomes strange when you find nearly identical representations of gods halfway around the world. According to the Mori legend, the god Perengahoa flew on his magical bird from his legendary dwelling, Hawaii, to New Zealand. Curiously, according to legend, this ancient god came from heaven riding a silver bird. These two cases are reminiscent of the Mayan Lord Pakal's sarcophagus. In this depiction, Pakal appears to be operating a machine with a flame coming out of the bottom of it. It is so named Lord Pakal's spaceship because of these features. How is it possible that the ancient Egyptians and ancient pre-Incan cultures developed on opposite sides of the globe yet mysteriously share so many similarities? Not only are we referring to architecture and symbolism, we can easily include religion and science into the list of shocking similarities that are not only being ignored by mainstream scholars, they are even being repressed as if a giant cover-up in ancient history is occurring before our very eyes. How can we explain these striking similarities? Well, either ancient civilizations were interconnected and came into contact with each other in the past, or a long lost culture predates all the ancient civilizations on earth, and it's from this ancient culture that ancient civilizations such as the Aztecs and Egyptians inherited their knowledge. Imagine for a second that this was true and by the way it does seem plausible, an ancient forgotten civilization inhabited our planet. Were these beings the Atlanteans that taught all other civilizations similar traits, we can explain the striking similarities of worldwide culture with this theory. We have to start taking into account the possibility that such a mother culture existed in the past and predated all ancestral civilizations. This however would mean that history as we know it today is only partial, incomplete, and points to the fact that human beings are in fact a species with amnesia. While today's scholars reject the notion that a mother culture existed on earth in the distant past and reject with even greater notion the idea of Atlantis, there are many indicators that cannot be overlooked while studying ancient civilizations around the globe. Agriculture is known to have started near the Mount Ararat. This is also where the arc of Noah was said to have come to rest. This is where the survivors of the Great Flood started a post cataclysmic civilization that would spread out in every direction from this point and place. Could these bags represent seeds that were preserved on board the ark in some sort of vault? Maybe they were distributed across the world as we repopulated the globe after the Great Flood. So many different cultures built up massive complex pyramid structures in the remote and distant past that were eerily similar to each other as if to follow a direction from some sort of blueprint. Could the handbag also have contained information that allowed civilizations to excel and develop and the depictions are one culture to the next handing it forward to the next line people just a thought. Why is it that ancient civilizations in America, Egypt and ancient Mesopotamia depicting their deities carrying a mysterious handbag in their hand? Is it possible that all ancient cultures in America, Egypt, Australia and Mesopotamia were visited by the same gods? Another instance of the ancient handbag can be found in far away New Zealand. Amore myth tells of a hero who once ascended to the home of the gods and returned to earth carrying three baskets full of wisdom. Golbakli Tepe is one of the oldest ancient megalithic temples on our planet.  The massive stones were believed to have been carved some 14,000 years ago even though recent evidence points towards the fact that it is probably far older. Again we see the strange bag present here. Nobody really has any idea what these bags are. The oldest known depictions of the bags is found in Northern Australia and is thought to be 15,000 years old.  As she traveled far and wide she scattered her babies which gave rise to many different cultures and tribes. Even in North America there are petroglyphs that are roughly the same age as the Australian depictions. Again, we see the ancient handbag apparently being portrayed as carrying the seeds of life. Could the civilizations around the world all be scattered remains of the lost empire before the great flood? Each culture appears to be telling the same stories and all this is fitting in with a time in history of when the flood took place. What do you guys think of the idea that ancient cultures around the world were the survivors of the flood and knowledge was passed down from one civilization who survived on board the ark with all the equipment to make life flourish again on earth after the great cataclysmic flood? We told you the truth would seem strange. What are you thinking? Comments below and thank you for watching.


Actor-entrepreneur Will Smith is trending like anything on Twitter and all social media handles right now because of the fact that he slapped comedian Chris Rock on stage during the Oscars. The reason this happened is that Rock basically made a comment. He passed a joke on Will Smith's wife saying that she should be part of GI Jane too for the ones who don't know. GI Jane was a very popular movie in the 90s starring Demi Moore in a bald look. Jada Pinkett Smith also, who is Will Smith's wife, sports a bald look and hence the joke. However, what most people might not know is the fact that Jada Pinkett Smith suffers from alopecia which is a hair related problem and that is probably why Will Smith got irritated, got emotional, got angry and slapped Rock on stage. The bottom line is that in today's social media age we need to look beyond the memes and beyond the controversial slaps and realize what the backstory is.