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BMW Speaker Upgrade/Installation | 2009-2015 F01/F02 7 Series | BAVSOUND Stage One

you alright ladies and gentlemen we are here in the dashboard Oh a 2012 fo.1 7 series this is the same from oh 9 to 15 maybe 16 I always give that up but nevertheless your car looks different from this you have a different car so here we are we’re knocking out the stage one speaker upgrade which in this particular car consists of fourteen submit’ speakers 7 mins 7 tweeters and 2 ghost subwoofers one of course under each front seat we’re tackling those as well in this particular car we’ve got the shop open because it’s hotter than Hades here in San Diego today so hence we hear some news and delivery trucks and such was something I was going to mention about the under seat woofers ah yes that was a bit hot with the under seat well first you got to trim the carpet back a little bit from the edges of the carpet on the outside of the car we just don’t want the carpet sitting on top of the woofers it’s all hidden under the grille it’s completely invisible it’s just – we need that extra excursion because our woofers throw that much further than the factory so moving on to the center channel I always like to start with this because why not it’s really easy it’s really satisfying and it takes just a few minutes so they have sound toolkit available in your cart just add it to the cart everything you need is in there we’ve got our plastic panel removal tool which you will be using do not use the metal tool here so there’s just it’s very simple there’s just a gap between the panel and the – we just take our tool and that one actually popped out real easy boy howdy alright so look at that speakers all brown and gross and dusty and disgusting so here’s what we’re gonna do we’ve got some Torx bits here here and here we’re gonna pull this whole assembly out because we’re gonna do the tweeter upgrade up on our workbench so it’s real simple you can watch or not basically one two three four pull it out we’ll swap everything up on the table that you’ll want to see this part is not going to be particularly exciting but nevertheless you can watch so using our right angle tool that is included in your Bab sound kit we just come up here you’re not gonna get these out any other way so you definitely want this guy then for the rear ones you can use the show you these are notorious for actually blowing and giving you guys lots of problems so again this is a critical part of the upgrade the image in these cars sits high and center thanks to this processing and the DSP amplifier and having the center channel right here so this is a very critical driver to have performing properly and most importantly to get these god-awful metal tweeters so to get those back ones we use our other screwdriver again it’s included in the kit come back here just pop this guy out of here you’re gonna get a bunch of schmutz on your windshield so it’s great time to clean the car after you’re done with the install anyway it’s always nice to get some good tunes and then have a nice clean car especially when you take the seats out and do the subwoofers and find french fries and M&Ms and money any other number of other things that have gotten wedged down in the seat we found a water bottle in this car wedged in the seat mechanism you never know although over the years I have found some very interesting things all right so they’re all that loose we can just oh he’ll maybe you do have to take this out first I honest eyes don’t remember let’s go ahead and pull the speaker out and just see I can’t it’s been so long since I’ve been in this car and one of them rather yeah it looks like they’re using Torx t25 on these speaker bolts on the you know so I’m using a 20 right now but you should probably use a 25 the 20 old grab just be conscientious of that usually you can take the pods out I know in the rear you can pop the pods out without having to remove the speaker the exciting stuff lefty-loosey righty-tighty if that’s the first time you’ve heard that or are unsure what I mean by that you do not need to be

performing this installation on your own and I highly suggest taking it to a dealer all right so with the mid out because an how you take this thing out of here yeah just stuff hell I could have taken it out it was just stuff so yeah don’t worry all right so we’re gonna go up to our workbench and cover the process of upgrading the tweeter and the mid-range after I get the center channel I’ll always come back and grab these rear speakers because we need to do them both at the same time because BMW has used adhesive to fix the tweeters to the housings and we need to do the same when we put our new tweeters so I just like to do it all at once so plastic panel removal tool just like the grille up front and the gap throw it up just like that you lift up and then out you can see there’s little tabs back here on the rear edge so like when we put it back in we go in and down so just wanted you to see that and then back here we’ve got four eight millimeters just securing everything down we’re gonna take the pot out just like the center channel and take that up to our workbench and we’ll do these all at once just a hell of a lot easier that way so you can actually just break them free with your fingers and then they were breaking free with the tool and use your fingers pull the rest of them out so well pause right here and then come back with the workbench alright guys so this was our center channel which you recall you do not in fact have to take this out in the car you just do it on the finish so put the mid out return to the tweeter which in this case you can see just snaps right in so we’re gonna use our right angle pick and we just sneak back in here there’s a couple little tabs and quite frankly it usually just pops right out of here just like that take a little the passenger ironist there out with that and then in with our new center channel tweeter which has the integrated capacitor harness on it so make sure you this is the tweeter you put in your Center debt and your Center and not one of these guys which looks the same but is not because as you’ll see it does not have the capacitor harness so this is the one we want it just snaps right in just like the factory it’s very tight however I always err on the side of caution and add a little adhesive to this because the tweeters in the rear deck are glued and I always like to put a little extra because why not and we’re up here so I just put a little dab it’s a little dab there may be a little dead there there that’s it done the top back on and we’ll revisit that in a moment with the rear doors so now we can just take this guy turns out the shotgun not very scary end of the day just like that factory we can put this in the same location it’s pretty tight just like that and then we’ll plug this into our mid-range which we have right here and drops in like so and then I actually went to get our Torx t25 so it would use it which is over here our screwdriver this time I actually kind of do want you to watch this because there’s a process to it he’s adhering these drivers to the car he’s looking tight man that is the dog snoring she is asleep over there so what we do is we don’t wrench one down and then the other down and then the other down we get them all started I think so and then we come get it snug good snug get it snug and then we just work our way around snug snug and snug and now it is perfectly flush it is perfectly gorgeous it is perfectly ready

now for this which is we pull off this rubber boot we have some spatial concerns this is just acoustic you can use it now as a koozie or whatever I don’t think it’s actually an issue in the front of this but I always take them off just in case I can’t ever there’s so many of them I can’t keep them all straight and then I just take this plug in my tweeter here and then we plug this back into the car when we get in there to to wrap this up so that’s our sinner Genesis super straightforward and now we’re gonna head over and knock out the rear deck driver which same thing I think they might have used what a weird thing they only use the t25 on that front driver so the t 20s on the rear again nothing exciting but the adhesive process is the same whereby we just go little by little we don’t just wrench it all down at once because we do not want any fitment issues she is out like a light all right so unplug our tweeter from our mid and this one the tweeter is actually glued and to the housing you can see there big blobs of glue here here here here and here good news is the dish it’s the glue usually has hardened at this point and so it’s pretty darn easy to get out of there so it is I’m just using my go in there and I’m just breaking the glue free it’s not a big deal and then I just take my angle pick and I just provide the tweeter like so then you just do the same thing where we just pull out the tweeter and this is where our tweeter this is the one BMW manufactured since the new generation started back in ho to where the tweeter is a perfect fit and it snaps in but it’s not as snug as as I’d like which means it tells me that because this is the only car I’ve ever seen glued this aggressively that BMW is aware of that so that’s why we’re gonna put a little more glue just like the factory one and the reason I’m doing this all at once is because I want you guys to let this stuff adhere this needs about 30 minutes to cure so that’s kind of the process here which is I wanted to do all the doors at once or all the drivers at once at least the center channel in the rear drivers so that we could let that set up and cure so that we weren’t rushing the installation or improperly putting it back together so this one you actually need to let sit for a bit because we’re working on it so again the the flip side of this is we just attach the driver and then go drop it in the rear deck it’s really straightforward we’re not even going to cover that that’s just what we do but this needs to Stephanie your rear deck drivers we didn’t touch on this earlier it is imperative much like every other tweeter in the car that we have the capacitor harness in line between the mid-range and the tweeter otherwise you will fry the tweeter and I will send you a new one because I will know you have done that and I just will know so let’s not do it so anyway put it in tie it up be done with it rock and roll oh one door panel very straightforward front and rear doors identical in every way except the front door has three bolts behind this panel and the rear door has two bolts the speaker’s mount the same way there is no difference so when we do the front door consider the rear door the same way because it is – that little caveat so to start with this is the unique I’ve never seen this and I’ve been doing this 20 plus years this piece comes off but to free this piece there’s a little pin a little plastic vertical pin that secures this to this to the subframe behind it we have to extract that first and to do that I’m gonna have first I’m gonna have you guys come in and get tight on it so you can see it you give us just a second we want to make sure you can see it it’s just this little guy right here so I’m gonna keep the camera tight on that while I do this so using your right angle pic which is in your tool kit the way this works there’s a little indentation on that side on the outside of the pin we hook this into so you get this up in there and then we just pull this little guy now that starts to come out [Applause] you’ll see when it comes out here you’re like what the hell cuz that’s what I

said you can see it’s just this little plastic bin strange right anyway so with that out I mean now put that on the somewhere we’re not gonna lose it although I question its long-term purpose but nevertheless so now again plastic panel removal tool do not use your metal tool here we prop this off so are we starting this back edge it’s kind of getting it until he’s a little scary at first but it will come then you just get your tool back here and believe me it’s scary and for those of you wanting to just do like a a swap I guess on your colors here’s how you do it if you want to put wood grain or something good so just go free you come it just comes right off so you can see it’s just a series of tabs a couple little hooks really well done just beautiful alright well we can look at that all day and wax poetic about the design language in the car but we shan’t that reveals one two three Torx T 30 s all of which are in your bath sound toolkit we are not gonna watch me remove those so we’re gonna pause here and come back and get the rest of the door off the screws are removed now the tweeter pod must come off before we take the rest of the panel off so the the process here there’s a seam right here and that’s where we separate the pod from the rest of the car and you’ll see you know it’s funny I usually do this a very specific way we might as well just show you guys just come up here in this gap perhaps this front edge it just comes right out I always forget that’s the easier way and then the tweeter pod just comes off you unplugged the tweeter voila we’ll take that up to our workbench we’re gonna take everything up to the bench at once and do it all at once this car it’s just better to do it that way because there’s a little bit of glue here and there again from the factory so all right with that off now the general flow is there’s a series of clips around this outer perimeter that we pop free and once those are free we then lift up we lift the door up off of the lock mechanism and then we start unplugging stuff so plastic panel removal tool I always start up here feels good to get that first one and then we just follow I see my fingers are behind the door and then I just use my fingers the rest of the way around like that and so as I was speaking to the door is now loose so we lift up and back so we go so now you’ll just watch me do that and look at that the door is off now we’ve got to show you this first and foremost right actually it’s probably better to shoot from back here there’s a very tight little plug right here we need to get that first I’ll show you how we do that it is really tight and if you’ve got big fingers you really do need your right angle pic and so I’ll show you the tab you just depress this little tab on the bottom edge of this and then it comes right out you see these little tabs just do you you you and then the way we take the door off in this car we pull down and unhook which brings us up to the front of the door so we’ve got a couple this is where we use our metal panel removal tool included in our tool kit so everything has a purpose because we’re taking this wiring harness off we’re just unplugging so we’ve got a couple of tabs here so we get a couple of these guys off and then we just start unplugging just pulling just come off you don’t have to watch this this is pretty boring I think our shop dog is about to start barking if I’m not mistaken because someone is here so again if you hear her she’s just protecting us if you don’t hear her then the intruder has finally won this eight-year long battle of coming into

the shop and threatening to kill everyone and the dog did not do her job just unplugging unplugging nothing exciting this process is repeated of course on the driver’s door and as I mentioned is the same on the rear doors as well slight variations but we’re just removing the harness from the door ok leave a little time and so now we’re gonna take everything up to our workspace and do it all at once and now lastly at least if your garage to bench work is our front tweeter which you’ve been doing these the same way forever again it pops right out just like that out with those harsh metal tweeters in with our butter smooth silk tweeters which are also super sexy but that’s just a function of design acoustically there you snap right in there brilliant in every way so you don’t have to put any glue up here if you don’t want because they’re never gonna move especially when you put the foam back in which we are gonna do you snap the phone back in and we are good to go with our tweeter and stimulation wait you took where they put this out well they’re bringing it out on the backside okay well you can do that too I suppose you don’t see this anyway in fact why don’t we just do this you can just have a blade up here you can do whatever you want but I just will slit in that and then we can just bring the wire right out through the front okay because it’s just fun cool now the wire comes off the backside so that’s how we do that front tweeters it’s super easy so that’s all the kind of the the grunt work if you will now I’m gonna clear a little spot here we’re gonna come through is that me 6,000 or boogers no I’m going with you six nails alright so now let’s start with the front door again we want to make sure your workspace is clean obviously so here if we’re gonna do a little bit of work the most complicated thing and it takes two minutes so we’re gonna talk about sound deadening because these because an interesting place to mount the speaker so it’s mounted directly to a hard plastic which is then riveted to another surface so our goal with the sound deadening is going to be bridging these two surfaces together where the woofer and the mid and the and the panels come together so we’re gonna get the speaker off its held down by the eight millimeter nuts which again the eight millimeter socket is in your bath sound toolkit we have indeed thought of everything okay so what we do is we take the sound-deadening that we gave you we cut it in half half for the right front door half for the left front door and that’s what we got that’s what we’re left with here and if there’s anything left you can put it elsewhere it’s not that important on the rear doors because the way the soundstage is skewed in these cars it’s heavily front biased so that energy in those rear doors is not as nearly as intense as the front so what with what’s left you can absolutely do the rear doors and there’s no harm in doing it but we won’t really focus on the front so what I’m doing when I say that what I mean by that so we’re gonna start like this and we’re basically just gonna eyeball it like this and then we’re just gonna kind of mop you know we’re gonna invisibly just go so for me what that means is this I’m just going to kind of draw then kind of follow this line loosely and that gives me almost a perfect cut for what I need which is bridging all these panels now what’s tricky here is that we’re not actually going to do it right here we can’t so what we do is we got you know we’re gonna lay it down we’re gonna get it hot we’re gonna lay it down then we’re gonna cut this out with the speaker mouth but we’re still gonna leave that gap bridge so I just flip there sound deadening over heat gun or hair dryer and probably have a hair dryer you probably don’t have a heat gun if you do cool if not if you’re bald like me then you’re single and you don’t have a hair dryer well now’s the great time to go talk to that pretty neighbor lady would be a hell of a weird first thing to say can I borrow your hair dryer but I don’t know maybe she’ll think you’re manly or something for

working on your car I look I’m not a dating advice specialist I’m just trying to help disorder these are like 20 bucks on the Amazon Plus every man should have one of these so just getting it hot you want it really tacky not like that shirt tacky that you’re wearing but tacky isn’t sticky alright don’t buy your shirts at Costco guys alright so with that nice and tacky you kind of lay this down perfect you know be careful this is aluminum it is hot it will it will burn yeah so now I take the panel removal tool and I work this edge don’t do this with your fingers you will cut yourself and you would Brett it big time you can see what I’m doing here because Ed all we’re doing is we just want to tie these panels together that is the key and as a head mentioned up here you kind of have to identify or are those guys one two three so we want to go just around them like this I’m gonna go back and then you tie that all together I tied this oh and then I just come through here like that we can just take this piece back here that’s just kind of big and dead and not really doing much of anything kind of come back here tuck it in and just add some more mass back here to control this piece of plastic up here so again I’m just tying things together that’s how you control resonance and this one I need to cut a little more cuz I left a little bit too close to the grill cut that that over here so now we need these surfaces flat with the speaker mount so don’t have anything in there but we’ve still got it nice and tied together all through and through and then with everything that’s left I just start adding it in these areas here with the rest of that or again if you want to take that and use it in the rear door you can see it’s cut exactly the same as the front it’s just the inverse so you could do that same thing in the rear door as I had mentioned I tend to focus it on the front so we’ve got the harnesses reconnected we’ve tied up our excess harnesses here that are tweeter harness ready for the tweeter install you’ll note that there were two male blue connectors coming into the speaker one from the amplifier and one just daisy chain back up to the tweeter so on both of the front doors just take note of the harness that has the female connector up here and then you’ll find its corresponding male connector here don’t use that one use the other one that’s been coming from the amp so a little brain power goes a long way here so there’s that so everything’s connected last thing we’re going to do plug this guy in and you’ll remember this tricky little part down here from before with me door handle so we hook it like so and then we pull down the white part and then we plug this guy in and then the trick is lining that up so as I had mentioned it just drops down perfectly and then the trick of course making sure your tweeter harness is up there lifting up and you’ll feel all the clips they just settle right in and then you just use the palm of your hand reinstall those let’s do the tweeter real quick while I got you here plug in the tweeter and this is the part where you actually do want to start on the other side because we need to hook this back lip a little may be a little tricky to show you guys but again a little brainpower a long way here so we settle this in like so and then we just clip in this front edge and it’s done look at that perfect

oh no it’s perfect so we just snapped in that front edge oh now it’s perfect I’m pretty sure it’s perfect now it might not be all right then we reattach the t30 s we snap the panel back on you might as well watch this because we can talk about that little tab the mystery tab as it has been dubbed I still don’t know why it’s necessary hey look at that that happened and again because you can see where it attaches but there’s absolutely no reason for it I’m reasonably sure the dog is gonna start barking here in a moment so we’ll get ready for that so with this guy again we just open the door handle kind of line these things up like so you’re just gonna snap on like so then we take our magic pin you’ll remember the little funny side down stick it up BAM that’s it we’re done we’re rockin and rollin the dogs going to start barking we’re gonna join you for the tuning process and get rocking and rolling from there we’ve done the install this is this is kind of how we adjust the equalizer the processing the bass treble balance fader etc this is a logic seven or a Harman Kardon or a top hifi if you’re overseas this holds true across all the cars three five six seven nine etcetera there are nine series well there is now I just I just made it up so tone bass and treble flat balance fader middle equalizer surround surround you can turn it on or off off it goes volume settings PDC that’s your parking distance control turn that down it’s annoying gong turn it down annoying speed volume all the way down what that does as you turn it up it pulls out the lower frequencies from the music as the RPMs increase turn that off we don’t want that so go back over then we go into our equalizer and you can see I’ve got this flat for the most part that’s where you want it for you older guys that might be a little bit harder of hearing above 10,000 Hertz you’re welcome to fuss with that a bit they’ll play up to 20,000 Hertz not that you’ll hear it but you can adjust this up or down one or two you younger guys want all these this five and 10k is specifically flat one to two K that’s going to be most this 500 1000 and 2000 is gonna be our mid-range drivers so our hundred millimeter drivers in the door I like those flat 200 Hertz starts transitioning and rolling off into the mid Wolfers under the seats again flat maybe maybe at higher volumes down a notch at lower volumes if you like a little bit tighter midbass + 1 + 100 Hertz this is where just think of it in your mind as sub base just because you do this doesn’t mean it’s going to sound better at lower volumes it will but at higher volumes it needs to be flat maybe even minus one and depends on the music you’re listening to a lot of this is based on the recordings so the key is to having everything for the most part flat with minor tweaks at 2 5 and 10 K maybe 1 and 2 K are 100 and 200 Hertz bass and treble flat and most importantly perhaps is considering your source so we want it connected via USB if possible and if you’re using an iPhone or an iPod in your music settings so you’ll go settings music go down to there’s a three bank category where there is volume EQ and sound check we want the EQ off we don’t want it flat we don’t want anything we want it off we want the volume set about half way maybe two-thirds way and then we want sound check on that ensures consistent output a irrespective of the recording so if you’re listening to some old Pink Floyd that might be a little bit quieter on the output side and you listen to something modern which I don’t know any modern music maybe Katy Katy sire Perry whatever you know that’s probably gonna be recorded louder so turn that you know sound check on and it keeps everything consistent so that’s how we tune our logic seven harman kardon top hi-fi systems from here we are ready to rock we’ve done in this particular seven series the full stage one the full ghost upgrade the car sounds incredible hopefully you’re enjoying yours as well if not go visit a local hearing specialist he or she can help you and we will see you guys next time

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TRACO 2016: TGF beta and topoisomerase

OUR FIRST SPEAKER TODAY IS SONIA JAKOWLEW SHE GOT HER PH.D. AT RUTGERS UNIVERSITY, THEN SHE DID A POSTDOCTORAL FELLOWSHIP IN FRANCE, SUBSEQUENTLY SHE CAME TO NCI IN THE 80s AND SHE STARTED WORKING WITH MICHAEL SPORN AND ANITA ROBERTS ON TGF-BETA AND SHE IS GOING TO TALK TO US ABOUT THAT TODAY CURRENTLY SHE IS IN THE CANCER TRAINING BRANCH AND THE TITLE OF HER TALK IS TRANSFORMING GROWTH FACTOR BITA IN LUNG TUMORIGENESIS SONIA >> THANK YOU IT’S GOOD TO BE HERE TODAY I’LL BE TALKING TO YOU ABOUT TRANSFORMING GROWTH FACTOR BETA IN LUNG TUMORIGENESIS AND I WILL JUST TOUCH BRIEFLY ON THE LUNG CANCER DISEASE AND LEAVE THE HEAVY LIFTING FOR LUNG CANCER DISEASE TO THE SPEAKER COMING IN A FEW WEEKS TO THE CLASS SO LIKE MOST PAST YEARS, LUNG CANCER WAS DETERMINED TO BE THE MOST COMMON CANCER DEATHS AMONG BOTH MEN AND WOMEN IN THIS COUNTRY LAST YEAR THERE WERE OVER 221,000 NEW CASES OF LUNG CANCER THAT WERE DIAGNOSED IN THIS COUNTRY LAST YEAR AMONG BOTH MEN AND WOMEN, AND OVER 158,000 DEATHS DUE TO THIS DISEASE SO IT’S A SIGNIFICANT DISEASE BUT BECAUSE LUNG CANCER IS PRIMARILY CAUSED BY TOBACCO SMOKING, AND THE INCIDENTS OF SMOKING HAS DECREASED IN THIS COUNTRY, THANKFULLY, MOST CASES OF LUNG CANCER NOW OCCURS IN FORMER EXSMOKERS HOWEVER, THERE ARE MANY, MANY PATIENTS WHO HAVE LUNG CANCER WHO HAVE NEVER SMOKED SO THERE IS LUNG CANCER WHICH INCLUDES EXPOSURE TO RADONS, EXPOSURE TO COAL, AND COAL DUST AND THINGS THAT — ENVIRONMENTAL CONDITIONS THAT PRECIPITATE INFLAMMATION IN THE LUNGS SO, FIVE-YEAR SURVIVAL RATE FOR LUNG CANCER IS STILL NO LESS THAN 15% SO A LOT OF WORK HAS YET TO BE DONE ON THIS DISEASE NOW, I STARTED ON TRANSFORMING GROWTH FACTOR BETA OR TGF-BETA AND BEGAN IN THE EARLY 80s AND TGF-BETA HAS BEEN SHOWN TO BE A MULTI-FUNCTIONAL REGULATOR OF CELL GROWTH IT IS A RATHER POTENT INHIBITOR OF THE GROWTH OF MOST NORMAL EPITHELIAL CELLS AND WIDESPREAD EXPRESSION TGF-BETA HAS BEEN SHOWN TO PLAY A PIVOTAL ROLE IN MAINTAINING EPITHELIAL HOMEOSTASIS AND HAS COME TO BE ASSOCIATED WITH VARIOUS TYPES OF CANCER, INCLUDING LUNG CANCER SO, AND FINALLY, TGF-BETA HAS BEEN SHOWN TO HAVE WHAT WE CALL CONTRACT-DEPENDENT, INHIBITION OR STIMULATION OF CELL PROLIFERATION AND NEOPLASTIC TRANSFORMATION, DEPENDING ON THE ENVIRONMENTAL CONDITIONS SO TGF BAIT AT IS AN ATTRACTIVE CANDIDATE FOR NEW THERAPEUTICS INTERVENTIONS IN CANCER NOW TO UNDERSTAND TGF BAIT AYOU HAVE TO GO BACK TO THE BEGINNING AND THIS HAS ITS ROOTS IN ANOTHER GROWTH FACTOR SO IT WAS IDENTIFIED IN THE EARLY 80s AS BEING A POLYPEPTIDE THAT WAS SECRETED BY [ INDISCERNIBLE ] AND STIMULATED AND PERFORMED

COLONIES THIS WAS USED AS THE ASSAY FOR THE GROWTH FACTOR BY JOE AND GEORGE AT THE NCI IN THE 70s AND SHORTLY AFTER THE GROWTH FACTOR WAS IDENTIFIED, IT WAS DETERMINED THAT THERE WERE ACTUALLY TWO DIFFERENT PROTEINS THAT MADE UP THIS GROWTH FACTOR AND THEY WERE CALLED — ISOLATED FROM THE SARCOMA VIRUS TRANSFORM CELLS THAT THEY USED TO FIGHT SARCOMA GROWTH FACTORS AND ONE CLASS WAS SHOWN TO COMPETE WITH EPIDERMAL GROWTH FACTOR, EGF RECEPTOR BINDING AND THIS CLASS WAS PDF ALPHA AND ANOTHER – TGF ALPHA AND ANOTHER CLASS WAS HOPE IS NOT TO BE ABLE TO HAVE TGF BINDING BUT DID FORM COLONIES IN THE PRESENCE OF EGF AND THIS WAS CALLED TGF-BETA SO SARCOMA GROWTH FACTORS REALLY IS A GROWTH FACTOR THAT IS REALLY COMPOSED OF TWO DIFFERENT PROTEINS THAT FORTUNATELY OR UNFORTUNATELY, CARRY THE NAME OF TGF THEY ARE VERY DIFFERENT PROTEINS AND DO VERY DIFFERENT THINGS SO THIS WAS REPORTED BY ANITA ROBERTS AND MICHAEL SPORN IN THE EARLY ANXIETY SO FOLLOWING THIS — IN THE EARLY 80s SO FOLLOWING THIS THE LAB HASTENED TO PURIFY DFG BETA FROM ROVARIETY OF DIFFERENT SOURCES, INCLUDING HUMAN PLATELETS, HUMAN A ASENTA AS WELL AS BOVINE KIDNEY NOW TO DEMONSTRATE THE PURIFICATION FROM BOVINE KIDNEY, IDNEYALLY OBTAINED FROM THE FLORIDA HEALTH AND EXTRACTED FOR SEVERAL HOURS WITH ETHANOL AND THEN SENTRI FUSED TO GET RID OF THE WASTE AND THEN PRECIPITATED NVERNIGHT WITH ABOUT 50 LITERS 0 ETHER ETHANOL IN THE COLD ROOM NEXT MORNING IT WAS THEN DISSOLVED IN ONE MOLOR ACID AND THEN APPLIED TO 80 LITERS PE LITER WAS THEN SELECTED AND WHAT AW WHAT A ONE LITER IS AND THESE WERE THEN REDISSOLVED 100 GRAMS OFURIFICATION TISSUE THEY WOULD GET — MICOGRAMS OF TGF-BETA THIS IS THE ENORMITY OF THE COLUMNS NEEDED TO PURIFY TGF-BETA AND ON THE RIGHT, LOWER WORK, THIS IS 55-GALLON BARREL TO GIVE YOU SOME IDEA OF SIZE NOW THE ASSAY FOR TGF-BETA INCLUDED THE ROTH OF NORMAL RED KIDNEY CELLS AND RK CELLS SO TYPICALLY, TO PERFORM THIS ASSAY, A PLATE WAS MADE AND THEN A MIXTURE OF MEDIA SERUM AND RK CELLS, EGF AND SAMPLE, WHICH MAY OR NAY NOT HAVE CONTAINED TGF-BETA WAS APPLIED AND THEN ALLOWED TO INCUBATE AT 37 DEGREES FOR A WEEK AND THEN STAINED AND THE COLONIES WERE THEN COUNTED ON THE PLATES USING AN IMAGE ANALYSIS SYSTEM, WHICH IS SHOWN AT THE BOTTOM OF THE SLIDE HERE THAT WAS STATE-OF-THE-ART AT THE TIME SO, IF NO TGF BAIT IS PRESENT IN THE SAMPLE AND NO COLONIES WOULD BE COUNTED IF TGF-BETA IS PRESENT IN THE SAMPLE, THOUGH, COLONIES WOULD APPEAR OVER THE ONE WEEK, AND IF THEY WERE A SPECIFIC SIZE OR LARGER, THEY WOULD BE COUNTED SO, AFTER THE FINAL HIGH-PRESSURE CHROMATOGRAPHY PURIFICATION, SAMPLES WERE THEN

PUT IN A GEL AND RAN AND PURIFIED TGF-BETA, MIGRATED AS A 25,000 MOLECULAR WEIGHT PROTEIN ON SINGLE BAND ON THE GEL SO, DOCTORS MICHAEL PORN AND ANITA ROBERTS ARE CREDITED WITH THE DISCOVERY OF TGF-BETA AT THE NCI AND I HAD THE GOOD FORTUNE TO WORK WITH BOTH OF THESE INVESTIGATORS FOR SIX YEARS AND IT WAS ONE OF THE HAPPIEST AND INFORMATIVE AND EXCITING TIMES OF MY LIFE, MY RESEARCH LIFE AND THEN FORTUNATELY, WE LOST UNFORTUNATELY WE LOST ANITA FROM GASTRIC CANCER WHEN SHE PASSED ON, SHE HAD THE ENORMITY OF BEING THE SECOND-MOST HIGHLY-PUBLISHED FEMALE INVESTIGATOR IN THE WORLD AND MICHAEL S PORN, HAS SINCE LEFT THE NIH AND SHE ENDOWED PROFESSOR OF MEDICINE AT DARTMOUTH COLLEGE AT NEW HAMPSHIRE AND HE IS AT THE RIPE YOUNG AGE OF 84 STILL GOING AND STUDYING TGF-BETA LET’S HEAR IT FOR THE YOUNGSTERS SO FOLLOWING THE IDENTIFICATION AND PURIFICATION OF TGF BAIT ATHE SEQUENCE WAS DETERMINED AND ACTUALLY IT WAS FOUND THAT TGF-BETA WAS OR IS INITIALLY FORMED AS WHAT IS CALLED A PREPRO MOLECULE OF 391 AMINA ACIDS IT INCLUDES THE SEQUENCE AT THE END TERMINIS, A MIDDLE PORTION, WHICH WAS THEN CALLED THE LATENCY ASSOCIATED PEPTIDE OR LAP FOR SHORT, AND THEN TGF-BETA SEQUENCE AT THE SAME TERMINIS OF 112 AMINO ACIDS NOW THE AMINO ACID SEQUENCE HAS BEEN DETERMINED AND I GIVE YOU A PRETTY DOLLAR PICTURE DENOTING THE 9 CHARACTERISTICS SISSTEINS THAT HIGHLIGHT THIS CLASS OF PROTEINS AND I’LL TALK ABOUT THAT A LITTLE LATER SO, IT CONTAINS A SINGLE PEPTIDE, THEREFORE THIS IS A SECRETED PROTEIN NOW THE CRYSTAL STRUCTURE OF TGF-BETA WAS DETERMINED IN THE EARLY 90s THIS WILL HAPPENS TO BE A REPRESENTATIVE — REPRESENTATION OF TGF-BETA TWO BECAUSE FOR UNKNOWN REASONS, TGF-BETA TWO IS MUCH EASIER TO CRYSTALLIZE THAN TGF-BETA ONE SO TGF-BETA 2 AND 1, OCCURS AS A DIMER OF TWO IDENTICAL MONOMERIC CHAINS HELD TO GO BY A BOND WITH A HYDROPHOBIC POCKET, THAT OTHER PROTEINS CAN INTERACT WITH NOW, IT GF BETA IS A CONIKEAL MEMBER WHAT HAVE IS CALLED THE SUPER FAMILY THIS SUPER FAMILY CONTAINS, IN ADDITION TO THE TGF-BETA ISOFORMS, THE BMPs, GDF, GROWTH DIFFERENTIATION FACTOR, AS WELL AS ACT EVAPORATES AND INHIBITTANTS NOW TO SUMMARIZE WHAT I HAVE BEEN TALKING ABOUT TGF A 25,000 MOLECULAR DISULFIDE-BONDED HOMODIMER THERE ARE THREE HIGHLY HOMOLOGOUS ISOFORMS IDENTIFIED IN HUMANS AND THESIS ARE CALLED TGF-BETA, 1, 2 AND 3 THEY ARE SEPARATE PROTEINS BUT THEY ARE HOMOLOGOUS AND IN ADDITION, TGF-BETA 4 HAS BEEN IDENTIFIED IN BIRDS TGF-BETA 5 HAS BEEN IDENTIFIED IN AM PHIBIANS AND TGF-BETA 4 AND 5 HAVE MOST CLOSELY HOMOLOGY TO TGF-BETA ONE THE PRINCIPLE SOURCES IS TGF-BETA IN THE HUMAN ARE PLATELETS, BONE AND SPLEEN MOST CELLS HAVE BEEN SHOWN TO EXPRESS TGF-BETA AS WELL AS ITS RECEPTORS AND TGF BAIT IS – USUALLY SECRETED IN A LATENT, IN ACTIVE FORM WHICH MUST BE ACTIVATED BEFORE TGF-BETA CAN ACT ON EXPRESSION OR DO ANY OF ITS ACTIVITIES

AND WILL THE SUPER FAMILIES ARE TGF-BETA ENCOMPASSES NOT ONLY THE TGF-BETAS BUT ACTIVINS AND BMPs AND GDFs MEMBERS OF THE TGF SUPER FAMILY CONTROL CENTRAL CONTROL MODULES FOR A NUMBER OF BIOLOGICAL PROCESSES, INCLUDING DEVELOPMENT IMMUNE SYSTEM REPRODUCTION, ANGIOGENESIS, AGING, TISSUE REPAIR, METABOLIC REGULATION AND HOMOSTAYSIS SO IT EXPANDS A WIDE RANGE OF ACTIVITIES TGF-BETA CAN INHIBIT PROLIFERATION OF CELLS AND REGULATE ISOTOSIS AND STIMULATE ACCUMULATION OF EXTRACELLULAR MATRIX AND PROMOTE CHEMOTAXIS OF CERTAIN CELLS SO ANYTHING ELSE TGF-BETA CAN’T DO? THAT WAS THE QUESTION IN THE EARLY 80s SO, THE PATHWAY HAS BEEN DEVELOPED OVER THE YEARS AND THIS WE WILL CALL THE DEPENDENT PATHWAY BECAUSE OF THE INVOLVEMENT OF THE PROTEINS IN THIS PATHWAY SO THIS BEGINS BY THE LIGAND SHOWN IN BLUE AT THE TOP AND IT CAN BE TGF-BETA ITSELF AS WELL AS ACTIVIN INHIBINS AND BMPs, BY THE TYPE — LOST MY MOUSE SORRY THE TGF-BETA LIGAND BINDS FIRST TO THE TYPE II RECEPTOR, WHICH IS PHOSPHORYLATED ONCE THIS BINDS, IT THEN FORMS A COMPLEX AND RECRUITS ANOTHER MOLECULE CALLED THE TGF-BETA TYPE I RECEPTOR TO FORM A COMPLEX THE TYPE II TGF-BETA RECEPTOR THEN TRANSFERS TYPE I RECEPTOR AND THAT ENABLES IT TO THEN BE TRANSDUCED TO OTHER PROTEINS CALLED THE SMAD PROTEIN DEPENDING ON THE LIGAND IS, THE SMADS ARE SPECIFIC TO THE LIGANDS TGF-BETA ACT VINCE THEY ARE SMAD 2 AND 3 FOR THE BMPs, WE HAVE SMAD 1 AND SMAD 5 AND SMAD 8 SO THESE CARRIES THE SIGNAL OR TRANSPHOSPHORYLATED AND THEN DELIVERS THE SIGNAT TO ANOTHER SMAD CALLED SMAD 4, WHICH IS CALLED A — SMAD SO THIS SMAD IS UNABLE TO TRANSDUCE A SIGNAL INTO THE NUCLEUS WHERE IT CAN THEN PARTICIPATE AND AFFECT TRANSCRIPTION SO THE WHOLE PROCESS CAN BE CIRCUMVENTED BY WHAT ARE CALLED IN HIM TORY SMADS SMAD 6 AND SMAD 7 WOULD CIRCUMVENT THE WHOLE PROCESS SO THERE ARE REGULATORY SMADS THAT MAKE THE TGF-BETA PATHWAY GO FORWARD AND THERE ARE INHIBITORY SMADS THAT STOP THE PATHWAY ITSELF SO, IN ADDITION TO THE LABORATORY, CLINICALLY, TGF-BETA HAS BEEN STUDIED IN THE CLINIC AS WELL IT’S BEEN SHOWN THAT TGF BAIT AT IS A TYPE OF TUMOR SUPPRESSOR FOR EXAMPLE, GERMLINE MUTATIONS HAVE BEEN SHOWN TO OCCUR IN VARIOUS COMPONENTS OF THE TGF-BETA PATHWAY TO CAUSE FAMILIAL PREDISPOSITION TO CANCER AND THIS HAS BEEN SHOWN QUITE NICELY WITH SMAD 4 IN JUVENILE POLYFOCUS SYNDROME THE SECOND EXAMPLE IS TGF-BETA PATHWAY COMPONENT MAYBE SOMATICALLY MUTATED OR DELETED IN SOME HUMAN CANCERS NOW THE TYPE II TGF-BETA RECEPTOR HAS BEEN SHOWN TO BE MUTATED IN HUMAN, COLORECTAL CANCER WHILE SMAD 4 HAS BEEN SHOWN TO BE DELETED IN MANY CASES OF PANCREATIC CANCER

THIRDLY, REDUCED EXPRESSION OF TGF-BETA SIGNALING PATHWAY COMPONENTS ARE OVEREXPRESSION OF THE INHIBITORS HAVE BEEN SHOWN TO BE ASSOCIATED WITH CANCER DISEASE PROGRESSION AND FOR THIS IT IS DEMONSTRATED AS A TYPE I AND TYPE II TGF-BETA RECEPTORS HAVE BEEN OR CAN BE ORE EXPRESSED WHILE SMAD 7 CAN BE AS AN INHIBITOR CAN ALSO BE OVEREXPRESSED IN SEVERAL TYPES OF CANCER NOW, IN ADDITION TO BEING A TUMOR SUPPRESSOR, CLINICALLY, TGF-BETA HAS BEEN ALSO SHOWN TO BE A POSSIBLE TUMOR PROMOTOR IN THE CLINIC SO, ELEVATED LEVELS OF TGF BAIT AT 1 HAS BEEN SHOWN TO BE OCCURRING IN MANY ADVANCED TYPE OF HUMAN TUMORS AND TO CORRELATE WITH METASTASES AND OFTEN WITH CORE PROGRESSION AND I HIGHLIGHTED A NUMBER OF TISSUES IN WHICH WE SEE ELEVATED LEVELS OF TGF-BETA 1 IN TUMORS, INCLUDING THE LUNG AND SHOWN ON THE RIGHT IS A PATHETIC ADENOCARCINOMA THAT HAS BEEN HISTORICALLY STAINED FOR TGF-BETA 1 WITH A VERY SPECIFIC ANTIBODY AND TGF-BETA SEEMS TO SIT AT THE INTERFACE BETWEEN THE TUMOR CHROMATIN AND MICROENVIRONMENT — PA REVEREND MA — WHAT IS GOING ON? WHAT IS THE ROLE OF TGF-BETA IN CARCINOGENESIS? IS IT A HERO LIKE WE ALL THAT THE IN IN THE BEGINNING? OR HAS IT TURNED INTO A PART-TIME VILLAIN? WELL, THE ANSWER IS PROBABLY BOTH TGF-BETA IS A PROXIMAL EFFECTOR OF THE MALIGNANT PHENOTYPE IT’S ALSO A POTENT GROWTH INHIBITOR AND TUMOR SUPPRESSOR, ESPECIALLY EARLY ON IN THE PROCESS AND IT IS ALSO A PRO METASTATIC FACTOR NOW HOW DOES THIS OCCUR? WELL, OVER THE YEARS, A UNIFYING HYPOTHESES HAS BEEN DEVELOPED WHEREBY TGF-BETA CAN SWITCH FROM BEING A TUMOR SUPPRESSOR TO PRO-ONCOGENIC FACTOR AS CANCER PROGRESSES SO WHEN WE START IN THE NORMAL EPITHELIUM, THE NORMAL EPITHELIUM AND NORMAL CELLS, TUMOR SUPPRESSOR ACTIVITIES OF TGF-BETA DOMINATES OVER ANY PRO-ONCOGENIC ACTIVITIES BUT, AS CHANGE IS OCCUR IN THE GENETIC AND EPIGENETIC CONTEXT, THE LEVEL OF TGF-BETA RESPONSIVENESS GOES DOWN AND AS TUMORIGENESIS OR CARCINOGENESIS PROGRESSES, THE EXPRESSION AND/OR ACTIVATION OF TGF-BETA INCREASES SO WHEN WE HAVE A INVASIVE METASTATIC CANCER, THEN THE PRO-ONCOGENIC ACTIVITIES OF TGF-BETA PREDOMINATE OVER THE TUMOR SUPPRESSOR ACTIVITIES SO WE HAVE ESSENTIALLY A TUMOR SUPPRESSOR SWITCHING AND TURNING INTO A PRO-ONCOGENIC MOLECULE NOW, IN ADDITION TO THE SMAD PATHWAY, THERE ARE A NUMBER OF TGF — WHAT IS CALLED TGF-BETA SMAD-INDEPENDENT PATHWAY THAT IS ENTER INTO THE PICTURE AND THIS INCLUDES A NUMBER OF PATHWAYS SUCH AS THE MAP KINASE PATHWAY, THE REST AND THE PP2A PATH WAY WE WERE PARTICULARLY INTERESTED IN THE RAS MAP KINASE PATHWAY BECAUSE THE KRAS PROTOONCOGENE IS VERY IMPORTANT IN CANCER MUTATION IN AT LEAST 25-50% OF HUMAN LUNG TUMORS MUTATION OF EVEN ONE ALLELE OF KRAS CAN INCREASE APPEARANCE OF LUNG LESION EARLY ON AND IT’S BEEN DEMONSTRATEED

THERE IS CROSS TALK THAT OCCURS BETWEEN THE SMAD-DEPENDENT PATHWAY AND THE RAS/MEK SIGNALING PATHWAY IN SEVERAL TYPES OF CELL TYPES AND ACTIVATION OF THE RAS PATHWAY CAN MODULATE TGF-BETA SIGNALING THROUGH THE SMADS ED AND LASTLY, IN-VITRO STUDIES IN CELL LINES HAVE SHOWN THAT TGF-BETA DOMINATES OVER THE MITOGENIC AFFECTS OF RAS BUT WHEN RAS IS ACTIVATED, THIS CAN OVERRIDE THE ANTI-PROLIFERATIVE AFFECT OF TGF-BETA AND TURN IT MORE INTO A PRO-ONCOGENIC FACTOR AND IS THIS A PROBLEM SO, THIS SLIDE REPRESENTS AT LEAST FOUR DIFFERENT WAYS THAT THE TUMOR SUPPRESSOR CAN TURN INTO A TUMOR PROMOTOR HOW DOES THIS OCCUR? WELL, WE HAVE THE NORMAL CONDITION WHERE THE — WHERE ENORMOUS CELLS WERE TGF-BETA INTERACT WITH THE TYPE I AND TYPE II RECEPTORS THROUGH THE SMAD PATHWAY, AND OTHER PATHWAYS, AND TO RESULT IN TUMOR SUPPRESSION BUT IF SOMETHING OCCURS AND THE — SAY FOR INSTANCE THE DECREASE IN THE AMOUNT OR LEVEL OR EXPRESSION OF THE TYPE II RECEPTOR OR THERE MAY BE HYPERACTIVATION OF THE SMAD OF MAP KINASE PATHWAY WITH ACTIVATES RAS OR THERE MIGHT BE DECREASED SMAD LEVELS OR ACTIVITY, OR THERE MAY BE OTHER THINGS THAT MAYBE HAPPENING IN THE TUMOR SUPPRESSOR ARM OF THIS PATHWAY THEN YOU HAVE A TUMOR PROMOTOR SITUATION IN LIEU OF A TUMOR SUPPRESSOR SITUATION SO WE HAVE BEEN KIND OF INTERESTED IN THE HYPERACTIVATED MAP KINASE PATHWAY IN OUR STUDIES SO, THE BROAD GOAL OF MY LABORATORY WAS TO DETERMINE THE ROLE OF TGF-BETA IN THE DEVELOPMENT AND MA LIGNANT TRANSFORMATION OF LUNG EPITHELIAL CELLS AND THIS WORK WAS DONE IN THE EPITHELIAL CARCINOGENESIS SECTION OF NCI SO, WE HAVE THREE OBJECTIVES THE FIRST IS TO EXAMINE THE EFFECT OF TGF-BETA 1 DELETION IN KRAS MUTATION ALONE AND THEN IN COMBINATION ON LUNG TUMOR INCIDENCE AND PATHOLOGY THEN TO DETERMINE THE EARLY EVENT THAT MIGHT OCCUR IN THE DEVELOPMENT OF LUNG LESION AND HOW THEY PROGRESS AND THEN TO IDENTIFY ANY POTENTIAL SIGNAL TRANSDUCTION PATHWAY CHANGES THAT MAY OCCUR WITH INCREASES LEVELS OF TUMORIGENESIS SO TO DO THIS WORK, WE EMPLOYED FOUR MOUSE MODELS THE AJ, THE C57BL6TGF BAIT HETEROZYGOUS MODEL AND THE: [ READING ] WE GENERATED IN COLLABORATION WITH MIT SO, WE HAD TWO QUESTIONS DOES THE LUNG TUMORIGENESIS AFFECT THE TGF-BETA SIGNALING PATHWAY? AND THE RECIPROCAL QUESTION? DOES IT AFFECT LUNG TUMORIGENESIS? SO, WE TRIED THE AJ MOUSE MODEL BECAUSE THIS MOUSE IS VERY SUSCEPTIBLE TO CHEMICALLY-INDUCED LUNG TUMORS THE TUMORS DEVELOP IN A TIME-DEPENDENT FASHION THEY PROGRESS FROM HYPERPLASIA TO ADENOMA AND THEN CARCINOMA VERY SIMILARLY TO WHAT OCCURS IN HUMANS AND THE CARCINOMAS THAT DEVELOP ARE HISTORICALLY SIMILAR TO THE HUMAN LUNG ADENOCARCINOMAS, WHICH ARE THE CHIEF CARCINOMAS THAT APPEAR IN HUMAN LUNG CANCER AND THE SAME MOLECULAR MUTATIONS OCCUR IN BOTH HUMAN AND MOUSE LUNG TUMORS, SUCH AS OVER-EXPRESSION OF KRAS AND LOSS OF p53 EXPRESSION SO, WE USE ETHEL CARBAMATE AS OUR LUNG-SPECIFIC CARCINOGEN,

AND ETHEL CARBAMATE CAN GO THROUGH TWO PATHWAYS WHERE THERE IS A DETOX PATHWAY THAT RESULTS IN JUST HARMLESS ETHANOL ALSO A BIOACTIVATION PATHWAY THAT PROCEEDS TO ASSIST E1 IN THE LUNG AND THIS RESULTS IN VINYL CARBAMATE AND VINYL CARBAMATE APOXIDE WHICH ARE THEN ABLE TO BIND TO MACRO MOLECULES AND CAUSE NASTY AFFECTS SO, FOR PRODUCTION OF TUMORS IN THESE MICE, WE INJECTED ETHEL CARBAMATE IN 2-MONTH-OLD MICE AND THEN SACRIFICED THEM AT MONTHLY INTERVALS UP TO A YEAR WITH 20 MICE PER SACRIFICE FOR A STATISTICAL REASON SHOWN ON THIS SLIDE IS IMMUNOHYSTERICAL CHEMICAL STAINING OF TUMORS AT VARIOUS STAGES OF TUMORIGENESIS AND STAINED FOR SPECIFIC ANTIBODIES TO TGF-BETA 1 AND THE TYPE I AND TYPE II RESEPTEMBERRORS BEING AND YOU WILL SEE IN THE LEFT HAND AND RIGHT HAND MIDDLE PANEL THIS BROWN STAINING FOR TGF-BETA ONE AND THE TYPE I RECEPTOR THROUGHOUT THESE TUMORS HOWEVER, ON THE RIGHT FOR THE TYPE II RECEPTOR, STAINING FOR THIS PROTEIN IS ADMONISHED IN THE TWO MONTH TOMBORS AND ABOUT 4 MONTHS AND CATER MONTHS IT’S VIRTUALLY GONE SO WE SEE DECREASED EXPRESSION OF THE TYPE II RECEPTOR PROTEIN IN TUMORS WITH ADVANCING TUMORIGENESIS THIS IS SHOWN MORE CLEARLYO THIS SLIDE IF YOU’LL DIRECT YOUR ATTENTION TO THE LEFT SIDE OF THE SCREEN SPECIFICALLY PANEL D&D, THE RED ARROW SHOWS DISTINCT EXPRESSION OF THE BROWN STAINING FOR THE TYPE I AND TYPE II RECEPTOR VIRTUALLY IDENTICAL LEVELS IN TUMORS BUT IF YOU LOOK AT THE TUMORS IN COMPARISON, THERE IS INTENSE STAINING FOR THE TIME 1 RECEPTOR WHILE THERE IS VERY DIMINISHED STAINING FOR THE TYPE II RECEPTOR THIS IS ALSO WE LOOKED AT THE EXPRESSION OF THE mRNAs FOR THESE PROTEINS AS WELL AND USING FIVE CELL LINES, MOUSE LINE CELL LINES, WE SHOW EXPRESSION OF THE TYPE I AT VARIOUS LEVELS IN THESE CELL LINES WHILE THE EXPRESSION OF THE TYPE II RECEPTOR MESSAGE IS VIRTUALLY GONE IN THESE 2 CELL LINES AND I HIGHLIGHTED THE 5th, PCC4 BECAUSE THESE WERE DERIVED FROM A ETHEL CARBAMATE INDUCED MOUSE LUNG TUMORS THIS MIRRORS WHAT WE ARE SEEING IN THE AJ MOUSE SO WE ARE JUST SHOWING DECREASED LEVELS OF PROTEIN IN mRNA FOR TYPE II RECEPTOR WITH ADVANCING TUMORIGENESIS IN THESE MICE WE ALSO LOOKED AT LUNG TUMORS FROM BP INDUCED TUMORS WE SAW THE SAME THING IF YOU’LL DIRECT YOUR VISION TO THE RIGHT SIDE OF THE SCREEN, WE SEE DECREASED LEVELS OF EXPRESSION OF THE PROTEIN FOR THE TYPE II RECEPTOR AND DECREASED EXPRESSION OF THE MRNA FOR TYPE II RECEPTOR IN COMPARISON TO THE TYPE I RECEPTOR AND TGF-BETA LIGAND ITSELF WHEN WE STAIN FOR THE PROTEINS THEY ARE ALMOST IDENTICAL SO OUR MOUSE MODEL REFLECTS THERE IS WITH DECREASED EXPRESSION OF THE TYPE II RECEPTOR WE SEE CORRELATION OF LUNG TUMORS IN OUR MOUSE MODEL

SYSTEM OUR NEXT QUESTION WAS, DOES THE DELETION OF TGF-BETA WONG AFFECT LUNG TUMORIGENESIS? AND FOR THIS, WE SHIFTED TO THE BLACK TGF-BETA 1 MOUSE BECAUSE THE TGF-BETA KNOCKOUT MOUSE WAS ENGINEERED AND GENERATED IN THE EARLY 90s AND IT IS BORN IT THRIVES, BUT THREE WEEKS OF AGE, IT STARTS TO GET VERY SICK AND SUCCUMBS TO A GENERAL SYNDROME SO THIS MOUSE WON’T BE GOOD FOR CARCINOGENESIS STUDIES BUT THE KNOCKOUT MOUSE, THE TGF-BETA 1 HETEROZYGOUS LITTER MATE IS BORN, THRIVES AND SURVIVES T DOESN’T SUCCUMB TO A WASTING SYNDROME SO IN COLLABORATION, WE DID A STUDY AND LOOKED ON BECAUSE SHE WAS INTERESTED IN LIVER TUMORIGENESIS, WE DOSED THESE OR CHALLENGED MICE WITH DIMETHYL NITROSE MEAN, WHICH IS SPECIFIC FOR LIVER TUMORS AND WE SHOWED THAT THERE WAS AN INCREASE LEVEL OF TUMORIGENESIS IN THE LIVER OF HETEROZYGOUS MICE SHOWN IN RED COMPARED TO THE WILDTYPE MICE NOW UNEXPECTEDLY IF YOU FOCUS YOUR ATTENTION ON THE RIGHT SIDE, WE SAW EVEN GREATER NUMBER OF LUNG TUMORS FORMING IN THESE MICE SO THESE MICE HAD INCREASED TUMOR INCIDENTS OF LUNG TUMORS IN THEIR LUNGS SO THIS WAS SURPRISING SO IN ORDER TO BE ABLE TO LOOK AT IT IN A BETTER MOUSE MODEL, WE CROSSED WILDTYPE TO DERIVE TWO DIFFERENT PHENOTYPES, TGF BAIT AT HETEROZYGOUS MICE AND THE WILDTYPE LITTER MATE OUR PLAN WAS THEN TO TREAT THEM AS CARCINOGEN, ETHEL CARBAMATE AND LOOK AT THE LUNG TUMOR WE ISOLATED LUNGS AND STAINED THEM FOR TGF-BETA 1 ANTIBODY AND AS WELL AS LOOKED AT IN SITU HYBRIDIZATION FOR TGF-BETA 1 LIGAND AS WELL IF YOU LOOK AT THE TOP MANUAL, THE MIDDLE PANEL SHOWS DECREASED EXPRESSION OF TGF-BETA 1 PROTEIN AND MESSAGE IN HETEROZYGOUS MICE COMPARED TO WILDTYPE MICE AS WAS EXPECTED WE PERFORMED NORTHERN BLOTTING ON THESE MICE LUNGS AS WELL AS COMPETITIVE RTPCR AND THE SHOWED REDUCED EXPRESSION OF THE TGF-BETA 1 LIGAND AND HEIGHT ROWZYGOUS MICE COMPARED TO WILDTYPE SO WE HAD THE RIGHT MICE WE WENT ALONG AS WE HAD DONE WITH THE AJ MICE THIS TIME INJECTING WITH ETHEL CARBAMATE AS BEFORE IN 2-MONTH-OLD MICE WITH HETEROZYG US AND WILDTYPE MICE AND SACRIFICED GROUPS OF 20 MICE OVER A 12 MONTH PERIOD AND WHEN WE DID THIS, WE EXTRACTED AND LOOKED AT THE MICROSCOPEICALLY HYPERPLASIA ADENOMA AND CARCINOMA AND IN PANEL A, YOU SEE THAT IN PANEL B, YOU’LL SEE THAT IN THE TGF BAIT 1 TET ROWZYGOUS MICE DRAWN IN RED, HERE IS DEFINITE OCCURRING BY ONE MONTH WHEREAS IT TAKES 2-4 MONTHS BEFORE WE SEE ADENOMAS AND HYPERPLASIA OCCURRING IN THE WILDTYPE MICE

NOW THE STRIKING THING IS WE SEE CARCINOMAS APPEARING BY 4 MONTHS IN THE TET ROWZYGOUS MICE BUT TAKES ALMOST ONE YEAR AND 12 MONTHS BEFORE WE SEE ANY CARCINOMA STARTING TO APPEAR IN THE WILDTYPE LITTER MATES IN THESE MICE, WE SEE INCREASED TUMOR INCIDENTS BUT A DECREASE TUMOR LATENCY IN THE TGF-BETA ONE HETEROZYGOUS MICE COMPARED TO WILDTYPE MICE THIS IS WITH BORN ALLELE TGF-BETA SO WE THEN PROCEEDED TO STAIN FOR TYPE II RECEPTOR AND UNLIKE THE ORIGINAL MICE, WE SIGH REDUCED STAINING FOR TYPE II RECEPTOR PROTEIN AND CARCINOMAS BUT NOT IN THE ADENOMAS OR HYPERPLASIA SHOWN IN THE RIGHT PANEL SO IT SEEMS LIKE THE LEVEL OF OR TYPE II RECEPTOR IS MAINTAINED IN EARLY TUMORIGENESIS BUT IT IS OVERTAKEN WITH INCREASING TUMORIGENESIS SO WE ALSO LOOKED AT THE RELATIVE LEVEL OF THE TYPE II RECEPTOR mRNAs WITH INCREASING TUMORIGENESIS IN THESE MICE AND SEEMS LIKE DECREASING LEVELS OF THE TYPE II RECEPTOR mRNA WITH INCREASING LUNG TUMORIGENESIS FROM HYPERPLASIA TO ADENOMA AND THEN TO CARCINOMA SO OUR NEXT QUESTION IS DELETION OF TGF-BETA ONE AND MUTATION OF KRAS COMBINED AFFECT LUNG TUMORIGENESIS? AND FOR THIS, WE GENERATED THE TGF-BETA ONE HETEROZYGOUS KRAS LIKE R. MOUSE NOW I WON’T BORE YOU WITH THE DETAILS ESSENTIALLY WE CROSSED MICE AND C7 BLACK 6 STRAIN BECAUSE WE HAD TO GO BACK TO THE ORIGINAL BECAUSE WE HAD DEFINITE PROBLEM WITH NUMBER OF OFFSPRING GENERATESSED FROM JUST THE AJ MICE WITH — ACTIVATABLE MICE AND THIS IS HAVING WE GENERATED FOUR DIFFERENT PHENOTYPES, THE DOUBLE MUTANT TGF-BETA 1 WILDTYPE WHICH WE CALLED THE KRAS SINGLE MUTANT TGF-BETA 1K KRAS WILDTYPE WHICH WE CALLED THE SINGLE MUTANT AND THE LAST WAS THE WILDTYPE COMBINES SO WE LET THESE MICE GROW AND AT 6 MONTHS LOOKED — STARTED LOOKING AT THESE 4 MONTHS BUT THESE ARE 6 MONTH LUNGS AND IN A AND B, YOU SEE THESE PEARLY WHITE TUMORS THAT ARE FORMING ON THE EXTERIOR OF THE LUNG AND THESE ARE VERY EVIDENT IN THE DOUBLE MUTANT SHOWN IN PANEL A AND THE KRAS SINGLE MUTANT SHOWN IN PANEL B WHEREAS IN PANEL C AND PANEL D, THE TGF-BETA SINGLE MUTANT AND THE WILDTYPE DO NOT REALLY SHOW ANY TUMORS ON THE LUNG, JUST AN OCCASIONAL ONE VERY INFREQUENTLY WE LOOK AT THE AFFECT OF TGF-BETA ONE DELETION ON MOUSE SURVIVAL AND PARTICULARLY ON MORTALITY AND LIKE TO DIRECT YOUR FOCUS ON A PLAT FOR A&B A IS A DOUBLE MUTANT AND SIGNIFICANT DECREASE IN MORTALITY, AND LIFESPAN, AND B, WHICH IS THE KRAS SINGLE MUTANT IS ALSO AFFECTED BUT LESS SO COMPARED TO THE TGF-BETA 1 HETEROZYGOUS SINGLE MUTANT SHOWN IN C LIFESPANS IN THE DOUBLE

MUTANT AND SINGLE KRAS MUTANT MICE WE LOOKED AT PATHOLOGY OF LUNG LESIONS AND SHOWED INCREASE HYPERPLASIA AND ADENOMA IN THE LATE KRAS SINGLE MUTANT SHOWN IN GREEN BIIT SEEMS IN THE SINGLE MUTANT A PROTECTION UP TO A CERTAIN POINT BUT IN THE DOUBLE MUTANT THAT PROTECTION IS GONE AFTER CERTAIN TIME WE STAINED FOR TGF-BETA IN LUNG TUMORS AND SHOWN BY THE PINK ARROW, YOU SEE DECREASED EXPRESSION OF TGF-BETA ONE IN THE TYPE II RECEPTOR IN THE DOUBLE MUTANT TUMORS AND THEN DOUBLE MUTANT ON THE RIGHT WE SEE DEFINITE EXPEDITED REDUCTION OF TYPE II RECEPTOR AND DEFINITE INCREASE IN PRODUCTION OF SMAD 3 OCCURRING BY TWO MONTHS COMPARED TO 3 AND 4 MONTHS IN THE WILDTYPE SO WE PERFORMED A NUMBER OF SIMILAR WESTERN BLOTS IN ADDITION TO THE SMAD 3 WE SEE REDUCED EXPRESSION OF THE SMAD 4 AND SMAD 7 PROTEIN AS WELL AS INCREASED EXPEDITED EXPRESSION PRODUCTION OF KRAS AND RAV 1 WE ALSO LOOKED AT THE RAF 1 WHICH IS INTEGRAL IN THE KRAS PATHWAY AS WELL WE PERFORMED OR LOOKED AT APOPTOSIS INDEX IN THE SINGLE MUTANT AND DOUBLE MUTANT AND SHOWN IN RED WE SEE DEFINITE REDUCED APOPTOSIS IN THE DOUBLE MUTANT ADENOMA COMPARED TO THE SINGLE KRAS MUTANT SO, OUR MOUSE MODEL SHOWS AND SEEM TO CORRELATE THE DECREASED EXPRESSION AND PRODUCTION OF THE TYPE II RECEPTOR CORRELATES WITH INCREASED LUNG TUMOR PROMOTION, ACTIVATED KRAS MAP KINASE SEEMS TO CORRELATE NICELY WITH INCREASED LUNG TUMOR AND COMPROMISED APOPTOSIS CORRELATES WITH INCREASED LUNG TUMOR PROMOTION AND I DIDN’T SHOW HERE WHAT DECREASED SMAD 4 ALSO SHOWS CORRELATION WITH INCREASED LUNG TUMOR PROMOTION IN OUR MOUSE MODEL SO I’D LIKE TO ACKNOWLEDGE THE PEOPLE INVOLVED WITH THIS STUDY AND I’D LIKE TO THANK TYLER FOR THE KRAS DISPOSABLE MOUSE THAT ENABLED THIS STUDY SO I’D LIKE TO TAKE ANY QUESTIONS YOU MIGHT HAVE AT THIS TIME [ APPLAUSE ] [ OFF MIC ] >> PEOPLE ARE WORKING ON IT THE PROBLEM IT’S VERY TIME CONSUMING TO PURIFY AND VERY EXPENSIVE ABOUT 10 YEARS AGO, THERE WAS A GREAT PUSH ON TO TRY TO GET BETA IN WOUND HEALING AND THERE WERE CLINICAL TRIALS THAT WERE STARTED TO DO THIS BUT THE PROBLEM IS GETTING ENOUGH

FOR LONG TERM STUDY AND THAT IS A PROBLEM HERE AND THAT IS THE PROBLEM WITH MANY OF THESE GROWTH FACTOR STUDIES SO UNFORTUNATELY, IT’S STILL IN THE PIPELINE >> WHAT IS THE BEST TARGET? RECEPTOR, LIGAND OR SMAD >> TAKE YOUR POISON IT DEPENDS ENTIRELY ON THE SITUATION IN SOME SITUATIONS, IT IS THE LIGAND AND REMEMBER ALTHOUGH I CALLED IT TGF-BETA ALL THE TIME, WE ARE TALKING ABOUT THE TGF-BETA SUPER FAMILY SO THERE IS A LOT OF INVOLVEMENT OF MANY DIFFERENT MOLECULES HERE SO WHAT MAY WORK IN ONE TYPE OF CANCER OR DISEASE MAY NOT WORK IN ANOTHER TYPE OF DISEASE SO IT’S KIND OF A LOT TO KIND OF FUSS OVER IN THE BASKET OF GROWTH FACTORS YVES POMMIER RECEIVED NIH MERIT AWARD FOR LOUISIDATION OF SUMMERAISE AS A TARGET FOR ANTI-CANCER DRUG HE IS ALSO EDITOR ON CANCER RESEARCH, ONE OF THE DISTINGUISHED JOURNALS HE RECEIVED A PAUL EHRLICH AWARD AND PUBLISHED HUNDREDS OF MANUSCRIPTS AND WILL TALK TO US ABOUT DNA TOWNSHIP OTHERWISE SUMMERAISES AND POISONING AND ANTIBACTERIAL DRUGS TOPOSOMBER ACES SO WHAT I DID AT GIPPING OF THE THIS IS NOT A FIELD THAT WILL SHRINK ANY TIME SOON IT GOT A LOT OF — AND DNA COULD BE LOOKED UNDER DIFFERENT CONFIGURATIONS WHEN IT WAS UPGRADEIENT AND FAST — AND THAT WAS CALLED FORM ONE PLASMID DNA AND SOMETHING SLOW THAT WAS CALLED FORM TWO AND THEN WHEN BROMIDE CAME ABOUT FOR YOU IT IS GIVEN BUT IN THOSE DAYS IT WAS NOT A GIVEN AND GELS WERE DEVELOPED IT WAS REALIZED THAT FORM 1 WAS A SUPER TWISTED DNA WHICH THEN WAS CALLED SUPER COIL DNA WHEREAS THE FORM 2 WAS RELAXED, FULLY RELAXED DNA AND THEREFORE THERE WAS A NEED TO UNDERSTAND WHAT THE PROCESSES WERE TO GO FROM ONE TO THE NEXT FORM AND IN THE FIRST TOPOISOMBER ACE WAS ISOLATED FROM BACTERIA AND VERY SOON AFTER FROM MURINE TISSUE SO TO KEEP TRACK, I PUT SOME RETCHESES THAT ARE THE ONES THAT I WILL USE DURING THE PRESENTATION THAT IS WHY THEY ARE MINE BECAUSE THEY ARE REFERRED TO THIS SLIDE MY INTEREST HAS BEEN LONGSTANDING INTEREST IS TO TAKE ADVANTAGE OF THE TOPOICESOMER ACES FOR THERAPEUTIC PURPOSES AND DRUGGING THEM AND THIS IS PRETTY MUCH UPDATED AND MORE RECENTLY A TOOK ANOTHER CHALLENGE WHICH WAS TO WRITE A REVIEW WHICH IS JUST COME OUT SO IT’S NOT EVEN — THERE ARE NO PAGES YET BUT THAT WAS UNIVERSITY OF CHICAGO AND JOHNNY IS MORE EXPERT ON TOPO2 AND ON YEAST AND SO TOGETHER, WE WROTE THAT REVIEW, WHICH COVERS NOT THE BIOLOGY NOT SO MUCH THE DRUGS REALLY BECAUSE THIS WAS COVERED, BUT THE EMERGING NEW RICS MERGING ROLE OF TOPOISOMERASE IN TRANSCRIPTION, REPLICATION AND NOW VERY MUCH IN THE MIND OF ALL OF US IN GENOMICS STABILITY I’LL USE SOME OF THE SLIDES

IF YOU WANTED TO GO DEEPER, IS THERE A BOOK IN THE LIBRARY WHICH I PAINFULLY EDITED NOW BUT TWO 3 YEARS AGO I SWEAR I’M NEVER GOING TO EDIT ANOTHER BOOK BECAUSE IT STAY LOT OF WORK AND I’M NOT SURE MANY PEOPLE READ THE BOOK IT’S GREAT HAVE A BOOK BUT NOWADAYS EVERYBODY WANTS SOMETHING ON THE FLY BUT I LOVE BOOKS AND THIS IS ONE NICE TO HAVE IF YOU WANT TO LOOK AT IT THE CHAPTERS ARE ALL DIFFERENT ASPECTS OF TOPOISOMERASES IF YOU LIKE THAT SO LET’S GET INTO THE TOPIC SO NOT COUNTING SPO11, WHICH IS IN GERMLINE CELL, WHICH IS ESSENTIAL TO TO GENERATE DIVERSITY OF WHO WE ARE WHICH IS A TOPOISOMERASE PROTEIN SO NOT COUNTING THIS IN ANY OF YOUR CELLS, ESPECIALLY REPLICATING CELL, THERE ARE 3 TYPES OF ISOMBER ACES AND 6 TOPOISOMERASE GENES IN THE HUMAN AND THERE ARE THREE GROUPS THERE IS THE TYPE — WE CALL THEM 1, 2, 3 AND IT STAYS SIMPLE BECAUSE IN EACH GROUP THERE ARE TWO ENZYMES AND TWO GENES WHERE IT GETS A LITTLE MORE COMPLICATED IS THE FACT IF YOU LOOK AT THE TYPES, THERE ARE TWO TYPES TYPE I AND TYPE II, AND TYPES DEFINES THE FACT THAT THE ENZYME CLEAVES ONE STRAND AT THE TIME SO IT’S TYPE I, AND WHEN THEY CLEAVE TWO STRANDS AT A TIME, IT’S TYPE II NOT SO HARD TO REMEMBER AND THEN YOU GOT AN A HERE BECAUSE HUMANS WHEN THEY HAVE TYPE II A BUT IF YOU GO TO RK BACTERIA, THEY HAVE A LOT MORE TOPOISOMERASES THAN WE DO HAVE A DIFFERENT KIND IN HUMAN, THERE ARE ONLY TWO WAYS BUT HUMAN HAVE ONE A AND ONE B AND ONE A WAS NAMED BECAUSE IT WAS THE FIRST TYPE OF TOPOISOMERASE DISCOVERED IN E.COLI ANYWAY, SO TYPE I, TOPO1, IN THE TOPO1 ENZYME, I’LL GO BACK LATER ON THE DETAILS OF THIS THE ENZYME THIS IS DUPLEX DNA SO ENZYME CLEAVED THE BACKBONE AND THAT IS A WAY TO RELAX THE DNA OR DO WHATEVER NEEDS TO BE DONE TO CHANGE STRUCTURE OF DNA IN TOPO1, THERE ARE TWO ENZYME, TOP 1 AND TOP 1MT TWO DIFFERENT GENES AND CHROMOSOMES IN THE TOPO2, SO TOPO2 CLEAVED TWO STRANDS SO INSTEAD OF ONE BREAK ON ONE STRAND, YOU GET A BREAK ON BOTH STRANDS BUT EACH SIDE IS CLEAVED BY ONE HOMODIMER SO IT’S A HOMODIMER IN ALL CASES FOR THE TOPO2 AND THERE ARE TWO TYPES OF TOPO2 TOPO2 ALPHA AND TOPO2 BETA WE WILL COME BACK TO THAT THESE PROTEINS ARE RATHER BIG A SINGLE MONOMEROF TOPO2 IS 170 KILODALTON AS A DIMER IT’S TWICE THAT SO BICKER THAN A NUCLEOSOME AND THEN A TOPO3 CLEAVES ONLY ONE STRAND AS 2 GENES AND 2 ENZYMES, 3 ALPHA AND BETA WE WILL COME BACK AT THE END BECAUSE THIS IS THE EXPANDING FIELD IN THE TOPOISOMERACE ESPECIALLY THE LAST ONE IT HAS BEEN WORKED ON FOR MANY YEARS BUT IT’S COMING UP AS BEING VERY CRITICAL THE TRANSACTION FOR CHANGING DNA STRUCTURE GO THROUGH THE NIX OR BREAKS IN THE DNA, WHICH ARE CALLED CLEAVAGE COMPLEXES AND THE ENZYME MAKES THE BREAK BUT ALSO LIGATES BACK THE BREAK SO THIS NORMALLY VERY TRANSIENT AND THEN THERE ARE DRUGS THAT POISON THE BREAKS FOR TOPO1 AND TOPO2 THERE ARE DRUGS WE COME BACK TO THAT SO IF YOU LOOK AT THE BIGGER PICTURE NOW, IF YOU COMPARED HUMANS VERSUS E.COLI, SO IN HUMAN YOU GET THE TYPE I, WHICH IS TOP 3 ALPHA AND BETA, THESE ARE THEIR SIZE IN KILODALTON THE LINKAGE TO THE DNA WHICH IS COVALENT AND I’LL COME BACK TO THAT WHEN THEY DO THEIR TRANSACTION HOW MANY TIMES PAY PASS ONE STRAND AROUND THE OTHER? ONE TIME AT THE OTHER FOR THE TOPOTYPE I A AND TYPE I B, ABOUT SAME SIZE YOU SEE THE LINKAGE HERE, THE 3 PRIME END, THIS IS A VERY DIFFERENT FROM THE 1A AND 2A AND ONLY PASSES ONE STRAND AT A TIME AND THEN THE TWO A. YOU GET THE TWO ALPHA AND BETA LINKAGE 5 PRIME E.COLI ON THE OTHER HAND IS SIMPLER IN A WAY T DOESN’T HAVE 1B ONLY 1A AND IT HAS A TOPO1 IN

E.COLI AND TOPO3 E.COLI THAT’S WHERE IT GETS CONFUSING IT’S HISTORICAL ON THE TYPE II SIDE, ON THE OTHERP OTHER HAND, E.COLI IS MORE SOPHISTICATED IT GETS TWO ENZYMES AND TOPO4 EACH ENZYME IS MADE OF TWO GENES AND IT’S A TETRAMER SO ABSOLUTELY MORE COMPLEX BUT THE MECHANICS IS VERY MUCH THE SAME IN TERMS OF MEDICINE AND IMPACT ON THERAPEUTICS, YOU SEE ALL THESE ENZYMES, THE TOPO1 IS AN ANTI-CANCER TARGET OF DRUGS THAT ARE PRESCRIBED EVERY DAY THE 2S HAVE BEEN USED AS A TARGET BEFORE WE KNEW THAT DOXORUBICIN WAS TARGETING TOPO2 BUT THERE ARE VERY PROMISCUOUS DRUG IN CANCER TREATMENT DOCKSY RUBE SIN IS PRIMARILY USED AND TOPOSAID IS WIDELY USED AND USED IN CANCER AND IMMUNE DISORDERS AND A BIG CHURCHING OF TARGETING TOPOIS ON THE BACKSIDE GIANT RAYS AND TOPO4 ARE THE TARGET OF KWINN OHLONES IT’S A HUGE IMPACT IN BUO MEDICINE INCLUDING TB THOSE TARGET ONLY THE BACTERIAL AND THEY DO NOT TOUCH THE HOST TOPOISOMERASE GIVING THEM SELECT ACTIVITY THE DRUGS THAT TARGET THE HOST DO NOT TARGET THE BACTERIAL TOPO SO INSIGHT OF VERY SIMILAR STRUCTURES, THE DRUGS ARE VERY SELECTIVE SO DIFFERENCES BETWEEN TOP 1 AND TOP 2, YOU HAVE REGROUPING BECAUSE THEY HAVE THE SAME MECHANICS SO TOP 1S WORK AS MONOMERAND TOP 2S WORK ADD DIMERS OR TETRAMERS WHEN THEY MAKE THEIR TRANSACTION IN DNA, THE TOP 1 CLEAVES ONE STRAND AT A TIME AND LINKS THE TOW 3 PRIME END SO DRAWN HERE AS IT USES TYROSINE TO ATTACK DNA AND MAKES A COVALENT LINKAGE AND THEN A NICK IN THE DNA THIS IS THE OH AND THEN THIS CAN COME BACK AFTER THE DNA HAS BEEN RELAXED IF YOU HAVE A DRUG THAT GETS POISONED BY THE — [ INDISCERNIBLE ] AND OTHER DRUG ON THE TOPO2 SIDE, SO DIMER, LINKAGE TO THE 5 PRIME END AND 4 BASE PAIR STAGGER IS ALWAYS A 4-BASE PAIR STAGGER NOT LIKE A BLUNT DOUBLE STRAND BREAK LINKAGE TO THE 5 PRIME END, 3 PRIME HYDROXYL THE DRUGS ARE SPECIFIC TO TOPOTWO AND DOXORUBICIN AND TOPO4 KWINN LONES AND THE DIFFERENCES IS THAT TOPOONE IS A VERY ECONOMICAL ENZYME AND DOESN’T BURN OR NEED ATP TOPO2 NEEDS IT T REQUIRES MAGNESIUM TOPO1 WORKS EVEN ON ICE SO IT IS VERY EFFECTIVE TOPO2 DIFFERENT WORK AT ZERO DEGREES AND DRUGS ARE SPECIFIC FOR EACH TYPE OF ENZYME SO A GOOD DIVISION IN TERMS OF THE BIOCHEMISTRY AND THE PHARMACOLOGY FOR THESE TWO TYPES OF ENZYME IF YOU COMPARE THE MOST CHROMOSOME LOCATION SHOWING YOU THEY ARE INDEPENDENT IN THAT WAY AND THEIR NAME AND THEIR SIZE AND NOW PAY ATTENTION TO THIS BECAUSE OUR GENOME IS NOT JUST OUR NUCLEASE OUR GENOME IS THE MITOCHONDRIA AS WELL WHICH IS CALLED CHROMOSOME M AND CHROMOSOME M YOUR MOMMY CHROMOSOME, IS VERY ABUNDANT IN THE CELL AND TOPOISOMER IS — IN CASE OF TOPO1, THERE IS A SPECIFIC NUCLEAR ENZYME AND THEN MITOCHONDRIAL ENZYME, FOR TOP 2A AND B, THEY SERVE BOTH COMPARTMENTS AND CAN GO TO BOTH NUCLEUS AND MITOCHONDRIA AND THEY DO THEIR DEED ON THE CHROMOSOMES WHETHER IN THE

MITOCHONDRIA TOP THREE IS IN NUKE LAWS AND CYTOPLASM THE DRUGS YOU CAN SEE THAT SOME OF THE ENZYMES ARE STILL NOT BEING TARGETED AND WE DON’T HAVE ANY DRUGS FOR THE 3S AND THEN THE MECHANISM YOU UNDERSTAND BETTER AT THE END OF THE LECTURE BUT TOPO1 SWIVELS THE DNA AND MAKES ROW TAIN WHERE AT TOPO2 MAKE A STRAND PASSAGE WITH DOUBLE-STRANDED DNA AND THE TOPO3S MAKE A STRAND PASSAGE WITH SINGLE STRANDS AND THIS IS THE POLARITY, LINKAGE OF ENZYME AND THESE ARE THE REACTION LATER OW CAN GO BACK TO THIS ONE WHO HAS GONE THROUGH THE LECTURE SO THE WAY THE ENZYMES, WHAT THEY HAVE IN COMMON, IS THE WAY THEY BREAK DNA THEY ALL USE A TYROSINE AS THE NUCLEO FILE TO TAG THE PHOSPHOBACKBONE AND DEPENDING ON THE ENZYME IT HAS A DIFFERENT POLARITY OR GEOMETRY IN THE CASE OF TOPO1, THE GEOMETRY WILL LINK IT TO THE 3 PRIME END AND IN THE CASE OF TOPOTWO AND TOPO3, THE LINKAGE WILL BE THROUGH THE 5 PRIME END AND THIS SETS TOPO1 APART FROM ALL OTHER TOPOICE OMER ACES AND BRINGS IT TO THE FAMILY OF THE CRE RECOMINATE AND IT’S BELIEVED THAT THE TOP 1B EVOLVE FROM A RECOMBINASE USED FOR RELAXING — BUT MOST BASIC TOPOISOMERASES ARE THESE, THE 5 PRIME LINKAGES SO THIS IS GOING TO TALK ABOUT MORE DETAIL ABOUT TOP 1 AS I SAID THERE ARE TWO TOP 1 THERE IS NUCLEAR, WHICH IS THE ONE WE MOST COMMUNITY IS REFERRING TO WHEN WE SAY TOP 1 YOU HAVE TO UNDERSTAND THIS IS NUCLEAR TOP 1 AND WHEN WE DISCOVER TOP 1MT, WE MADE THE DECISION AT THE TIME NOT TO CHANGE — THIS REMAINED TOP 1 BUT JUST TO ADD M TO THAT ONE ONE TO SAY THIS IS MITOCHONDRIAL THIS DOESN’T GO THROUGH NUCLEUS THIS IS FOR CHROMOSOME M AND THIS IS FOR THE REST OF THE GENOME THE RELAXATION OF DNA BY TOPOISOMERASE WAS DISCOVERED EARLY ON WHEN RUBBING IN THE BROMIDE IF YOU TAKE THE DNA, USUAL AT THE RUNS FAST AND WHEN YOU PUT A DROP OF NUCLEAR EXTRACT YOU CAN DO IT IN YOUR LAB ON THIS DNA, IT WILL BE MIRACULOUS, YOU SEE YOUR DNA BECOMES RELAXED AND THE ENZYME DOING THAT WAS CALLED THE DNA TWISTING ENZYME BECAUSE THE DNA AND OVER TWISTED HERE SEE THE HELICAL STRUCTURE BUT IT’S OVERTWISTED AND THE TOPOISOMBER AS 1B WITHOUT ATP AND EVEN ON ICE, WILL RELAX IN DNA AND CONVERT IT TO A FULL CIRCLE NO BREAK FULL CIRCLE AND THE MANAGE SICK IN THE CLEAVAGE COMPLEX WHEN TYPO ISOMBER ACE SURROUNDS THE DNA, CLEAVES ONE STRAND AND THEN CONTROLS ROTATION OF BROKEN STRAND ANDCATHCULATE IN THIS BUILDING THE SPEED IS QUITE IMPRESSIVE 6000RPM SO IT IS ESSENTIAL AS IMAGINE FOR TRANSCRIPTION AND REPLICATION WHY? BECAUSE WHEN YOU TAKE A PIECE OF DNA, THE BINDING TO MOLECULAR MATRIX AND NEED TO TRIBE AND REPLICATE AND YOU WILL UNDERSTAND THIS IF YOU TAKE A ROPE AND THEN I HAVE ANOTHER ONE AND THEN ONE OF YOU IS GOING IN THE MIDDLE TO START TO SEPARATE THE STRANDS OF THE ROPE ON EACH SIDE YOU’LL GET SORT OF OVER WINDING AND UNDER WINDING ON EACH SIDE SO ONE SIDE YOU GET SUPER COALING AND ON THE NEGATIVE SIDE SUPER COALING IF YOU CAN NOT RELIEF THE TWO POSITIVE SUPER COALING, NO WAY YOU CAN GO BACK TO THIS BECAUSE IT WILL BE KNOTTED AND SO YOU NEED SOMETHING TO TAKE AWAY THE SUPER COIL THAT’S WHAT TOPO2 WILL DO WITHOUT MUCH EFFORT AND THIS IS ACHILLES HEEL OF TOP 1 NORMALLY IT’S VERY TRANSIENT BUT IF IT GETS STUCK THESE COULD BE EXTREMELY DANGEROUS AND THAT’S WHAT WE TAKE ADVANTAGE OF WAS ANTI-CANCER DRUG AND WE REVIEW

LATER THAT DNA DAMAGE CAN DO THAT IT CAN TRAP THE TOPO ISOMBER ACE 1 AND COMPLEX IS FORMED AS A WAY TO DESTROY CHROMATIN SO A FEW WORDS ABOUT SUPER COILING ALSO IN REVIEW IN THE CONTEXT OF CHROMATIN WHERE THE ROTATION OF DNA CAN STRAIN, DNA SUPER COILING UNDER TWISTING, IMAGINE OVER TWIST OR UNDER TWIST IF YOU GO IN A SENSE OF EXLICKS YOU OVER TWIST, YOU TIGHTEN LIKE YOU SQUEEZE THE WATER OUT OF SOMETHING AND THEN THE OTHER WAY YOU’LL JUST UNTWIST SO, ON THE RIGHT IS THE WHENEVER YOU HAVE MADE SO MUCH TWIST THAT THE STRANDS FLIP ON TOP OF THE OTHER SO TOP 1 AND MT REMOVES SUPER COILING BY DNA UP TWISTING THEY SWIVEL DID THE NA WHEREAS TOP 2 ALPHA AND BETA REMOVES — THERE A WORK AT THE CROSSOVER POINT AND YOU SEE WHY LATER AND SOME BASIC CONCERNING SUPER COILING SUPER COOLING TIGHTEN DNA AND NEGATIVE SUPER COILING OPENS HELIX AND THAT IS VERY IMPORTANT BECAUSE FOR ANYTHING TO GET INTO THE DNA YOU FIRST NEED TO OPEN IT A LITTLE BIT AND NEGATIVE SUPER COILING IS A FACT OF LIFE ABSORB AND RELEASES SUPER COILING SO YOU CONSTRAIN SUPER COILING WHEN YOU TAKE A NUCLEO SEEM AWAY YOU GET NEGATIVE SUPER COIL AND POLYMERASE GENERATE NEGATIVE POSITIVE SUPER COILING AHEAD AND TIME TO ACCUMULATE THE SUPER COILS AND NEGATIVE SUPER COILING BEHIND TOO MUCH POSITIVE SUPER COILING THAT WILL ARISE YOUR TRACKING ENZYME AND SUPPRESS TRANSCRIPTION, STABILIZE NUCLEOSOME NEGATIVE SUPER COILING FACILITATE MELTING OF THE STRAND AND THAT IS NECESSARY TO INITIATE REPLICATION AND TRANSCRIPTION IF YOU HAVE TOO MUCH NEGATIVE SUPER COILING, DNA CAN FLIP AND MAKE AN R LOOP, IT CAN MAKE A QUADROPLEX OR MAKE CDNA OR AT A VERY STRANGE STRUCTURE THAT ARE NOTED ALWAYS DESIRABLE Y SO NEEDS TO BE REGULATED SO THESE OTHER TWO TOP 01s, THEY ARE VERY SIMILAR IN THEIR BIOCHEMISTRY THE ONLY DIFFERENCE IN THE END TERMINIS IT HAS A VERY SHORT TARGETING SEQUENCE AND TOP 1 AS NUCLEAR LOCATION SIGNAL AND THE THING THAT TALKS TO PROTEINS, IT NEEDS TO BE DRIVEN TO WHERE IT HAS TO WORK IT CAN’T WORK EVERYWHERE IT WILL BE EXTREMELY DAMAGING IF IT WERE TO LET FREE THESE ARE PRIMITIVE IN — TOPO1B SUCH AS VACCINIA VIRUS AS OWN TOPO1B SO TOPO1S ARE SELECTIVELY TARGETED BY A CLASS OF DRUG ROUTINELY USED IN CANCER TREATMENT SO TOPOT CAN USED FOR OVARIAN CANCER AND SMALL CELL LUNG CANCER AND SECOND LINE, WHICH IS ALSO USED IN SOME PEDIATRIC CANCERS AND THEN BROADLY USED FOR COLON CANCER AND IN ASIA AND JAPAN AND OTHER INDICATIONS SUCH AS LUNG CANCER AS WELL SO BOTH OF THEM IN PEDIATRIC CANCER AND IN ASIA ALSO DRUGS AND THIS IS A NATURAL ON PRODUCT WHICH IS DERIVED FROM A TREE DISCOVERED AT THE NCI BY THE NCI AND THEN THE DRUG COMPANY JUST EVOLVED THIS TODAY SO THIS DRUG HAS BEEN APPROVED FOR 10 YEARS NOW SO THEY ARE NOT MOVING SO MUCH STILL VERY USEFUL THEY ARE NEW TOPO1 INHIBITORS BEING DEVELOPED 3 OF THEM ARE IN CLINICAL TRIAL HERE AT THE NCI WE MAKE THOSE DRUGS AND THEY ARE REFERRED TO NP744 AND 76 AND 400 AND IT JUST FINISHED PHASE 1 WE HAD ACTIVITY IN ONE PATIENT AND THOSE LIMITING TOXICITY WITH BONE MARROW NOBODY DIED THE QUESTION IS PHASE II OR NO PHASE II 766 IS FINISHING PHASE I NOW AND 744 WE BELIEVE IS ABOUT TO START PHASE I HERE SO THESE DRUGS, THE WAY THEY WORK IS QUITE REMARKABLE SO THIS IS A STRUCTURE AND TOP 01

REACTION TOPO1 FIRST DNA BY MAKING COVALENT LINKAGE 3 PRIME END NORMALLY DNA REALLIANCES AND THIS REVERSES VERY READILY THIS IS THEN IN THE BREAK SITE AND NO THE ANYTIME BRAKE SITE THAT’S HOW WE DISCOVERED OR HYPOTHESIZED THIS MECHANISM THEY WERE THE BRAKE SITES THAT TURNED INTO HAVE A 5 MINUTE MINUS ONE AND ONE PLUS ONE SO SUB SET OF THE TOPOONE CLEAVAGE SITE AR TRAPPED AS THE DRUG BINDS IN HERE SO WE PROPOSE THIS IN 1990 SOMETHING AND IT TOOK ABOUT 10 YEARS TO REALLY CONFIRM THIS THIS IS THE CRYSTAL STRUCTURE SO IN THE CRYSTAL STRUCTURE, THE DNA IS GREEN, THE BREAK IS HERE, THE DRUG IS THERE AND YOU CAN SEE EXACTLY LIKE HERE SO WHAT HAPPENS THE DRUG PREVENTS THE RELYINGATION OR LIGATION AND THEREFORE THE TOPOISOMERASE TRAPPED IT’S NOT COVALENT BUT IT’S TRAPPED FOR LONG ENOUGH THAT THE TOPOCAN’T GET AWAY AND THEN THAT LEADED TO COLLISION POLYMERASES AND DISASTER AND THEREFORE CANCER CELLS DIE THE NATURAL PRODUCT IT WAS FOR LONG TIME MYSTERRUOUS WIDE PLANT WHICH PRODUCED THE OTHER TREES AND WHY ARE THEY NOT KILLED BY THE TOX THANE THEY MAKE? AND INITIALLY WE THOUGHT IT WAS BECAUSE THE TOXIN WAS ONLY MADE IN THE BARK BUT THAT IS INCORRECT IT’S ALSO IN THE LEAVES AND WHAT WAS FOUND IS IN PRODUCING PLANTS, ENCODE A MUTATION ON THEIR TOPOISOMERASE 1 GENE, A SINGLE-POINT MUTATION AND THIS WAS REPORTED IN 2008 BY JAPANESE GROUP AND THE JAPANESE GROUP FOUND IN THE PLANT A MUTATION THAT WE HAD GENERATED OR SELECTED IN A HUMAN LEUKEMIA CELL LINE VERY RESISTANT WE HAD PUBLISHED THIS SOME YEARS EARLIER THAT TELLS YOU WHAT YOU DEFINE BY TARGETED THERAPY IS THAT IF YOU MUTATE THE TARGET AS ONE RESIDUE IT BECOMES IMMUNE TO THE DRUG THEREFORE THEY ARE ABSOLUTELY TARGETED AND THAT IS EACH MORE CLEAR IN THE YEAST BECAUSE YEAST YOU CAN DELETE TOPO1 AND YEAST CAN SURVIVE AT THIS STAGE, NOBODY HAS FOUND THAT HE COULD TARGET ANYTHING AND THE SELECT ACTIVITY IS DRIVEN BY THE FACT THEY JUST BIND INTO THE POCKET HERE INTO THE CLEAVAGE COMPLEX OF THE TOPOISOMBER ACE 1 AND THAT IS RESISTANT RESIDUE THERE NEXT TO THE DRUG BINDS AND INTERFERE WITH THE DRUG BINDING AND RENDERS ENZYME EFFICIENT AND DRUG RESISTANT WE HAD KNOWN FOR A LONG TIME THEY HAD LIMITATION YOU CAN CURE A MOUSE THERE WAS A PAPER IN SCIENCE IN THE OLD DAYS SHOWING YOU CAN REALLY CURE A MOUSE BUT YOU CAN NOT CURE A HUMAN BECAUSE YOU CAN NEVER GIVE ENOUGH DRUG BECAUSE THEY CAN’T PUT THIS IN CHEMICALLY UNSTABLE AND THE CHEMICAL STABILITY IS DUE TO THE FACT THAT THEY HAVE ALPHA HYDROXY — AND AS SOON AS — THESE AGENTS SO OTHER REASON TO

PUT THIS IN LIMITATION IS BECAUSE YOU DON’T WANT THE DRUG TO GO AWAY SO QUICKLY SO THAT IS WHY THESE DRUGS WERE DEVELOPED, THE ONE I MENTIONED BEFORE SO THESE ARE THE ADD NOWS THAT WE DEVELOPED AT THE NCI AND WHICH ARE IN PHASE I CLINICAL TRIAL THEY ARE HIGHLY POTEDDENT AND PURIFIED ENZYME AND ALL THE TEST HIGHLY SPECIFIC THEY ARE CHEMICALLY STABLE BECAUSE AS YOU CAN SEE FROM THE STRUCTURE, THIS IS ALPHA HYDROXY, AND OTHER DRUG RESISTANT SO THIS IS CLEAR AND ATTRACT A DIFFERENT SITE AND MORE STABLE SO THAT IS WHY THE CLINICAL TRIALS ARE GOING ON THERE IS ANOTHER CHANGE IN THE PARADIGM OF CLASSICAL CHEMOTHERAPY ONCE YOU FOUND A WARHEAD, WHAT NOW WE CAN REALLY START THINKING AND DOING IS TO DO TARGETED DELIVERY AND I’M A FIRM BELIEVE THER IS A NICE WAY TO GO YOU CAN TAKE A VERY TOXIC DRUG BUT NOT LETHAL AND MAKE SURE YOU CONCENTRATE IN THE TUMOR AND THIS HAS BEEN WORKED ON BY DRUG COMPANY YOU CAN SEE HERE 1, 2, 3, 4, 5 5 EXAMPLES OF ONGOING DEVELOPMENT OF WAR HED ASSOCIATED LIPOSOME TO PEG OR TO ANTIBODIES TO TARGET THESE DRUGS SELECTIVELY TO THE TUMOR AND MAYBE THEN YOU CAN DO WHAT WE — SO IN MICE, IF YOU INCREASE 10 TIME THE AMOUNT IN THE TUMOR, YOU PROBABLY GOING TO GET THE CURE WE SEE IN THE MICE SO I THINK A LOT OF CRIN CALL TRIALS NEED TO BE DONE TO LOOK AT THIS BECAUSE THEY MAY BE VERY HOPEFUL AND ONE WAS FDA APPROVED FOR PANCREATIC CANCER SO LET’S MOVE TO TOPO2 THERE ARE TWO THE ALPHA AND MAYBE BETTER NOW TO CALL THEM TUCKED AWAY BUT TIME WILL TELL OR TOPO2 BETA B THIS IS VERY SCHEMATIC BUT EASY FOR YOU TO REMEMBER TOP 2A IS REPLICATION ENZYME AND TOP 2B IS TRANSCRIPTION ENZYME ONE REASON TO KNOW THIS OR TO THINK THAT WAY IS THAT TOP TWO A. IS HIGHLY EXPRESSED IN REPLICATING AND CANCER CELLS AND IT’S NOT EXPRESSED IN NON-REPLICATING CELLS SO IF YOU TAKE YOUR HEART OR YOUR BRAIN, THERE IS NO TOPO2 WAY BUT IS THERE A LOT OF TOP 2BA IS DRIVING REPLICATION AND B IS THE HOUSEKEEPING TRANSCRIPTION ASSOCIATED ENZYME EXPRESSED IN REPLICATING AND DIFFERENTIATED CELL BETWEEN NEURONS AND MUSELS AND THIS YOU HAVE TOP 2B AND IT GOES BOTH TO THE NUCLEUS AND MIGHT ROW QUANDARYIA SO POLYPEPTIDES SHOWN HERE, THIS IS HUMAN TOP 2A AND TOP 2B AND WITH DOMAIN, THE CLEAVAGE RELYINGATION DOMAIN IS HERE, VERY SIMILAR YOU CAN SEE BUT THE CENTER IS HIGHLY CONSERVED IT’S HIGHLY CONSERVED WITH THE GYRATE AND TOPO4 — BINDING WITH A BUNCH OF RESIDUE AND THIS IS VERY CONSERVED WHEN YOU GO FROM HUMAN TO E.COLI STOW THAT IS THE WAY THEY WORK AND THEY WORK WELL THAT WAY SO WHAT DO THEY DO? HOW DO THEY DO THE TRICK? SO ONE WAY TO SEAL THE TRICK IS LIKE THIS YOU TAKE COMPLEXES OF DNA AND ONE IS THE GATE AND THE OTHER IS FOR T FOR TRANSPORT SO ONE IS A GATE AND THE OTHER GOES THROUGH YOU HAVE TO THINK OF THE NIH GATES BECAUSE IT WORKS THE SAME IT’S A TWO DOOR CAN’T GO ONE DOOR

SO FIRST GATE IS YOU’RE COMING TO THE ENTRANCE AND THIS IS CLOSED YOU GET INSIDE FIRST DOOR OPENS AND YOU GET IN THEN YOU’RE IN THE MIDDLE AND THEN YOU GOING THROUGH THIS OPENING ONCE YOU’RE IN IT, IT CLOSES BEHIND YOU AND THEN OPENS ON THE OTHER SIDE AND THEN IT CLOSES BEHIND YOU AND YOU HAVE GONE NOW YOU’RE INSIDE THE NIH SO THIS IS A TWO-GATE MECHANISM AND BURPS ATP AS IT DOES THAT WITH THIS MACHINE YOU CAN TAKE TWO CIRCLES, IF YOU HAVE A KNOT ON DNA, IT CAN UNKNOT IT BECAUSE IT WILL PASS THE STRANDS THROUGH THE OTHER IT CAN KNOT IT AND IT COULD RELAX BECAUSE YOU CAN IMAGINE THAT IF YOU RELAX SUPER COILING AND IT WILL DO THAT PRETTY WELL AND IT WILL RELAX POSITIVE AND NEGATIVE AND THE ENZYME THAT GENERATES NEGATIVE IS GIRAISE THE ONLY ENZYME, THE OTHER TOPORELAX THE SUPER COILING DO NOT GENERATE NEGATIVE 3 SO HOW DO THEY FUNCTION? HOW DO THEY INTEGRATE THE FUNCTION WITH THE BIOLOGY OF THE CELL? SO LET’S ASSUME YOU HAVE TRANSCRIPTION UNITED WHICH BARRIERS AND DNA HERE AND THE POLYMERASE IS THERE AND YOU HAVE THE RNA HERE SO AS THE DNA OPENS AND AS WE SAID, YOU HAVE TO GENERATE FIRST NEGATIVE SUPER COILING HERE NEGATIVE SUPER COILING WITH THE INITIATION OF POLYMERASE AND THE RNA WILL GET GOING AND THE POLYMERASE WILL MOVE BUT AS THE POLYMERASE MOVES AND I’M GOING TOWARDS — SUPER COILING ACCUMULATES TOPO1 WILL TAKE IT AWAY IN REGIONS WHERE NO NUCLEOSOMES, JUST USING THE SWIVELLING OF THE STRAND SO 2 AND 1 WILL TAKE AWAY THE POSITIVE SUPER COILS AHEAD OF THE FORK TOO MUCH NEGATIVE SUPER COIL WILLING GENERATE R LOOPS AND THIS WILL BE DONE BY TOPO1 AND 3 BETA WE COME BACK TO THAT THE PROMOTORS TO GET ACTIVATED RECRUIT TOPO2 BETA AND TOPO2 BETA IN THE PROMOTOR APPEARS TO MAKE TRANSIENT BREAKS NOT SO TRANSIENT THAT ENABLE INITIATION OF TRANSCRIPTION AND THAT IS WHERE TOPO2 BETA IS COMING BACK INTO FOCUS FOR NEURONS AND FOR OTHER SYNDROMES IN NEURONS OR AGAIN MUSCLE CELL, TAKE AWAY THE TWO ALPHA IT WILL BE SUFFICIENT AND WILL DO IT IF IT’S A CANCER CELL IT WILL USE BOTH TOPO2 ALPHA AND BETA TO DO THE TRICK IF YOU THINK OF REPLICATION AND ROLES THAT ARE VERY SPECIFIC IF YOU TAKE THE REPLICATION AND FIRST THOUGHT WAS THE INITIATION, AGAIN THE INITIATION OF REPLICATION NECESSITATED MELTING TO INITIATE THESE FRAGMENTS OF DNA AND WHEN YOU DO THAT, WILL YOU GENERATE NEGATIVE SUPER COILING BUT YOU NEED THAT NEGATIVE SUPER COILING THEREFORE IF YOU HAVE TOO MUCH TOPO1, YOU CAN NOT INITIATE REPLICATION AS YOU INITIATE THIS, GENERATE SUPER COALING IN THE FLACK AND YOU HAVE TO DISSIPATE THE SUPER COILING FOR THE FOLKS TO MOVE SO AFTER INITIATION WHEN THE HELICASE IS MOVING ALONG, WHEN YOU GENERATE THE SUPER COILING, IN FRONT OF THE REPLICATION FORT, THE TRANSCRIPTION TOPO1 WILL TAKE AWAY THE SUPER COILING AND NUCLEOSOME-FREE REGION AND TOPO2 WILL TAKE AWAY ALPHA IN THAT CASE MOSTLY IN THE CROSSOVER AROUND THE NUCLEOSOME VERY EFFICIENTLY

SO BEHIND THE REPLICATION FORK, YOU WILL GENERATE NEGATIVE SUPER COILING THAT COULD BE DISSIPATED BY TOPO1, TOP 2 ALPHA AND TOP 3 ALPHA AND THEN YOU CAN FORM — WHEN THE FORCE CONVERGING, YOU HAVE EXSAYS OF POSITIVE SUPER COILING AND IF YOU WOULD NOT TAKE AWAY THE SUPER COILING, YOU COULD NEVER GO ALL THE WAY SO THE TOPOICER MERWILL TAKE AWAY THE SUPER COALING AND TOPO1 AND 2 WHEN ALL IS FINISHED AND EVERYTHING HAS BEEN REPLICATED, YOU END UP WITH TOPO2 BY CLEAVING TWO STRAND AT A TIME WILL THEN DECAT ON 8 AND IN YEAST, IF YOU TAKE A TEMP SENSITIVE MUTE APT, IT WILL DIE BECAUSE IT CANNOT DECATENATE THE CHROMOSOME THE DRUGS FOR TOPO2, THERE ARE LOTS OF THEM TOPOAND TOX I RUBE SIN AND THEN YOU COULD SEE ALL THESE DRUG LOOKS SIMILAR AND DIFFERENT IN COMMON THEY HAVE THIS AROMATIC POLYCYCLIC THING AND SORT OF SIDE CHAIN BUT THEY REALLY LOOK DIFFERENT AND NOW IF YOU TAKE THE ANTIBIOTICS VERY SPECIFIC TO GIRAISE, AND TOPO4 BUT NOT THE HUMAN ENZYME, YOU CAN SEE THEY LOOK DIFFERENT AND THESE ARE ALL THE KWINN OHLONE THAT IS YOU HAVE ENCOUNTERED THESE ARE SPECIFIC FOR BACTERIAL ENZYME AND THOSE ARE SPECIFIC FOR CANCERS BUT NOT THE BACTERIA WHAT IS IN COMMON? WHAT IS IN COMMON IS INTERESTING IF YOU TAKE FOR EXAMPLE HERE TOPOSITE, WHAT IS CLEAR IS THE DRUG WORKING EXACTLY LIKE THE TOPO1 INHIBITOR THE STRUCTURE OF THE TOPOISOMER IS TO CLEAVAGE COMPLEX AND A TOPOSITE REVEAL THAT THE DRUG IN THE CLEAVAGE SITE LIKE THE BINDING NEW CLEAVE ANNUAL SITE OF THE TOPO1 CLEAVAGE COMPLEX AND ONE DRUG IN EACH OF THE TWO CLEAVAGE SITES YOU CAN SEE VERY INTRICATE GEOMETRY AND THE DRUG, THE PLANTERRING HERE TENDS TO CIRCULATE BETWEEN THE BASE PARIS AND THE SIDE CHAINS TEND TO REACH TO CONFER SELECT ACTIVITY SO HIGHLY SPECIFIC FOR ALPHA AND BETA THEY DON’T TOUCH TOPO1 AND IT IS BECAUSE THEY HAVE ALL THE INTRICATE INTERACTIONS AND THE ANTIBACTERIAL IS THE EXACTLY SAME PRINCIPLE IF YOU TAKE THE STRUCTURE RADIO – THIS IS A DIMER AND TOP 04 VERY SIMILAR MACHINE AND THE DRUG AGAIN IN THE CLEAVE ANNUAL SITE WITH THIS AGAINST THE BASE AND THEN HYDROGEN BOMBS TO SPECIFIC RESIDUES TO THE BACTERIAL TOPOISOMER AND THAT LED TO ONE CONSENT WHICH IS THAT THESE DRUGS MADE US REALIZE THAT NATURE DEVELOP A PARADIGM TO POISON THESE DNA ENZYMES TO BIND THE DRUGS AT THE INTERFACE OF THE ENZYME AND DNA SO THAT CAME OUT AND THIS IS WHAT WAS REFERRED TO AS THE INHIBITION PRINCIPLE VERY PROWL BECAUSE YOU CAN TARGET MICROMOLECULAR COMPLEXES YOU USE INTERFACES AS THESE THINGS MOVE AROUND AND THEN JAM THEM SO THAT’S WHAT — IF YOU LOOK AT THEM IN COMPARISON, YOU HAVE A SINGLE STRAND BREAK AND YOU STACK AGAINST THE BREAK WITH THE — AND TH EN HAVE HYDROGEN BOMB AND THEN TOPO2, YOU STACK AND HAVE DIFFERENT HYDROGEN BOMB SPECIFIC FOR THE PARTICULAR ENZYME THESE DRUGS DO NOT WORK AT PICO MOLAR THIS DRUG WORKS NOT VERY HIGH AFFINITY BUT HIGHLY SPECIFIC THEY WORK AT MICROMOLAR SO NATURE DOESN’T NEED TO REALLY GET PICO MOLAR WHEN IT HAS SUCH SELECT ACTIVITY SO TOPO3

WE CAN BE CONFIDENT IS THERE A CLEAR DIVISION OF LABOR TOP 3 IS REPLICATION AND IT’S A DNA TYPO ISOMBER ACE AND WILL WORK ON SINGLE STRAPPEDDED REGION BUT IT NEEDS TO HAVE DUPLEX DNA TO ENGAGE NOT ENGAGE ON SINGLE STRANDED DNA IT RESOLVES SOMETHING WHERE ONLY ONE IF I COULD SPLIT MY ARM, ONLY HALFWAY IS GOING THROUGH, NOT MY WHOLE THING AND THEN YOU CAN CUT ONE STRAND AND IT WILL RESULT AND PREVENTS RECOMBINATION BECAUSE AT THE OTHER REPLICATION — TOP 3 BETA IS THE HOTTEST NOW THE ONE WHERE I THINK WE LEARN THE MOST IT IS HIGHLY INVOLVED IN TRANSCRIPTION IT’S A DNA TYPO ISOMBER ACE RESOLVING R LOOPS AND ALSO RNA TOPOISOMER I DIDN’T TALK TOO MUCH ABOUT THAT SHOW HOW DOES IT WORK? IF YOU TAKE A REPLICATING CIRCLE WHEN YOU FINISH REPLICATION YOU HAVE THIS KATE ONIATED REGION WHICH CAN BE RESOLVED IF YOU HAVE TOPO2 BY JUST CUTTING ACROSS TWO STRANDS AT A TIME AND END UP WITH THE PRODUCT IF YOU DON’T DO THIS PERFECTLY YOU WILL END UP WITH SINGLE STRANDED REGION AND THESIS ARE HIGHLY RECOMGENIC SO IF IT DOESN’T DO THE JOB FULLY YOU NEED DO THE JOB BY TOPO3A AND IT WORKS WITH BLOOM SYNDROME PROTEIN OR WITH VERNER WITH THE — SO THIS IS TOP 3A AND THEN THE NEWCOMER IS THE 3 BETA AND OF AND THAT CAME TWO YEARS AGO, 3 YEARS AGO WHERE IT WAS FOUND FOR ONE NOBODY TALKED TOO MUCH ABOUT 3 BETA BUT TWO PAPERS CAME OUT IN NATURE AND NEUROSCIENCE AND THIS LIFTED THE WHOLE THING AND THERE IS A REGION OF PEOPLE WHO HAVE VERY HIGH FREQUENCY OF SCHIZOPHRENIA AND MENTAL RETARDATION A LOT OF THESE PEOPLE THERE AND WHEN THEY LOOKED AT THEIR GENOME AND LOOKED FOR WHAT WAS WRONG WITH THESE FAMILIES, THEY FOUND THERE WAS A DELETION IN THE TOPO3 BETA LOCUS AND THAT WAS A SURPRISE WHY WOULD TOPOTHREE BETA SO IMPORTANT FOR THAT? AND THEN AT THE SAME TIME, THE INSTITUTE OF AGING IN BALTIMORE WORKING ON TOPO3 BETA FORMED THAT IT WAS RNA TOPOISOMERASE SO THE IDEA IS THAT RNA IS SUSCEPTIBLE TO MAKE KNOTS AND TO BE UNTANGLED ESPECIALLY DURING TRANSLATION AND TOPO3 BETA IS IN THE CYTOPLASM AND ESSENTIAL TO RESOLVE RNA PROBLEMS AND IN NEURONS WHERE YOU HAVE VERY LONG GENES, THAT TENDS TO HAPPEN OR IF YOU HAVE R LOOPS TOPO3 BETA WILL TEP TO DISSOLVE THEM AND THERE ARE FACILITATE TRANSCRIPTION BUT IF YOU DON’T HAVE TRANSCRIPTION IN THE NEURON, THAT SHOWS IMMEDIATELY BECAUSE OF HIGH LEVEL OF SOPHISTICATION SO YOU END UP BEING SCHIZOPHRENIC OR MENTAL RETARDED IF SOME OF YOUR NEURONS DON’T FUNCTION RIGHT SO THE DIVISION OF LABOR BETWEEN THE TWO TOPO3s IS THAT THEY WORK OR DRIVEN TO THEIR SIGHT OF ACTION BY A DRIVER IN THE CASE OF TOPO3 ALPHA, IT IS RMI1 ALSO DISCOVERED BY IN BALTIMORE AND RMI1, TOGETHER WITH BLOOM AND GAUGE TOPO3 ALPHA IN REPLICATION IN TERMEDIATES AND RESOLVE THE JUNCTIONS AND THEREFORE IT IS CRITICAL IN TRANSCRIPTION, THE PARTNER IS TDR3 RNA BINDING PROTEIN AND THEN ENGAGES TOPO3 BETA ON THE RNA AND ON R LOOPS WITH THE FRAGILE X COMPLEX AS WELL SO THIS IS IT EMERGING AS BEING VERY IMPORTANT AND THAT TOPO3 BETA IS MUCH MORE IMPORTANT THAT WE HAD ANTICIPATED BEFORE AND THAT LEADS ME TO WHAT I WANT TO FINISH WITH IS THE TOPOISOMERASE AND GENOMIC

INTEGRITY AND HUMAN DISEASES WHICH IS SOMETHING THAT IS REALLY EMERGING NOW SO NOT ONLY DRUGS BUT DNA ALTERATION AND PHYSICAL LONG CALL PROCESSES LEAD TO THE PERSISTENCE OF COMPLEXES AND FOR ONE ENZYME, TOP 1 AND 2, WE HAVE MANY EXAMPLES WHERE PERSISTENCE LEAD TO GENOMIC DAMAGE SO I’M NOT GOING TO SAY ANYTHING ABOUT THE ANTI-CANCER DRUG BECAUSE WE HAVE COVERED IT AND WE US TO THERAPEUTICALLY BUT IF THE DNA IS VERY COMMON, THAT POISONS TOPO1 AND 2 AND BOTH IN THE NUCLEUS AND IN THE MITOCHONDRIA YOU HAVE 86 SITES SAME THING TOPO1 GETS SLUGGISH AND WILL GET COLD TOBACCO PRODUCTS EN USE AND TRAP THE COMPLEXES BOTH TOPO1 AND 2 NUCLEOTIDES ARE COMING BACK AND I’LL SHOW YOU SOME EXAMPLES WHY WOULD IT BE SO BAD? LET’S SAY YOU HAVE A TOPO1 CLEAVAGE COMPLEX WHICH NORMAL IS VERY REVERSIBLE AND NEEDS TO GO ON AND OFF AND VERY QUICKLY SO NORMALLY IT’S COUPLED WITH REPLICATION AND REPLICATION FOLLOWS BUT IF THIS IS TALK THE REPLICATION CAN COLLIDE AND THAT WILL GENERATE WHAT WE CALL REPLICATION RUNOFF T JUST COLLIDED AND NOW TOPO1 CANNOT LIE GADE ANYWHERE AND THAT LEADS TO GENOMIC DAMAGE AND I’LL TELL YOU AT THE END, HOW THIS IS RESOLVED, OR, ANOTHER TRICK IS THAT THEY COLLIDE BUT IT CAN REVERSE WE COULD TAKE ONE OR THIS ONE AND THIS IS A TOPO1 CLEAVE ANNUAL SITE WHICH ARE VERY FREQUENT AND IN THE GENOME MANY, MANY DNA BOUND LET’S ASSUME YOU HAVE A NICK TOPO1 IS OBLIVIOUS TO THE PRESENCE T WILL STILL CLEAVE HERE IF YOU HAVE QUICKED HERE AND TOPO1 MAKING HERE, THEN THIS MAKES DOUBLE STRAND BREAK AND THE DOUBLE STRAND BREAK AND DNA COMES APART SO WHAT HAVE YOU DONE IS SINGLE TOPOISOMERHAS CONVERTED INTO A DOUBLE STRAND BREAK AND ANOTHER THING LIKE THIS YOU IF HAVE A NICK ON THE SAME STRAND, TOPO1 CLEAVES UP STREAM AND THAT COULD FLY-OFF BECAUSE IT’S NOT HELD BY TOPO1 NOW SOUBED LOOK THERE AND THE MOST RECENT, STILL A REALLY ONGOING WORK IN OUR LAB IS THE INTERESTS THAT WE THINK OF THE GENOME BEING ALL DNA AND BUT WHAT IS COMING SUPTHERE IS A LOT OF RNA THAT GETS INCORPORATED INTO THE GENOME ON PURPOSE OR BY ACCIDENT AND THIS IS VERY FREQUENT IT’S BEEN ESTIMATED IN YEAST FOR INSTANCE THEY COULD BE 1-1,000 AND IN YEAST THIS RIBONUCLEOTIDE IS REMOVED BY RNAH2 IS ESSENTIAL SO IF YOU HAVE PATIENT YOU WILL GET A LOT OF THIS HOW BAD IS THIS? PRETTY BAD BECAUSE YOU HAVE THE POLYMERASE THAT GETS STUCK AND WHAT IS WORSE IS IF TOPO1 COMES AND HERE BEFORE RNA2, IT WILL IMMEDIATELY CONVERT THIS INTO A NICK AND TOPO1 BINDS TO THE END HERE OF THE DNA AND BECAUSE IT IS A RIBOWITH A TWO PRIME HYDROXYL, THEN ATTACKS THE FIRST AGAIN AND THEN LIBERATES THE TOPO1 BUT GENERATES A TWO PRIME, 3 PRIME CYCLIC WITH A NICK SO WHAT TOPO1 DOES IS ACTS AS RIBONUCLEASE AND THAT COULD BE PRETTY BAD AND BEEN SHOWN IN YEAST NOW THAT IF THAT IS GENERATED A SECOND TOPO1 SITE COULD BE FORMED RIGHT UP STREAM THAT IS WHAT I SHOWED YOU RIGHT BEFORE AND WHEN THIS FORMS, THIS COULD THEN DISASSOCIATE AND TOPO1 WILL RELY GATE ACROSS AND THIS WILL FORM TWO-BASE DELETION AND IN

REPEATED SEQUENCES, YOU END UP WITH THIS DELETION COULD BE REPAIRED BUT THAT’S A PROBLEM THE OTHER ONE WHICH I ALLUDED TO IS IF YOU HAVE MADE A NICK ON ONE STRAND, AND RELEASED TOPO TOPOCOULD JUST JUMP ACROSS AND GO TO THE OPPOSITE STRAND AND IF IT FINDS A SITE THERE IT WILL CONVERT INTO A DOUBLE STRAND BREAK SO A RIBOIN DNA CAN GENERATE A DOUBLE STRAND BREAK JUST WITH TOPO1 AND WE COULD DO THAT BIOCHEMICALLY SO WHAT ARE THE HUMAN DISEASES LINKED WITH TOPOISOMER ACES SO FAR? THE LIST MIGHT BE EXPANDING IN THE CASE OF TOPO1, WE ARE LOOKING AT NEUROLOGICAL DISEASES DUE TO THE LACK OF REMOVAL OF TOPO1 CLEAVAGE COMPLEXES, AND I’LL GIVE YOU A LITTLE KEY AT THE END OF WHY THIS IS TOPO2B, WHEN TOPO2 CLEAVES DNA, IT NEEDS RETORELY GATE IN THE PROMOTOR IF IT DOESN’T RELY GATE QUICKLY, ANOTHER PIECE OF DNA CAN DO A TRANSLOCATION AND THE CAUSAL IMPLICATION OF TOPO2 BETA IS NOW PRETTY WELL ESTABLISHED AS IA SOURCE OF LEUKEMIA, SECONDARY LEUKEMIA AND PRIMARY, AND IN TRANSLOCATION SITE IN PROSTAT CANCER WHEN THE ANDROGEN RESPONSIVE GENES A TOPO2 BATA SALE IN THE PROMOTOR AND KNACKED LEAD TO RECOMBINATION IN THE CASE OF 3 BETA, AS INDICATED EARLIER, THE LACK OF 3 BETA LEADS TO NEURODEVELOPMENTAL DISORDERS SCHIZOPHRENIA AND COGNITIVE IMPAIRMENT AND THEN, IF YOU HAVE A LACK OF REPAIR OF THESE CLEAVE ANNUAL COMPLEXES, YOU END UP WITH THIS TYPE OF DISEASE NEUROLOGICAL DISEASE IN THE CASE OF THAT ENZYME AND WILL — [ READING ] IF YOU CAN NOT REMOVE THE TOPOCLEAVE ANNUAL COMPLEXES, I’LL SHOW YOU HOW THEY DO IT, SO THE WAY THE TOPOISOMER IS REPAIRED AND BECAUSE OF THE DANGER, ALL SPECIES GOING ALL WAIT THROUGH YEAST HAVE BATTERY OF EP ZYMES TO REMOVE TOPOCOMPLEXES AND SO WHEN THEY ARE EFFICIENT, THAT’ST THAT’S WHEN YOU END UP WITH DISEASES HOW DO THEY DO IT? YOU CAN LOOK AT THE REVIEW OR FIND A LITTLE SCHEME THAT IS SAYING, SO IF YOU HAVE A TOPOISOMER, IT IS LINKED TO THE 3 PRIME END OF KNA HERE AND THIS IS THE DNA THERE IS A SPECIFIC ENZYME LINKED TO THE 3 PRIME END AND DNA THE ENZYME WILL CLEAVE IN THE MIDDLE HERE AND IT REMOVES TOPOONE AT THE SITE OF LINKAGE AND REGENERATES 3 PRIME PHOSPHATE IN THE CASE OF TOPO2, A SPECIFIC ENZYME IN HUMAN AND VERTEBRATES AND YEAST SAME ENZYME BUT IN HUMANS YOU GET A TDP TWO ENZYME SPECIAL AND SEE THE POE LATERY IS THAT THE TOPOTWO IS LINKED TO THE 5 PRIME END SO TDP2 WILL AGAIN REMOVES THE TOPO2 AND REGENERATE THE 5 PRIME FIRST FATE CELL HAVE A BACKUP AND ALTERNATIVE PATHWAY TO CHOP THE PIECE OF THE DNA A LITTLE BIT OF STREAM OF THE TOPOISOMBER ACE LINKAGE BY NIKE LACES AND THESE NUCLEASES ARE VERY COMMONLY KNOWN LIKE CTIP THAT COU3 KNOWN LIKE CTIP THAT COULD CLEAVE THESE AND THEN GAP REPAIR SO THE NCI DIRECTOR FOR TRANSITION RESEARCH IS DOING

CLINICAL TRIALS WITH HIM AND THEN THE DEVELOPMENTAL THERAPEUTICS PROGRAM HERE WERE VERY CONNECTED TO THEM SO IF YOU HAVE ANY QUESTION, NOW I AM HAPPY TO TAKE THEM [ APPLAUSE ] [ OFF MIC ] >> IT IS HARD TO TELL WHERE THE HETEROZYGOTES HAVE MINOR FORMS OF THAT AND IT’S VERY NEW IT WAS FORMED IN A PATIENT THAT HAS SYNTHETIC DISABILITY SEIZURES AND FOUND TO START TO SCRATCH THE TIP OF THE ICEBERG I THINK YOU ARE ALL BURNED-OUT? YOU CAN LOOK AT SLIDES AND SEND 01:51:08.328,00:00:00.000 ME E-MAIL IF YOU HAVE ANY

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Hirschsprung disease consequent to mutations in the RET gene regulatory … – Aravinda Chakravarti

Aravinda Chakravarti: Okay, good morning Thank you to the organizers for inviting me, and as I was telling a few people, it’s been a long time since I’ve attended a pure genomics meeting, and I’ve sort of forgotten — meaning I go to more human genetics than genetics-oriented meetings, and I’ve forgotten how much fun this used to be, or still is — I should say So, so given my current perspective, what I thought I was going to do is speak to you about a specific example that I think will exemplify many of the problems we already discussed yesterday, so I’m not going to speak specifically about ENCODE, but rather how ENCODE kind of data leading to the solutions of problems that we simply didn’t have in human genetics So to give you a perspective on disease, at least what the disease community or people who interest in the path of physiology of human disease, what it is that they want and why it intersects so strongly with genetics is, I sort of used a quotation from really in medicine a very well-known book by Barton Childs who used to be a colleague of mine and has passed away unfortunately a few years ago And I’ll recommend this book for you It’s called Genetic Medicine: A logic of disease, that’s what genetics can provide And he poses three very simple, but cogent questions that patients want to know, and of course we as scientists wish to answer, which is, “Why me? Why this disease? And why now?” And if you think about it, you would appreciate that the first one has to do with the question of genotype, the second one has to do with specificity, and the third with many aspects, but clearly epigenetics and environmental factors come into play So I think for a long period of time we’ve been preoccupied really with just mapping human disease genes, both simple and complex for a very simple reason This was a very, very slow task and you can’t do genetics without at least knowing what unique identifiers are And ENCODE has now pushed us into this much larger land of trying to understand how cis-regulatory elements or enhancers as just a shorthand can provide us a view of disease that we never really had And I think we all know that even though we are at the very beginning stages of just doing a simple intersectional of finding sequence variance and various kinds of motifs and cis-elements of some defined function that this is really very incomplete What we would want to know is answer the question of much more mechanistic and qualitative term and these issues have already come up in the meeting yesterday So I’m going to give you some reasonable speculation, some complex disease genetics say after 10 years or more A long history Mendelian genetics and 10 years or more of many, many groups around the world trying to do GWAS kind of mapping and probing the answers And the first one is that it’s quite clear that there are many, many genes as originally expected anywhere from hundreds to thousands; Height [spelled phonetically] already has 800 sort of identified SNPs or more in different locations, so each trait is going to be composed of many, many different components Even though we sometimes focus on the statistical aspect of how much of the phenotypic variance we’ve explained there’s a whole variety of analysis very well done that actually sort of emphasize the fact that common variance, though say with the frequency of about five percent, 10 percent, that in many cases they explain at least half of the trait and disease variation We haven’t found most of them, and this is where ENCODE, of course, in reverse, can be very, very important And so the question is if we don’t have the statistical power to map them individually, which is the third point, we might never be able to find all the individual SNPs that contribute individually to Height or diabetes or hypertension, let alone even the rare ones so we have to find other ways of doing it I think at least much of our data has suggested and clearly most of the mapping data that each locus been mapped by GWAS, they may in fact have more than one culprit gene in many cases And clearly they are many, many individual SNPs that contribute to the observed association And so one of the really big challenges is how do we get to the true genetic structure for each given gene and locus that we come across? And at the end I’m going to come back to this issue that we’ve done genetics, we understand the importance of the recombination map to focus on the bid of the genome where a particular part or a phenotype maps, but I would ask you to think, and I think we should

argue for a while that what ENCODE has brought to us is the idea of physical interactions between distal elements and a gene, and so we have to consider the physical distance We will have to step out of the genetic recombination haplotype, and haplotype, as I will show you in my example is often really defined by the functional piece of DNA that is relevant Not only what the recombination distances are that we statistically focused on Okay, so here’s the example We’ve studied this phenotype for many, many years for — as a model to understand what really happens in complex disease genetics And many of the things that or I should say aspects of complex traits that we take for — that we take as given today We’ve been able to show using Hirschsprung’s Disease as a model for actually many years I’m not going to talk much about the phenotype except to say that what it is a defect of an enteric of a system Most of us don’t think about the fact that we have an intact almost independent nervous system in our gut and this is a disorder in which these children are born with absence of the enteric nervous system — the gut that leads to a functional obstruction and needs to be repaired in the all kinds of consequent — both problems that comes with efficient repair as well as without repair As disorders go, it is relatively rare It’s about one in five thousand live births, but this of course is 10 times or more common than most Mendelian disorders It is clearly multi-factorial from a variety of evidence, it’s got a four percent recurrence risks, and many, many other features that has allowed us to probe the genetics At a molecular level, the study is done for a long time we know three essential bits And the three essential bits is much of the genetic defects that we’ve known arise because of defects in two major signaling pathways Is this the one — the one in the middle is the laser, right? They are cut through two major signaling pathways This is a receptor tarzine carnays [spelled phonetically] pathway mediated through RET, and this is through a G-protein couple receptor, the Endo Teelandtype B [spelled phonetically] receptor and all of the other components, and I’m not going to go into details There’s very strong epistasis between these two pathways and we have a crude understanding as to how this epistasis arises The second feature that is quite strong is that the disorder really occurs because of an interaction, or I should say failure of an interaction, between what these cells, called Mesenchyme do — they actually secrete menotrolophic [spelled phonetically] factors now we know they secrete other factors that are important for the maturation of enteric neurons and the enteric neurons are the neuroblasts themselves So we at least have a crude understanding of the mechanism is, that is if you have a defect here or you have a defect here that will lead to a failure of these two kinds of cells to communicate early in development, you get failure of these cells to develop and differentiate and proliferate and then differentiate and you get lack of these cells with is the A-genosis [spelled phonetically] that you see in the gut Third, they’re many genes that we have very definitively mapped or we find mutations in Hirschsprung Disease, but they don’t fit in this pathway, say that they’re — so of course there are many, many other portions that of the gut by the physiology that impacts on this that we’re trying to find out But basically, RET is the major gene, and I’m just going to tell you, and maybe in question and answer we can discuss it, that we now know that almost every Hirschsprung patient has a RET mutation Most of them are non-coding mutations, but almost every one of them have it The central feature for today’s discussion is that even it was found through severe, syndromic forms of this disorder, we now know that there are many, many mescense [spelled phonetically] mutations individually rare, none of them are of full penetrance, and of course we found analogous mutations in many other genes of the same pathway SOX10, which is its transcription factor; GDNF, its ligand; and GFRA1, a very important co-receptor RET is sort of an unusual RTK in that it requires its co-receptor The surprise came in trying to explain Hirschsprung Disease several years ago There’s a study that we did originally with Eric Green in the old days when you had to clone out pieces of DNA, you had to sequence, you had to do — we did multi-species, comparative genomics to identify regions which we then tested as enhancers and we found a very common variant It’s about 25 percent in the general European population or those of European ancestry,

but it is a gut-specific enhancer It is bound by SOX10 and this polymorphism disrupts this binding and leads to low penetrants, but still quite highly important mutation or variant for this disorder Interestingly, it shows this form, that is a coding, is involved in the long and severe forms, whereas the non-coding is involved in the short segment of the much more common forms Because of this finding, we argued recently because we knew of other enhancers in this region, that any regulatory variant that would reduce RET expression, which this one does, which clearly all these other coding mutations should have similar effects So the idea is if we can find one loss of function mutation any other loss of function mutation that would lead to loss of RET expression, even through non-coding, should lead to that And this is where this stands from this screening and you can image that ENCODE has played a — this is just a summary — has played a very big role in this We now have three such different non-coding polymorphisms Here are the RS numbers which I never remember RET+3 is the one that sits in the first lodge in front of [spelled phonetically] RET — these two are further away as you can — I think there is a distance here, this is roughly 10KB from the transcription start site RET-1 is the non-coding polymorphism that’s much further away, it’s about 125 kilobases upstream This is about 18 kilobases upstream, so closer to RET They have very large allele frequencies, and here are the odds ratios And these odds ratios, by any count, four or close to two, are really phenomenal so far as complex disorders go is why it makes this as a model But if you did the genome-wide association study as we did this would clearly be recovered in fact which is why we found it early This one was discovered, was recovered from a genome-wide association study, you would not take this, because you would say well this is just another polymorphism that is high in LD in some sort, and of course it is not genome-wide significant And the point that I’d like to make is once you get close to a locus, you have to try and interrogate the effect of many other polymorphisms that actually do interrupt the known elements So, here is what we did in order to find these three that is throughout the region We did a much more forward kind of screen by looking at — we looked at 125 SNPs that were all of major frequency that were peppered through this region in ENCODE defined elements of various sort Thirty-four of them were Hirschsprung Disease-associated, that is by a conventional GWAS-B value Eight of them lay within ChIP-seq peaks In this specific cell line, this is a human neuroblast cell line which is sort of relevant, really a relevant cell type in that sense And all of these were enhancers, these Grice enhancers, the enhancers we really identified the hard way in this collaboration with Eric Green about 10 years ago, and what we find is three of them also showed allelic differences by doing various kinds of recorder assays I think the effect sizes here are all substantial, not only individually, but combinatorially, so these — they are in a region in which there is some residual linkage to saquillibrilium [spelled phonetically], but as I will show you that the effects are not all at the same time in development They act in different times is development, in different combinations, and that’s the basis for finding genetic interactions, even between these non-coding elements You know, these SNPs show significant levels of LD in the sense that r2 is small, but they all lie in a pattern, and therefore they are relevant to saquillibrilium and all large, and therefore what we can do is to identify a major source of missing heritability at this locus So I’ll very quickly go through; I think I’ve generally explained this So here are the three polymorphisms In this particular case, many of these DHS sites and other such as histone [spelled phonetically] marks that came from the — both ENCODE, as well as some from the road map project, and this is an intersection of all the SNPs that I told you about, that anyway, that led to those three If you looked at the transcription factors as well if you looked at the sequence, that was right under any of those SNPs we found, we’ve known before that RET+3 interrupted a SOX10 binding site and this one suggested that it was a RET not-acid [spelled phonetically] receptor site, later that we proved was RER Beta and this one was a GATA [spelled phonetically] site, this later shown to be either GATA 2 or 3 — in fact, we find effects on both So these are just luciferase assays to show that these three elements, at least apparent

controller wild type [spelled phonetically] form, they do show an effect, an enhancer effect So here’s — I will keep calling this other geneticit [spelled phonetically] a wild type versus mutants forms or the control versus variant forms, and as you can see it actually scales fairly well with the odds ratios And we have now done many more of these studies — by the way, some of those studies involve only two of the SNPs, some three, because we’re in the middle of completing all of this, but even when we take all three, we have synthetic constructs in which we can access reporter activity of combinations of these SNPs, and here is the haplotype specific odds ratios that we can measure in a population with of about 600 to 700 patients, and what you find is a log ratio or a log relationship as one would expect if you assume some aspect of mass action So the first two elements that we discovered, not only short reporter activity [spelled phonetically] in vitro that was done in a mouse neuroblast cell line called neuro-2A activity [spelled phonetically], the new one is much — the human line is much better And what you can see, hopefully you can see that the enhancers in fact are found both at 11-and-a-half and 12-and-a-half, but RET+3 which is the SOX10 bound enhancer, it’s — by the way, it’s found in not only the gut, but in other cell types that’s of importance in the development and where RET is expressed; that is in portions of the forebrain and in fact in the dorsal root ganglion What you find is the first enhancer is active both at 11-and-a-half and 12-and-a-half, the Distal-1 enhancer is found at 11-and-a-half and not at 12-and-a-half In fact, if you do measure gene expression of the cognate transcription factor, Rarb is expressed at 11-and-a-half and not at 12-and-a-half So these, obviously, do act as enhancers in vivo as well and this is to show that the effects are specific That is, I told you that the SOX 10 form and the Rarb and the Gata2/3 are transcription factors this is doing knockdowns, in doing siRNA knockdowns and this all done in neuro 2A And what we found is the expected result that the positive control or the negative controls in this case work if you knockdown Rarb, obviously gene expression of Rarb goes down as it does in all of these cases But what’s interesting is when you knockdown SOX10, RET goes down as it should because it’s a transcription factor, but when you knockdown RET, what you find is SOX10 goes down, so this suggests not only the same is true for Gata 2 and 3, so this suggests that not only are these, the transcription factors that regulate RET, but RET has at least has a feedback effect at least on the Gata 2s and the SOX10 transcription factors These are just not artifacts of any particular cell line We control these results by looking at a RET null and a RET wild type mouse embryo from the same early time in development, and I’m not going to go through the details, this is just to show that this effect that we found in cells can be recapitulated in vivo as well So I want to make a final point that one of the kinds of tests that we do in order to show specificity is that the transcription factor effect when in fact you do knockdown is specific for the so-called wild type or the native form of the enhancer, and this again shows in a number of cases, let me see, my eyesight is failing me, that I’ll take the big one here, that even though the, I think this is the, this is the control that when you do the SOX10 that what you find is a difference that, you find it on SOX10 and siRNA and, I’m sorry, on RET, but you don’t find it on these ones because you find that the difference that you see on knockdown is equalized for the wild type form So in this particular case what you find is that, quite interestingly, in other data that I haven’t shown you is that all the variants that lead to Hirschsprung Disease in this case that they affect all components of RET signaling So this is sort of written in the form of these are the three transcription factors, obviously, that make the cognitive proteins, they regulate RET This is the part of RET production that is important and I gave you some evidence that RET, in fact, signals back and in some ways controls at least these two transcription

factors We also know that if you knockdown RET if you look at the RET null mouse embryos that they are negative regulators of its own ligand as well as its co-receptors, so this fact is — so this is another level of control on RET Finally, RET is only active in its dimerized phosphorylated form, and this of course you don’t want during early times of development You want tight control of this to control proliferation within the gut and so this form of RET is rapidly in fact cleared the signal is terminated through the action of an E3 ligates is called Cbl and what we find is this is positively regulated by RET So RET is a particular case in which its production, its activation, and its signal termination are all controlled through various kinds of enhancers, and one of the interesting things is that we find that genetic variation in all of these components through its non-coding elements is an important part of this disease So, and the total effect therefore depends on the total degree of loss of function So, I’m just going to end with a couple of sort of pleas really that in order for us to understand even this relatively simple disorder, I think it required us to understand the rest of the network that is just finding a non-coding variant outside of RET was not sufficient for us to explain the kinds of odds ratios, the kinds of patterns of risks that we see that are specific to not only to haplotypes, but also in the groups of patients that we have So, I think it goes without saying that since no gene acts alone, understanding the functional context of its expression is actually very crucial for us to get eventually quantitative answers, and that’s what I mean by modeling and predicting what the consequences are going to be And for this there’s a lot of extensive literature that suggests that the gene regulatory network is in fact a fundamental unit to consider because of many, many reasons indicated here is modular parts of this network is in this particular case very highly conserved and there are of course a number of classical studies that have compiled much of this information At least, Eric Davidson argues that this comes in a defined number of sub-circuit classes, so it’s possible that we might be able to classify them in some ways They do provide some system level explanation of the development and physiological functions, and I think there are many opportunities for mathematical modeling that can provide then the quantitative answers And I think this finally gives us a way for understanding complex inheritance in a way that we haven’t We’re still doing it on a gene-by-gene basis Many of you in this audience would know the work of Eran Segal when he originally tried to model and explain gene expression along the entry of posterior access [spelled phonetically] during fly development, but this is a model, it’s a thermal dynamic model, it’s fairly straightforward and I think this gives us ways not only in this case to look at gene expression, but there are various ways in which what we are learning from ENCODE can be used to build mechanistic models for gene activity, for trade values, and some sense you’ll hear Nancy Cox speak about it, and eventually for categorical diseases what matters, which is penetrance So, I’m just going to end with a series of acknowledgements of many people that have helped us in these studies, and I know that I was asked to answer some questions which I’ll just keep up while I sort of point them out as we discuss All right, thanks [applause] Eric Boerwinkle: Questions, a couple of questions from the audience Ewan? Ewan Birney: At that frequency and those kind of odds ratios — Eric Boerwinkle: Mic Ewan Birney: Oh, okay At that frequency in those odds ratios that, for me, suggests that this is on the balance and selection, a postural [spelled phonetically] something Something must be pushing that allele, those alleles that high, right? Keeping them high Is that the — have you looked at that? Is there an argument for that? Aravinda Chakravarti: Yes, so the original one, which has the biggest odds ratio, clearly that SNP has been published on this before is almost all the features of balance and selection One thing that I should tell you though is Hirschsprung Disease was an effective lethal about right now 70 years ago So it’s gone from seeing only new cases or cases that were perhaps very, very mild and that had expression to now people living in obviously reproducing and passing the phenotype

along So whatever selection we’re seeing obviously had to be — I mean, so in order to explain it’s not a recent phenomenon There has to be a long term phenomena and in fact the allele frequency shows all of the features with surrounding, at least some of the surrounding, you know, within the recombination haplotype Ewan Birney: Any idea what the, what the beneficial aspect — Aravinda Chakravarti: So, I only speculated on this I mean the gut is a favorite place for doing various kinds of selection The second is we know of receptors that are null or nearly null that have, like the chemokine receptor and the CCR5 that can act as or prevent the entry of various kinds of pathogen RET was considered not to be only neuronal be now expressed in other cell types including the epithelial, so it could be it has nothing to do with Hirschsprung Disease per se, but selected simply because of things like that Eric Boerwinkle: So we might be [spelled phonetically] real short Everyone, we’ve lost the desk mic volume, so please speak up with, or project, when you ask a question Male Speaker 1: I’m just curious So did ENCODE or other data help you find a series to give — Aravinda Chakravarti Oh, ENCODE clearly — Male Speaker 1: What fraction of that do you find? Aravinda Chakravarti: So, ENCODE clearly helped us in the last two The first one has been known for a very long time, and — but ENCODE also suggested in many others, I think what we finally helped by was the fact that originally we had a set of, I forget, 10, like a dozen pieces of DNA that previously that also shown were enhancers, so I think the combination of that just give us much more confidence of which ones to follow on Thanks [end of transcript]

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Ras raf mek erk signaling pathway (map kinase pathway)

okay guys let’s talk about the map kinase pathway it’s also called as a map k pathway so let’s let us talk about it so let me take a color first here map kinase pathway is a pathway which is related to cell proliferation and cell division it is ultimately giving the cell the different proteins and all these enzymes ultimately cell to divide and proliferate in a particular area so in a map kinase pathway you can see the different enzymes and different different signaling molecules working together to finally make a cell growth and divide properly now if anything wrong happen with the map kinase pathway even it gets activated too much so you know the functionality is the cell growth and the division and also cell proliferation right so if both of these things happen very positively I mean extra positively in that case it may turn into the cell as a malignant cells I mean without the control of cell growth so ultimately map kinase will lead to cancer in that case we see all these things later but actually map kinase pathways along with the cell proliferation and growth and if you begin with the with this with the very basic thing about any pathway it should have first the cell signaling molecule one signaling molecule and obviously it should have a receptor of that signaling molecule so in this case the signaling molecule for a map kinase pathway most of the cases it they are hormones they are hormones like growth hormones especially growth hormones so there are different types of growth hormones like there one of them is EGF epidermal growth factor right another one is they can be v DGF this is also another kind of growth factor so these are also called as growth hormone or growth factor whatever we can say EGF is epidermal growth factor v DG f is vascular endothelium I mean PDGF sorry not vdg platelet-derived growth factor so these are the different types of growth factors that can figure the map kinase pathway so the idea here and there should be a receptor for it and the receptor name naming is very easy you just take the name of the signaling molecule that is let us say the epidermal growth factor or EGF and simply put the receptor as a suffix ter so EGFR is the receptor for EGF so this is a EGF the signaling hormone the signaling molecule EGFR is the receptor that is embedded in the cell membrane now this EGFR or this receptor that is present here this receptor is nothing but this is a transmembrane protein and once EGF is on contact with this EGFR it finally it gets phosphorylated is an enzymatic receptor it gets phosphorylated and it is now attached with the accessory proteins out there in the cytosolic section which are grb-2 and is OS so these things grb-2 and so is once those things are attached with this phosphorylated EGFR they have a huge property to activate one of the most important mediators of this whole pathway that is called wrasse now wrasse is a protein which can activate further which can actually activate the map kinase because rest of the things we are going to see will be called as map kinase whereas is different and wrasse should be active for making map kinase activated so wrasse usually is in inactive form when it is bound with gdp now this grb-2 with SOS they can activate vas by substituting the gdp of grass with GTP so now it is substituted with GTP once it is substituted with GTP the gdp is released now the wrasse is attached with GTP and it is termed as a Razzie TP which is active this is the active form of GTP and once the grass is active now this wrasse can activate the map kinases now it first activate what is called as map kinase kinase kinase it is also known as RAF RAF is nothing but map kinase kinase kinase so this map kinase kinase kinase is activated by the active protein once it is activated this map kinase kinase kinase RF activates map kinase kinase that is also known as make so make is also known as map kinase kinase once RAF activates make that means map kinase kinase kinase activates map kinase tiniest and all those things as their kindness proteins you know they have the tendency to phosphorylate other proteins to activate it so in each of these stages they require the phosphate group and usually the phosphate group

donor is nothing but ATP so ATP donates the phosphate group to RAF RAF is phosphorylated active it donates it to make make gets phosphorylated active now make finally activates what is called as map kinase so we started with map 3 kinase is then map to kinase now finally map kinase so once the map kindness is phosphorylated it is in active form this map kinase can bring in the actual cellular effect that’s why we call it a map kinase pathway instead of anything else so once map kinase is activated map kind is we activate another proteins like mnk this is also kind of times our SK another type of kindness or Mik and finally it can activate CREB Mik s6 protein so these proteins like krebs cs6 make these things are nothing but transcription factors you know transcription factors can actually start a transcription process now once the transcription process is initiated due to the binding of transcription factor to the promoter sequence it will start initiating the transcription of the gene once this is transcribed into mRNA that can be brought to the cytosol and can produce proteins now the proteins usually produced after this transcription factor activation are all of those proteins necessary for cell growth and division like cycling’s cyclin CDK s and many more different types microtubules and all those constructive materials for cell cycle and division so once those proteins are activated the cell will get the signal to grow and divide rapidly and then proliferate so now let’s imagine if the situation occurs like that that this signaling pathway remains on throughout the time because like all of the signaling pathway is an on and off switch and in this case of map kinase pathway we also have that on an off switch now if this pathway remains on continuously it can activate this whole process of cycling’s and cdk proteins and finally it can turn a normal cell into a malignant cell because the cell will lost the control of cell division it will grow and grow and grow and divide and divide who ultimately gave us cancer now that thing happens if there is any problem with this grass protein because we know Raz is a very important mediator because if grass is activated all of these downstream mediators will be activated sequentially so normally after the activation of rass there is a cycle of inactivation also to stop this signaling pathway and that thing is simply by by hydrolysis of the gtp into GDP so this GTP bound with wrasse gets hydrolyzed into gdp inorganic phosphates frost rate is released and now the wrasse becomes only attached with gdp becomes inactivated so that thing is also necessary now let’s say in certain cases of rass protein mutation the gtp can bind with rash permanently in those cases the rash will not revert back to the inactive form and in that case the whole signaling pathway remains on throughout the time and it can convert the cell into a cancer Swan so there is map kinase pathway is related with cancer

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Homemade QUAD 6×6 WHEELS With CAR ENGINE ?! Part 1

As you can see, we will use existent naves that I used on tank, but they are welded inside so we will bring them to the turner so he can make hole in the middle

so we can place them here and weld, we’ll be back shortly Regarding front wheels turning, I decided to make it like this: fiber optic bar Fi 30mm will be welded on front machinery for turning and put 3 bearings and big shell

which will be weld on nave and it will look like this. We will leave this to the turner and lets get back to work On little thing I forgot to mention, this shaft will be drilled in here and weld from inside and out so it will be firmer Regarding rear double shaft, I decided to double this with iron because at the back you will have like mini trailer, of course, that adds weight and with just 4 bearings,

it will be not firm as I would like so I decided to put 4 more Now that we cut this part of shatf in half, we got 4 shafts. I did that because I will be turning easier with fronts. For example, if you turn with 2 full shafts to the left, all the wheels are turning with same power and speed, you won’t go in this direction you wanted to go. While you have 2 connected wheels on the left and right, for example, if you are turning on the left, left side of rear wheels is stopping and slows down while right side speeds up or have the same speed as it had and that really helps I did the same thing with kart and tricycle, you always ask me why only one side of my vehicle is working. This is the cheapest possibilty, of course, you have plenty of differentials, I will use one here, also I will connect all of this as it needs to be. Just naves and that’s it Meanwhile, my turner drilled a hole here and we came to better idea. He found in his workshop this nave from Yugo or Golf Mk1, it fits the nave that I brought him

and that’s great, look how many space we saved, now we can put here bracket and weld it much better. I like it, this is much better because it’s taken from a car, much simpler. Let’s weld now Now that we strenghten naves, now it’s time to work on handling system So, this shaft need to be welded here in the middle so it will come to turning by turning the steering wheel, but I took this PTO just in case so the shaft doesn’t bend. And that’s it. Let’s get back to work Now we came into problem, this is a steering system but this bearing is too big and I need to get smaller one, it will come in few days, but I will post this video without it So, this should have looked like this But we will make this without bearing so will connect naves with steering wheel and that’s it Now we came to the interesting situation, usually tractos clamps for tractors like my IMT 539, on one side it has this bearing with right angle, and on the other side is

little bit stronger than 90 degrees, but as you can see, if we connected all that, we couldn’t screw this right way so we must change this two joints Now that we connected clamps with naves and steering system, we still don’t have this bearing which will hold this shaft for turing so we will connect it with a tie and finally put wheels on naves And guys, I hope you liked this video, hit that like, share and subscribe button, write in comments what do you want to see on this project, that’s it regarding this episode, and in the next episode you can expect disunion of a car. Yes, you heard it right, that will be a real machine. We will use engine, clutch and shifter, all the installation from the car and put it on this construction. Enjoy and bye! It even has a parking pilot

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Activation of PI3Kalpha by Physiological Effectors and by Oncogenic Mutations

>>> GOOD AFTERNOON, EVERYBODY WELCOME TO THE WEDNESDAY AFTERNOON LECTURE ON I’M HERE TO INTRODUCE OUR SPEAKER TODAY, DR. MARIO APPLESEL HE IS A WORLD RENOWNED LEADER SHE IS PROFESSOR AND DIRECTOR AT THE DEPARTMENT OF BIOPHYSICS AND BIOPHYSICAL CHEMISTRY AT THE JOHN’S HOPKINS UNIVERSITY SCHOOL OF MEDICINE HER RESEARCH FOR UNDERSTANDING MECHANISMS OF ENZYMES INVOLVED IN REACTIONS AND PHOSPHORALATION TRANSFERS USING VARIOUS TECHNIQUES, INCLUDING X-RAY DEFRACTION, BIOCHEMICAL AND BIOTYPICAL METHODS HIS LAB ALSO DOES COMPUTATIONAL METHOD DR. APPLES SELL IS NOT ONLY A GREAT SCIENTIST BUT ALSO A GREAT TEACHER MENTOR — AMZEL HE WON THE GRADUATE STUDENT TEACHER OF THE YEAR AWARD AND UNIVERSITY TEACHING AWARD IN FACT, AFTER THE TALK, HE IS ON HIS WAY OUT TO RECEIVE A PRESTIGIOUS AWARDXñ/ç BY THE ARG TEENIAN MINISTER OF SCIENCE AND TECHNOLOGY THE GROUP DESCRIBED THE STRUCTURE OF P13 KINASE ALPHA ABOUT WHICH HE WILL BE TALKING TO US TODAY THANK YOU >> THANK YOU VERY MUCH FOR THE INTRODUCTION THANK YOU FOR THE INVITATION TO PRESENT MY DATA HERE I HAVE HAD NICE CONVERSATIONS WITH POSTDOCS THERE ARE PROJECTS IN THE LABORATORY AND THE ONES I CHOSE TO TALK TODAY INVOLVES THE STRUCTURE OF THE TERMINATION AND INTERPRETATION OF THE MECHANISM OF ACTIVATION OF P13K-ALPHA THIS IS AN ENZYME WHICH IS NOT MEMBRANE BOUND, IT’S MEMBRANE ASSOCIATED HERE IS THE LOCATION, AND IT IS RESPONSIBLE THROUGH A SERIES OF RECEPTORS OR THE SUBSTRATES AND IT’S FORMED WITH 2 SUBUNITS, ONE THE UNIT IS 10 KILOS WHICH CONTAINS THE CATALYTIC SITES AND 85 UNITS OF KILLADALTONS THAT IS ASSOCIATED WITH BRINGING THE PROTEIN TO THE MEMBRANE THE PROJECT IN THE LAB STARTED WHEN A GROUP AT HOPKINS3W)5f DIRECTED BY FOGELSTEIN, PUBLISHED IN 2004, THAT THE GENE THAT CAUSED 120 PROTEINS IN THIS KINASE WAS HIGHLY MUTATED IN SEVERAL CANCER MUTATIONS IN THIS PROTEIN — AND ASKED WHY ARE THESE MUTATIONS INVOLVED? SINCE THEN, MORE DATA HAS BEEN COLLECTED AND MORE CANCERS HAPPEN TO HAVE THESE MUTATIONS AND BY NOW, THEY ARE ABOUT 1,500 MUTATIONS THAT HAVE BEEN DESCRIBED IN THE GENE CODING FOR THE 120 KILLADALTON SUBUNIT OF THE PROTEIN SO, MOST OF THE MUTATIONS INCREASE THE ACTIVITY THE ONES WE DON’T KNOW ARE THE ONES — NOT TESTED SO THIS IS WHAT PEOPLE WANT TO CALL TYPICAL ON COJEAN YOU CREATE ACTIVITIES — ON COJEANS THE ENZYME, THAT ISP) KINASE WHICH HAS A VERY,á<Uv VERY SIMPLE REACTION THISlnG IS A BIG PHOSPHATE IT PHOSPHORALATES, THAT'S ALL THIS REACTION CAN BE REVERSED BY P10, A PHOSPHATASE WHICH IS A TUMOR SUPPRESSOR FACTOR AND THAT GIVES YOU THE IDEA WHY THEY STARTED LOOKING AT THIS GENE IN PARTICULAR BECAUSE THE REASONING WAS IT'S GOING IN THIS DIRECTION IF YOU FAIL TO HAVE THIS REACTION, YOU GET TUMOR SUPPRESSOR FACTOR HERE AND THEN THIS COULD BE AN ONCOGENE AND THAT'S WHAT THEY ENDED UP BEING

SO, THERE ARE SOME QUESTIONS WE CAN ANSWER AND SOME QUESTIONS THAT WE CAN’T THAT SOME OTHER PEOPLE CAN AND THIS IS, WHY IS SO MANY MUTATIONS THAT HAVE INCREASED P13K ACTIVITY? THIS IS A VERY GOOD QUESTION AND WHAT SORT OF ANSWERED BY OTHER PEOPLE AND THEN, HOW DO THESE MUTATIONS INCREASE THE ACTIVITY BY PHYSICAL MECHANISTIC QUESTION? THAT IS THE GENERAL INTEREST WE HAVE IN THE SYSTEM SO, THE WHY IS VERY SIMPLE PI3K IS VERY SIMPLE THESE ARE RECEPTOR TYROSINE KINASES OR GROWTH FACTORoYj RECEPTORS WHEN THEY BECOME PHOSPHORYLATED, THE SUBUNIT P85 BINDS TO THEM THROUGH THE PHOSPHORYLATED SUBSTRATES AND THENG Ox ACTIVATED FROM THE MEMBRANE, THIS IS THE 110, AND REDUCE THIS PHOSPHATE AND THAT IS THROUGH PHOSPHORYLATION CONTROLS ALL THESE FUNCTIONS — [INAUDIBLE] MIGRATION, MOTILITY, THOSE ARE CHARACTERISTICS THE TUMOR CELL WILL TRY TO DEVELOP, WILL DEVELOP THROUGH THE NUMBER OF REPUTATIONS THAT ARE VERY, VERY LAST NUMBER BEFORE TUMORS BECOME DEDUCTIBLE JUST TO GIVE YOU MORE DETAIL, THERE ARE TWO CLASSES OF P110 CHANGE ALPHA, BETA AND DELTA BELONGS TO CLASS 1A AND THEY USE P85 AND THEN P110 GAMMA HAS DIFFERENT REGULATORY SUBUNITS AND ALSO IS NOT RESPONSIVE TO THE RECEPTIVE TYROSINE RECEPTORS BUT RESPONSIVE TO G PROTEIN COUPLE RECEPTORS SO RESPONSES TO CHEMOKINES I GET A LITTLE MORE DETAIL ABOUT THIS ONE THING THAT IS INTERESTING FOR PEOPLE WORKING IN5/>, MANY OF THESE THINGS, TO GET AN IDEA OF WHAT CONTROLS THIS PROCESS, IS THIS IS A COMPLICATED DIAGRAM NOT COMPLETE THAT WILL GIVE YOU AN IDEA THIS IS WHAT PI3K IS, IT BRUISES PIC3 AND RECRUITS TO THE MEMBRANE — PRODUCES — PLUS AKT AND PDK1 THAT CONTROLS THE PHOSPHORYLATION OF THIS PROTEIN SOME OF THE ACTIONS ARE MIG TORY AND SOME OF THE ACTIONS ARE ACTIVATING BUT MOST OF YOU RECOGNIZE MANY OF THESE PROTEINS THESE ARE THE PROTEINS THAT I SEE INVOLVED IN TRANSLATION THESE PROTEINS ARE INVOLVED IN APOPTOSIS SO, THROUGH THE ACTIVATION OF SOME OF THE PATHWAY, PI3K CONTROLS MANY OF THE ACTIVITY IN A LITTLE BIT MORE DIRECT, ALL MY TALK IS GOING TO BE CENTERED AROUND PI3K ALPHA AND THAT IS JUST FROM THE THINGS THAT I SHOW YOU, IT IS INVOLVING SEVERAL DISEASE PROCESSES, TUMORIGENESIS BECAUSE IT’S AN ALPHA AS A HUMAN COJEAN AND IN VIRAL INFECTION, SOME REQUIRE THE FUNCTION OF THE PI3K IN CARDIAC FUNCTION, THEY ARE INVOLVED IN SUPPRESSION OF CARDIAC HYPERTROPHY AND VENTRICULAR DILATION AND FIBROSIS IN SITUATIONS UNDER STRESS AND OF COURSE IT’S PART OF THE INITIAL STEP AFTER THE RECEPTOR IN THE INSULIN PATHWAY SO AS A GENERAL IDEA OF WHAT HAPPENS TO THE DIFFERENT CLASSES, ALPHA, BETA, AND DELTA ARE CLASS 1, GAMMA IS CLASS 1A AND GAMMA IS CLASS 1B AND THIS ARE THE FUNCTIONS THAT THEY HAVE THEY ARE VERY, VERY SIMPLIFIED I SHOW YOU A LITTLE BIT MORE DETAIL IN THE PREVIOUS SLIDE ANDWIv THESE ARE THE SPECIFICS THAT CAN BE TARGETED BY TARGETING THE PROBLEM PI3K ALPHA IS ACT SIGHTING MUTATIONS AND THROUGH THE — ACTIVATING — THROUGH THE RECEPTOR PI3K BETA AND INVOLVED IN ACTIVATION AND IS NOT MUTATED IN CANCERS THAT IS HOW THEY FUNCTION IN CANCERS WHICH ARE — TUMORS WHICH HAVE DEFICIENT PK FUNCTIONS AND THEN THESE TWO, THEY BELONG TO DIFFERENT CLASSES, THEY ARE RELATED WITH A NEW SYSTEM AND THEY ARE NOT INVOLVED IN RESPONSE TO IMMUNE IMPORTANT CYTOKINES THAT RESULT IN IMMUNE CELL MOTILITIY AND THEY ARE TARGET FOR INFLAMMATION [email protected]

SO, ONE WOULD LIKE TO CONTROL SOME HOW CHEMICAL IN THIS MOLECULE FOR BOTH BIOLOGICAL EXPLORATION FROM LABORATORY TESTS BUT ALSO FOR’ZxCHEMICAL INTERVENTION SO, WE ARE — THERE ARE SEVERAL ISOFORMS THAT CONTROL SEVERAL PROCESSES FOR EXAMPLE, IF ONE IS THINKING IN CANCER CHEMOTHERAPY, ONE WOULD LIKE TO INHIBIT ONLY PI3K ALPHA BECAUSE THAT’S THE ONLY DEMONSTRATED ONCOGENE FOR THIS PROTEIN SO, IN PRINCIPLE, THE IDEA SHOULD BE ISOFORM SPECIFIC.ñ?]d BUT EVEN IN THE INITIAL PI3K WILL HAVE SOME TO DETECT SO IN THE CASE OF CANCER CHEMOTHERAPY, ONE WOULD LIKE TO HAVE ISOFORM SPECIFIC BUT ALSO MUTANTS SPECIFIC SO THIS IS THE GENERAL IDEA OF WHERE THE DEVELOPMENT OF THE INDIVIDUALIZED MEDICINE AT THE LEVEL OF THE GENOTYPING OF THESE INDIVIDUALS OF THE PATIENT WILL GO WE ARE — WE CAN DO IT NOW BUT FOR NO USE THE ONLY USE HERE IS WHEN ALL THE CELL BIOLOGY AND ALL THE BIOPHYSICS IS DONE AND WE KNOW WHAT TO DO ONCE WE KNOW WHICH GENES ARE MEW TRADED SO THE DIRECTION TO TRY TO GET ALL THE INFORMATION NECESSARY TO MAKE GENOTYPING IN INDIVIDUAL PATIENTS TO BE CLINICAL REALITY SO, IN THE CASE OF THE ISOFORM-SPECIFIC MUTANTS-SPECIFIC INHIBITOR, ONE THING THAT IS CRUCIAL IS BIOPHYSICAL AND !gÑ FACTUAL INFORMATION SO ACTUALLY THE MUTATION SPECIFIC ARE VERY CLOSE ISOFORM-SPECIFIC INHIBITORS THIS IS A CARTOON, SHOULDN’T BLAME ME TOO MUCH FOR IT, IF YOU HAVE A WILDTYPE ENZYME, YOU HAVE AN INHIBITOR IF YOU HAVEd” REPRESENTED BY3;; ADDITIONAL — IT WILL BIND TO THE MUTANT ALSO HOWEVER, IF YOU ARE ABLE TO ASSIGN AN INHIBj THAT, IT WILL — [INAUDIBLE] TO THE WILDTYPE AND THIS LOOKS LIKE A CARTOON THAT DEPICTS THIS BUT IN REALITY, WE ASSUME THAT KIND OF INITIATION IN THE CASE OF ONE OF THE MOST STUDIED FOR INHIBITORS, HIV PROTEASE THIS IS THE WILDTYPE PROTEASE BINDING TO ONE OF THE INHIBITORS AND THEN IF YOU LOOK AT THE MUTANT, THIS AREA, IS COMPLETELY DIFFERENT IT’S VERY CLEAR THAT YOU COULD MAKE AN INHIBITOR TO BE MORE SPECIFIC FOR THAT MUTANT THAN IT IS FOR THE WILDTYPE SO AGAIN, THIS DIFFERENCE ISc VERY DIFFICULT TO REALIZE THEY WILL REQUIRE BIOPHYSICAL INFORMATION SO, NOW LET’S LOOK IN MORE DETAIL ON THIS STRUCTURE OF PI3K ALPHA 1,068 AMINO ACIDS AND HAS FIVE DOMAINS AND THE BINDING DOMAIN, — [INAUDIBLE] THE REGULATORY PROTEIN ALSO HAVE FIVE DOMAINS AND THIS IS: AND THE DOMAIN WAS MEANING INTERVENING SH2 THAT IS NOT AN SH2.hV]Bç FOR THE STUDIES WITH OVER 50 OR SO DIFFERENT CONSTRUCTS AND THE ONE THAT WORKED IN CELLS WAS ONE IN WHICH WE HAD THE COMPLETE P110 AND TWO DOMAINS OF THE P85, WHICH ARE THE SH2 AND YOU WILL REALIZE THAT THIS WAS NOT COMPLETELY RANDOM THIS IS PROBABLY THE MOST IMPORTANT DOMAIN TO HAVE AND WE REPORT IN 3 STRUCTURES, THE WILDTYPE WITH THE NI WITH THESE TWO WE REPORT IN ONE OF THE ONCOGENIC MUTANTS AND WE WILL DISCUSS THIS ONE AND THE SAME

ONCOGENIC MUTANTS IN THE INHIBITOR AND I’M GOING TO JUST TALK ABOUT THIS IN GENERAL IN VERY FEW CASES IT WAS REFERRED TO SOMETHING SPECIFIC ABOUT THESE STRUCTURES SO, THIS IS THE STRUCTURE OF THE PROTEIN AND HERE ARE THE DIFFERENT DOMAINS AND IT’S A VERY COMP CALLED STRUCTURE THE WAY THE — COMPLICATED — IT HAS NOTHING TO DO WITH THE LIN AR SEQUENCE LI SHOW YOU THIS THIS IS THE DOMAIN THAT INTERACTS WITH THIS ONE, THE HEEL CALL DOMAIN INTERACTS WITH THESE TWO AND THE KINASE DOMAINS HERE IT IS A QUITE COMPLICATED STRUCTURE — TO GET ANOTHER GENERAL VIEW IS THIS SUBUNIT THAT I SHOW YOU OF THE 120 STRUCTURE, THIS IS THE EXTENDED COIL AND THIS IS THE NSH2 AND I’M GOING TO TALKi : ABOUT THIS DOMAIN JUST IN THE SENSE, WHAT IS THEIR FUNCTIONAL IMPORTANCE SO, HERE WE COME BACK TO THE MUTATIONS AND YOU REALIZE THERE ARE TWO SPOTS ONE IN THE MEAL CALL DOMAIN, ONE — HELICAL DOMAIN AND ONE IN THE DOMAIN THAT HAS THE PHOSPHORYLATION ACTIVITY AND THERE ARE SOME MUTATIONS IN THE C2 DOMAIN AND OTHER MUTATIONS THAT I’M GOING TO TALK ABOUT ARE THESE THREEUHAAó MUTATIONS STARTING WITH THE PHENOTYPE AND THEN THE KINASE DOMAIN AND ONE THING THAT PROBABLY WOULD APPEAR IN ONE OF MY NEXT TALKS IS WE LOOK AT THE MUTATIONS NOT ONLY TO SEE WHAT THE MUTATIONS DO THEY ARE ACTIVATING THE ENZYME AND THAT IS THE FUNCTION TO ACTIVATE UP ON INTERACTION WITH THE RECEPTOR TYROSINE KINASES WE EXPECT THAT THE MUTATIONSR:V5 AN EASIER WAY TO LOOKING AT THESE ACTIVATION OF THE MOLECULES THIS IS THE QUESTION HOW DO THESE MUTATIONS INSIST IN ACTIVITY AND HOW DO THESE INCREASES INp%- ACTIVITY RELATE TO STRUCTURAL CHANGES? WHAT WE EXPECT IS WHAT I JUST SAID, INFORMATION WILL BE GIVING US INSIDE THE ACTIVATION ALSO SO, WE DISCUSSED MANY OF THESE MUTATIONS IN THE PUBLICATIONS AND THERE WOULD BE NO TIME TO WRITE THIS DOWN BUT THESE ARE PUBLICATIONS THAT PROBABLY DISCUSS MOST OF THE MUTATIONS THATgr WERE OBSERVED AND WE ARE GOING JUST TO DISCUSS A FEW OF THEM IN SOME DETAIL SO THE FIRST THING THAT HAPPENED HERE THE MUTATION THAT YOU SAW OCCUR HERE, HERE, THIS IS AN ACTIVE BINDING DOMAIN AND THIS IS THE C2 DOMAIN WITH ANOTHER SET OF MUTATIONS AND THIS IS THE KINASE DOMAIN WITH ANOTHER SET OF MUTATIONS HERE SO, BESIDES THE MUTATIONS IN THE KINASE DOMAIN, ALL OTHER MUTATIONS OCCUR AT DOMAINS SO, ONE THING WHICH !’i IS ALSO IMPORTANT, I HAVE TO TURN THIS 90 DEGREES TO ([b YOU WHERE THE BINDING SITE IS BECAUSE THE SITE IS HERE AND HERE AND ALL THE MUTATIONS HAVE A VERY FAR FROM THE SITE YET THEY ARE ALL ACTIVATED SO, HOW DO THEY DO IT? FOR SOME OF THE MUTATIONS, THE ANSWER LIES ON THIS SUBUNIT HERE WHICH IS THE NSH2 SUBUNIT OR DOMAIN OF THE REGULATORY SUBUNIT SO, IT WAS KNOWN FROM BIOCHEMICAL DATA THAT THIS DOMAIN HAS A PANEL IN — PARTIAL INHIBITORY AFFECT SO IF YOU LOOK AT THE FIGURE, NEW NEED THE REGULATORY DOMAIN FOR THE PROTEIN BUT WITH THE DOMAIN, THE PROTEIN IS LESS ACTIVE THAN WITHOUT IT SO AND IT WAS DETERMINED THAT THAT WAS USED TO THE NSH2 DOMAIN SO THIS IS SLIGHTLY INHIBITORY SO ONE WAY THAT SOME MUTATIONS MAY WORK IS BY RELIEVING THE INHIBITION BY THE NSH2 DOMAIN SO, NOW WE ARE LOOKING INTO THE

STRUCTURE STRAIGHT INTO THE NSH2 DOMAIN HERE IS THE MORE REALISTIC SHOW AT THE SURFACE OF THE NSH2 DOMAIN AND YOU REALIZE THERE ARE THREE DOMAINS OF THE LARGE CATALYTIC SUBUNIT THIS IS WHERE THE BINDING SITE IS SO HERE IS WHERE THE NSH2 DOMAIN EXPRESSES, FAR FROM THE BINDING SITE BUT IT SEEMS TO INTERACT WITH THREE OF THE SUBUNITS AT THE VERY, VERY CRUCIAL POINT WHERE THEY JUST [email protected]á POSITION OF EACH OTHER ONE OF THE STRONG INTERACTIONS OCCUR BETWEEN THE HELIX OF THE NSH2 DOMAIN AND THE POSITION OF THE HELICAL DOMAIN AND THE KINASE DOMAIN SO IN THIS GROUP, THIS HELIX IS THERE THAT’S ONE OF THE IMPORTANT INTERACTIONS AND WE ARE GOING TO LOOK AT THIS INTERACTION FROM TWO PERSPECTIVES ONE IS FROM THE PERSPECTIVE OF THE BINDING OF THE PEPTIDE WHICH WILL BE THE EQUIVALENT OF BINDING TYROSINE KINASES PHOSPHOTYROSINE SIGNALING< GROUPS AND THE THE OTHER IS THESE MUTATION THAT IS ARE FREQUENTLY MUTATED IN CANCER ARE THOSE RELATED TO THE ACTIVATION SO WE WERE NOT ABLE TO GET THE STRUCTURE OF THE PROTEINS WITH THE PEPTIDES, BUT OVER TEN YEARS AGO, THE STRUCTURAL OF THIS IDENTICAL DOMAIN, ISOLATED, IN THE PRESENCE OF PHOSPHOSEEN PEPTIDES WAS DETERMINED THAT -- THAT THIS IS THE ONE FROM THE GROUP -- [INAUDIBLE] AND THIS IS THE NSH2 OF THE SAME MOLECULE WHEN A PHOSPHOTYROSINE PEPTIDE BOUNCED HERE IF WE ALIGN THIS NSH2 WITH THE ONE FROM OUR STRUCTURE, THEY ARE IDENTICAL SO INés5XjT)T&E BY THIS, WE KNOW THE POSITION OF THE PHOSPHOTYROSINE PEPTIDE IN OUR STRUCTURE SO, THIS IS WHY IT WOULD BIND THIS IS THE NSH2 DOMAIN AND THIS IS WHY THE PHOSPHOPEPTIDE WILL BIND IF WE LOOK AT THE COMPLETE STRUCTURE, THAT SAME GROUP IS OCCUPIED BY A LOOK AND THAT LOOK CARRIES THE TWO HOTSPOT MUTATIONS OF THE HELICAL DOMAIN A GRADUATE MATE 542 AND GLUTAMATE 545 SO, IT ESSENTIALLY THE PHOSPHOTYROSINE SOMETHING SIMILAR IS BEING DONE BY THIS MUTATION THEY ARE NOT IN THE SAME PLACE SO, AFTER THE STRUCTURE WAS PUBLISHED, A GROUP OF COMPARISON HAD THE FOLLOWING DRAMATIC DATA ON THE ONE SIDE AND SOME OF THE MUTANTS AND HERE IS THE ACTIVITY -- [INAUDIBLE] THIS IS THE ACTIVITY OF THE WILDTYPE THIS IS THE ACTIVITY OF ONE OF THE HELICAL DOMAIN MUTANTS AND THIS IS THE ACTIVITY OF THE OTHER AND HERE ARE THE DATA THE ACTIVATION IS ALWAYS IN SIGNALING MOLECULES, THERE ARE TWO OR 3 VERY, VERY SMALL ACTIVATION BUT IT IS IMPORTANT FOR INITIATING SIGNALS IF WE NOW NORMALIZE THESE THREE ACTIVITIES TO 1 AND THOSE ARE THE -- [INDISCERNIBLE] THEN THIS IS THE ACTIVITY WITHOUT ANYTHING ON IT IF WE LOOK AT THIS BLACK BOX, IT IS HOW MUCH THE ACTIVITY INCREASED IF YOU ADD PHOSPHOTYROSINE PEPTIDES AND THE ONE SIDE GETS INCREASING ACTIVITY BY THE FACTOR OF 2, IT'S THE CORRECT ACTIVATI THE TWO MUTANTS THAT I DESCRIBED DO NOT INCREASE THE ACTIVITY THAT MEANS THEY USED UP THE SAME ACTIVATION MECHANISMS, REMOVING THE NSH2 FROM THå y INHIBITORY POSITION SO THIS IS MORE OR LESS WHAT HAPPENS AND THAT IS THE DOMAIN HAS AN INHIBITORY AFFECT ON THE KINASE DOMAIN AND THAT, INHñÑ4O BINDING PHOSPHOTYROSINE PEPTIDE WHICH WILL MOVE THIS SOMEWHERE

ELSE, NSH2 DOMAIN OR BY FOLLOWING THIS MUTATION IN THE SAME SURFACE THAT WEAKEN THE INTERACTION OF THE HELICAL DOMAIN WITH THE NSH2 SO THIS SEEMS TO LOOK MORE OR LESS LIKE THIS THIS IS THE MOLECULE AND WE ARE LOOKING ON MUTATIONS HERE AND THIS GROUP, THIS DOMAIN, IS IN HIBITORY OF THE ACTIVITY, SO HERE THE INHIBITION IS SHOWN BY THIS ARROW THE FACT THAT THE MUTATION HERE ALWAYS BINDING PEPTIDES, WE DISLODGE THE NSH2 SUBUNITS AND THAT RESULTS IN ACTIVATION SO THAT WOULD BE THE GENERAL SCHEME FOR THE MECHANISM OF ACTIVATION BY THE RECEPTOR TYROSINE KINASES OR BY MUTATIONS SO, THERE IS A CATCH TO ALL OF THIS IN THE MUTATION THEY ARE VERY SIMPLE BUT WHAT HAPPENS WHEN WE TRY TO LOOK AT HOW THIS ACTIVATION OCCURS? THIS HAS TO BE LOCATED THERE IS NOT ENOUGH BINDING FREE ENERGY CLOSE BY TO BIND THEM AND MOVE AND PUSH SOMEBODY AWAY THAT DOESN’T HAPPEN THIS SITE HAS TO BE AN&@#Jú OCCUPIED PART OF THE TIME SO, THAT MEANS MR. IS SOME SORT OF DYNAMIC EQUILIBRIUM BETWEEN THE HELICAL DOMAIN AND THE NSH2 BEING BOUND TO EACH OTHER AND SOME PARTS OF THE NSH2 DOMAIN — [INDISCERNIBLE] ONE HAS TO LOOK AT DYNAMIC AFFECT AND TO LOOK AT DYNAMIC EFFECT, WE WENT IN TWO DIFFERENT WAYS THE PROBLEM IS TOO LARGE AND WE DON’T KNOW ENOUGH SO WE TRY TO DO IT IN AS WE KNOW ONE WAY IS TO JUST DO COMPUTATIONALLY AND TRY TO SEE WHAT KIND OF CONFORMATIONS — [INDISCERNIBLE] BECAUSE THE MOLECULE IS VERY LARGE AND ONE WANTS TO HAVE INDICATIONS OF MOTION, WE WENT TO DO NORMAL ANALYSIS THIS IS ONE OF THE ANALYSIS AND I SHOW YOU MORE IT EXAGGERATED JUST FOR THIS SO WE DID NORMAL IN.MPY THELG TÑ WILDTYPE AND IN THE MUTANTS, DOUBLE MUTANTS, IN THE POSITIONS OF THEkm<% MUTANTS OF THE HELICAL DOMAIN, WHICH ARE ONES -- [INDISCERNIBLE] SO THE FIRST THING THAT HAPPENS IS THAT WHEN YOU TRY TO MODEL THAT MUTANT, THE7t [INDISCERNIBLE] JUST PUSH THE STRUCTURE AWAY AND WHEN YOU LOOK AS IT COMPARED WITH THE WHILE TYPE, THE DOMAIN IS ALREADY SLIGHTLY MOVED AWAY WHEN WE TRIED TO ANALYZE ITS WHAT THE EFFECT OF WEAKENING THIS INTERACTION, DOESN'T DISAPPEAR ON THÄ* GENERAL DYNAMIC MOTIONS OF THE MOLECULE SO, WHAT WE ARE SCRIBING AS THIS DISLODGING OF THE NSH2 BINDING TO THE LARGE DOMAIN, IT IS MORE THAN ANYTHING ELSE, IT IS WEAKENING THE BINDING AND THAT GIVES ACCESS TO DYNAMIC STATES WHICH ARE A WAY FROM THE POSITION OF NORMAL SO, ONE THING WE LOOKED AT IN THIS, IS DYNAMICS OF WHAT THE NSH2 DOMAIN DO AND HERE THE LAST NUMBER OF NORMAL MOUSE WHAT IT IS, IT REPRESENTS UNITS THE MOVEMENT OF THE INDIVIDUAL ATTRIBUTES THE NSH2 DOMAIN IN DIFFERENT NORMAL MODES AND HERE IN THE MOVIES THEY ARE BEING REPRESENTED THE ONE OF THE FIRST NORMAL MODES AND IF YOU LOOK AT THIS OR WHAT THIS SAYS IS THE WILDTYPE GENERIC -- IT'S A GRAY LINE AND IT HAS A VERY, VERY SMALL AMPLITUDE OF MOTIONS FOR THE NSH2 ONCE YOU WEAKEN THE INTERACTION OF THE NSH2 BY THE MUTATIONS YOU GET LARGE FLUCTUATIONS THAT ARE POSSIBLE AT LOW ENERGY SO HERE IS THE NSH2 OF THE WILDTYPE AND HERE IS THE NSH2 OF THE MUTANT AND THEY ARE MUCH LARGER MOTIONS IN THE MUTANT THAN THEY FOR WILDTYPE AND THOSE MOTIONS CHANGING THE INTERACTION OF THIS REGION WITH RESPECT TO THE KINASE DOMAIN

SO WE LOOK ALSO A VERY, VERY GENERAL IDEA — [INDISCERNIBLE] I SEE TWO GROUPS OF NORMAL MODES IN ORANGE IS THE WILDTYPE AND THESE ARE GENERALLASMITUDES OF MOTIONS AS A FUNCTION OF AMINO ACID NUMBER IN GREEN IS ONE OF THE MUTANTS AND IT IS CLEAR FOR THE NSH2 DOMAIN, THE AMPLITUDE IN EVERY RESPECT OF MOTION BECOMES MUCH, MUCH LARGER THAN THAT OF THE WILDTYPE THAT IS WHERE THE PEPTIDE IS GOING TO GO IN THIS CASE, THIS LARGE MOVEMENTS ARE THE ONES THAT DIRECTLY ACTIVATE THE ENZYME THIS LARGE MOVEMENTS IN THE WILDTYPE IN THE ORANGE ARE THE ONES THAT WE WILL ALLOW TYROSINE KINASE TO BIND THE ISH2, THE LONG HELICAL DOMAIN, FOR EXAMPLE, IT DOESN’TT-÷ EXPERIENCE THE SAME KIND OF MOTION THEY ARE MOTIONS BUTINATE AND I HAVE MUTANTS ARE EXTREMELY SIMILAR YOU CAN NOT RECOGNIZE HERE WHICH ONE IS GREEN, WHICH IS ORANGE AND WHICH IS GRAY THE SAME IS TRUE, THERE ARE TWO LOOPS IN THE MOLECULE WHICH FOR THE MOMENT I SAID WITH THE INHIBITION AND WITHIN ACTIVITY BUT THERE SHOULD BE A MECHANISM OF ACTIVITY AND THAT PART OF COURSE IS WE DON’T KNOW YET THAT IS WHAT WE ARE TRYING TO DO BUT ONE THING THAT IS CLEAR IS THERE ARE TWO LOOPS CALLED THE ACTIVATION LOOP AND0 THE — [INDISCERNIBLE] THAT IS VERY, VERY VALUABLEÓ CONFIRMATION WHEN YOU LOOK AT THE LARGE NUMBER OF KINASES HERE, WE BUILD MODEL OF THE ACTIVATION LOOP AND THEN WE LOOK AT THE SITUATION IT’S THE SAME COLOR THE WILDTYPE IS ORANGE AND THE MUTANT IS IN GRAY AND THERE ARE MANY MOVEMENTS THAT ARE LARGE AMPLITUDES FOR THE MUTANT WHICH ARE NOT LARGE AMPLITUDES FOR THE WILDTYPE WE EXPECT, WHEN WE ANALYZE THEM, THAT SOME OF THIS MOVEMENT WILL SHOW SOME POSITIONING OF RESIDUE THAT INCREASE CATALYTIC ACTIVITY WE ARE NOT THERE YET HERE, YOU CAN SEE WE ARE REPRESENTING AGAIN THE ONE WITH THE RED DOT AND THERE ARE SOME LARGE MOVEMENTS IN THE WILDTYPE BUT HERE, IT IS VERY SMALL AND IF YOU LOOK HERE IN THE ACTIVATION LOOP, IT IS VERY LARGE SO AGAIN, THE DYNAMIC EFFECTS WILL MAKE CONFIRMATION ACCESSIBLE, PARTS OF THE TIME CONFIRMATIONS MORE ACTIVE CATALYTIC AFFECTS THERE ARE NOT CONFIRMATIONS, JUST BECOME ACCESSIBLE THERE ARE MANY STRUCTURES THAT HAVE BEEN DONE BY US AND BY OTHERS AND IT DOESN’T SEEM TO BE A HEMOGLOBIN TYPE STRUCTURE WHERE YOU HAVE ONE INSTRUCTOR HERE AND ANOTHER STRUCTURE HERE — [INDISCERNIBLE] CHANGES IN THE DYNAMIC LIKE THIS ONE WHO SEEMS TO CONTROL THE CATALYTIC ACTIVITY REMEMBER THIS ACTIVITY IS ONLY A FACTOR AT THE MOST OF 4 SO THEY ARE VERY, VERY SUBTLE CHANGES IN ACTIVITY SO, YOU HAVE TO GIVE AN IDEA, WHAT IS SAID HERE IS THE MUTATIONS WEAKEN THE INTERACTIONS AND CHANGING THE CHARACTERISTICS OF THE — [INDISCERNIBLE] AND THEN PHOSPHOTYROSINE PEPTIDES ON THE MUTANTS HAVE SIMILAR EFFECT ONE THING WE ARE DOING IS TO TRY TO SEE IF WE CAN GET ANY INFORMATION ABOUT BINDING OF PEPTIDES TO THE MOLECULE THE MOLECULE PROBABLY BECAUSE NSH2 IS DISLODGED AND IF KEPT MOVING, IS RESISTING THE PEPTIDES, SO WE WENT TO A TECHNIQUE CALLED — [INDISCERNIBLE] SO THE KIND OF INFORMATION YOU GET IS THEN FROM THE MOLECULE THE ENVELOPE HERE IS SHOWN AT THIS EGG CRATE IT IS USED TO CALCULATE THAT ENVELOPE AND HERE IN THIS ENVELOPE IS THE FITTING OF THE MOLECULE THE FITTING LOOKS MUCH SMALLER THAN THE ENVELOPE ITSELF BUT THIS ENVELOPE INCLUDE WATER WHICH IS IMMOBILIZER AND TRACKING THE PROTEIN SO REFLECTS WATER AS ASSOCIATED WITH THE MOLECULE WHEN WE HAD PHOSPHOTYROSINE PEPTIDE FROM THE INSULIN RECEPTOR 1 THAT WE KNOW BINDS TO

THEE PORTION OF THE ENVELOPE CAN JUST DRAMATICALLY — THERE ARE OTHER PLACES WHERE IT CHANGE [INDISCERNIBLE] I CONCENTRATE IN THIS PORTION THIS IS WHERE THE NSH2 IS THIS IS THE NEW PORTION OFuWmlñ THE ENVELOPE SO, WHAT IS HAPPENING IS THAT THIS NSH2 NOW CAN SAMPLE ALL THE CONFIRMATIONS BETWEEN THESE TWO POINTS AND NOW THE ENVELOPE BECOMES BIGGER HERE SO NSH2 SAMPLES FROM HERE TO HERE THAT IS CONSISTENT AND SIMILAR TO THE OBSERVATIONS THAT WE HAVE FROM THE NORMAL THE DATA, EXPERIMENTAL DATA IS HERE YOU REALIZE INFORMATION CONTENT OF THE DATE DATA ISqH # VERY SMALL BUT IT’S GOT ENOUGH INFORMATION THIS ENVELOPE AS I’M COMPLETELY CONVINCED DO NOT REFLECT ANYTHING OF THIS STRUCTURE THEY ARE JUST BASED ON THE EXPERIMENT OF DATA AND THEN WE DID IT WITH ANOTHER PEPTIDE JUST TO MAKE SURE IT WAS NOT A FLUKE LIKE WE DID HERE ALL THE PEPTIDES, THESE ARE ALL EXPERIMENTAL HERE IS THE ENVELOPE WITH ANOTHER PEPTIDE FROM ANOTHER AND RECEPTOR TYROSINE KINASE ASSOCIATED PROTEIN AND AGAIN THIS REGION HAS GROWN WITH RESPECT TO THE WILDTYPE EACH OF THESE ENVELOPE CALCULATED ENZYMES WITH TEN DIFFERENT DATASETS SO THERE ARE THINGS WE WILLD+ñ SIGNAL THERE SO THAT’S MORE OR LESS WHERE WE ARE WE ARE NOW TRYING TO LOOK WITH MOREe0/Z SOPHISTICATED WAYS OF MATCHING THE EXPERIMENTAL DATA THAT TAKES A BIGGER ADVANTAGE OF THE POSSIBLE COORDINANTS OF THE STRUCTURE OR KNOWING THE DYNAMICS HAVING ALL THE?k CONFIRMATIONS THAT ARE BEING SAMPLE IN SO, JUST TO SUMMARIZE THIS TALK, THE BINDING OF THIS PHOSPHOTYROSINE PEPTIDE RELEASES THE INHIBITION BY WEAKENING THE INTERACTION WITH THE NSH2 DISLODGING THE NSH2 FROM THE CENTER POSITION CHANGES THE STATES OF THE DYNAMICALLY ACCESSIBLE IT’S NOT A NEW CONFIRMATION IT’S JUST CONFIRMATION THAT SOMETHING BECOMES JUST — SPENDS MORE TIME AWAY THIS CHANGES, ONCE THIS HAPPENS, THEY ARE CHANGES THAT IS IN THE CONFIRMATIONS ACCESSIBLE TO THE CATALYTIC GROUP OF THE KINASE DOMAIN, THAT PROBABLY WILL BE ABLE, SOMEBODY WILL BE ABLE TO FIND THEY WILL HAVE CHANGES IN THE CATALYTIC ACTIVITY WHICH IS SOME OF THIS CONFIRMATION THAT HAS THE LIMITATION OF CATALYTIC RESIDUES AND THE PHOSPHOTYROSINE BINDING SEEMS TO BE THE SAME AS THE ONCOGENIC MUTATIONS IN THE HELICAL DOMAIN SPOT AND ANOTHER GROUP OF MUTATIONS OCCUR IN THE INTERACTION OF THE DOMAIN WITHIN THE HELICAL THIS IS THE PRESENTATION OF THAT BIG GLOB OR COIL, AND I DON’T HAVE TIME TO DISCUSS IT BUT THERE ARE A SERIES OF SPECIFIC> INTERACTIONS WHICH INVOLVEK]Ñ CHARGES AND HYDROGEN BOMBS BUT THERE IS A LARGE SURFACE OF INTERACTION THERE IS ONE MUTATIONqhD WHICH IS QUITE PREVALENT IN THE C2 DOMAIN AND THIS IS A — [INDISCERNIBLE][p6 MAKES HYDROGEN BOMBS OF THE COILED COIL OF THE IL2 DOMAIN — [INDISCERNIBLE] 5 SCOUR AND 560 SO WE REASON THAT CHANGING THIS RESIDUE WILL CHANGE THIS TO A — AND WILL HAVE SOME AFFECT FROM THE ACTIVITY OF THE — THAT SEEMS TO BE THERE WAS ANOTHER PREDICTION ONE COULD MAKE WHY DID THESE MUTATIONS — [INDISCERNIBLE] THEY WILL HAVE THE SAME AFFECT AND THE REASON IS THAT IT DIDN’T LOOK FIRST SO ABOUT TWO YEARS AFTER THE STRUCTURE, A LARGE NUMBER OF GLOWO BLAST OHMS CAME OUT WHEN THEY DID THAT FROM THEIR PAPER, THEY FOUND THE RESIDUE THAT WAS PRODUCED THAT WILL HAVE THE SAME EFFECT AS MUTATED FREQUENTLY IN GLIOBLASTOMA SO IT’S VERY CLEAR THAT WE ARE

COMING OR GETTING IT HANDLED ON WHAT THESE MUTATIONS DO NOW THESE LARGE — THE GENERAL IDEA IS THIS IS INTERACTION FOR SOME MUTATIONS HERE AND THIS IS A COILED COIL SO,qKQm ATTENDING THE POSITION OF THE MUTATION HERE OF THIS ROD WILL ALSO HAVE AN AFFECT ON HOW STRONG IS THE INTERACTION OF THE NSH2 WITH THE HELICAL AND KINASE DOMAIN SO MUTATIONS HERE WILL HAVE THE SAME AFFECT WITH WEAKENING INTERACTION OF THE NSH2 SO, TO DISCUSS THE LAST KIND OF MECHANISMS, I’M GOING TO TALK A LITTLE BIT ABOUT MEMBRANE INTERACTIONS THIS IS A LIPID KINASE SO SUBSTRATES AND MEMBRANE COMPONENTS THEY ARE NEVER IN SOLUTION THEY ARE A MEMBRANE COMPONENT SOCIETY PROTEIN HAS TO SOMEHOW INTERACT WITH THE MEMBRANE SOME OF THE THINGS IN HERE — IF YOU LOOK AT THE SURFACE OF THE MOLECULE, IT HAS THE PERCENTAGE THAN USUAL OF BLUE IS POSITIVELY CHARGED, POSITIVELY CHARGED HERE IT REMAINS BUT THERE IS A LARGE NUMBER OFwu LYSINES ANDAL JANINES SO WE ARE NOT PRO PEASING THIS SULPHATE INTERACTS WITH THE MEN BRAIN Tha ISH2 DOMAIN THAT IN REALITY HAVE NOT BEEN PREDICTED THIS DATA USING ANTIBODIES THIS IS THE ADAPTER BINDING DOMAIN THAT ALSO THE C2 DOMAIN INTERACTS SONGLY WITH THE ISH2 AND THE C2 DOMAIN IN MEMBRANE INTERACTION DOMAIN SO EVERYTHING MAKES GENERAL AND THE5÷5çñ CATALYTIC SIDE IS SOMEWHAT HERE AND I SHOW IT TO YOU IN MORE DETAIL, ACCESS TO SUBSTRATE THIS IS AN OTHER REPRESENTATION OF THE SAME AND HERE IS WHERE THE CATALYTIC SITE IS I CANNOT SEE IT FROM HERE BUT MAYBE IN THE SCREENING RIGHT THERE THERE IS AN ATP BUILT IN THE SITE OF THE CATALYTIC SITE AND HERE IS A DIRECT CONNECTION TO A MEMBRANE WHERE THIS COULD HAVE BEEN A LITTLE BIT MORE IN THE MEMBRANE AND THEN THE ACCESSIBLE DIRECT PHOSPHORYLATION ONE THING THAT IS INTERESTING, THESE ARE ONE SET OF INTERACTIONS WHICH ARE NOT SPECIFIC INTERACTIONS WITH THE MEMBRANE BY DOMAINS OF THEóBñ PROTEIN HOWEVER, THERE IS STRUCTURE OF THE SAME PROCESS OF ANOTHER ISOFORM WITH RAS SO RAS BINDS TO THE BINDING DOMAIN WE TRANSFER THAT BINDING TO+fÑ MODEL AND THEN THIS IS THE DERMINOUS AND THIS IS MORE OR LESS WHAT THE LENGTH OF THE PATHWAY IS NOT PRESENT IN THE STRUCTURE BECAUSE — [INDISCERNIBLE] AND THAT’S WHERE THIS ONCOR FOR6ISU(j IS IT’S POSSIBLE THAT SOME CONDITIONS, THE FUNCTION OF RAS IS TO AID THE PROTEIN IN THE MEMBRANE AND THERE ARE SOME STORIES YOU CAN SEE RELATED TO THAT SO AGAIN, THIS IS WHERE THE BINDING SITE IS AND THIS>=Ñ IS WHY THE LIPID SUBSTRATES WILL BE SO, WHAT HAPPENS WHEN THIS MUTATION AND WHY AM I MENTIONING THE MEMBRANE WHEN I TALK ABOUT THAT MUTATION? THIS IS ONE OF THE MOST PER VALIANT MUTATIONS AND IT IS CORRELATING QUITE STRONGLY WITH AGGRESSIVENESS OF THE TUMOR IT IS LOCATED CLOSE TO THE END OF THE MOLECULE THE MOLECULE HAS 1,068 AMINO ACIDS MOST OF THE TIME IT IS AN ARGININE IN THE MUTANT LOOK AT THE STRUCTURE OF THE MUTANT AND THERE ARE SOME SMALL DIFFERENCES I SHOW YOU IT IS ALMOST IDENTICAL TO THE ONE OF THE WILDTYPE.N IN THE RIGHT PLACE

SO, WHAT IS DIFFERENT? SO IF WE LOOK AT THE PATH RESIDUE 478, HERE IS WHAT 1047 IS IN THE WILDTYPE THE WILDTYPE IS A HIS TEEN AND THEN THE CELL CHAIN IS COMPLETELY DISORDERED AFTER ONE AMINO ACID, 1047, NEVER SAW ANYMORE OF THEM WHEN WE LOOK AT THE MUTANT, MANY THINGS HAPPEN THIS CONFORMATION CHANGES BECAUSE THE ARGININE NOW FUNCTIONS IN THAT DIRECTION AND NOW THIS WHOLE LOOP UP TO AMINE ON ACID 1062 BECOMES ORDER FOR THESE LOOP TO BE ORDER, THIS OTHER LOOP HAS TO CHANGE CONFORMATION”í>Q÷ AND ESE TWO LOOPS NOW FORM A SULPHATE HERE YOU LOOK AT OUR SUGGESTION, THE MEMBRANE PORTION IS, WE CHANGE COMPLETELY ONE OF THE REASONS WHY THIS INTERACTS WITH THE MEMBRANE AND HERE ARE THE TWO SITUATIONS THIS IS THE WILDTYPE AND THIS IS THE MUTANT WITH THE TWO LOOPS INTERACTING WITHxxv THE MEMBRANE WE NEED SOMEñ&NK CALCULATIONS AND THE CALCULATIONS INDICATE NOT ONLY THAT BUT THERE IS CHANGING ORIENTATION AND THE SMALL CHANGING IN ORIENTATION WITH RESPECT TO THE MEMBRANE, THIS ONE WILL MOVE IN THAT DIRECTION TO MAKE A COMPOUND AND MORE OR LESS, IT SAYS THAT WEHvb [email protected] INCR EASING THE INTERACTION ENERGY WITH RESPECT TO THE FACTOR OFg 6-8 SO, THIS MUTANT INTERACTS A LITTLE BIT BETTER WITH THE MEMBRANE IF THAT IS THE CASE, THAT MEANS THAT IT MAY BE CHANGING THE ACTIVITY BY JUST HAVING MORE ACCESS TO — [INDISCERNIBLE] IF THAT IS THE CASE, THIS MUTANT SHOULD BE ACTIVATABLE BY THE ACTIVATOR, PHOSPHOTYROSINE PEPTIDE AND [INDISCERNIBLE] THE SAME SLIDE I SHOW YOU BEFORE THE WILDTYPE HAS NORMAL ACTIVITIES THESE ARE THE ONES I DISCUSSED BEFORE AND THIS IS THE MORE OR LESS THE SAME IF WE NORMALIZE, THOSE ARE THE WHITE BARS, TO ONE, AND THEN WE TAKE THEM IN THE PRESENCE OF ACTIVATING PEPTIDES, THEI it WILDTYPE INCREASES, AS IT SHOULD, THESE TWO DON’T BECAUSE THEY USE THE SAME MECHANISM BUT THIS ONE IS VERY ACTIVATED BY PEPTIDES WHICH MEANS WHATEVER MAKES THIS ACTIVATION IS NOT RELEASED IN THE ACTIVATION — INHIBITION SO CLEARLY THEY ARE TWO MECHANISMS AT PLAY ONE IS RELEASE OF NSH2 ACTIVATION AND THE OTHER IS IN CREASE OF THE — [INDISCERNIBLE] WE ARE NOT GREAT MEMBRANE CHEMISTS SO WE HAVE TO DEVISE AN EXPERIMENT THAT EVEN WE COULD DO AND WHAT WE DID IS, THIS IS OPERATIONAL DIFFERENTIAL ACCESS TO THE MEMBRANE WE SHOULD DETECT IT SOMEHOW SO, WE DID THE ASSAY IN THE PRESENCE OF — THAT CONTAINED DIFFERENT PHOS TO LITTICS — [INDISCERNIBLE] LIVER AND BRAIN LIP KIDS AND LIPIDSjx THEY WERE USED AS THEY WERE AVAILABLE NOT BECAUSE WE WANTED TO PUT ANY EMPHASIS ON THE FACT THAT WE ARE TUMOR CELLS WE CHECKED FOR THE INCORPORATIONlómD OF P CELL FORMATION INTO PIP3 AND WHEN YOU DO THAT, IFu LOOK AT THE WILDTYPE AND THE MUTANT, HERE IS THE NORMALIZED TO ONE FOR BASAL ACTIVITY, IF WE CHECK THE DIFFERENT LIPIDS, THEY RESPOND TO THE CHANGE IN LIPIDS IN VERY DIFFERENT WAYS SO, I HAVE EVERYTHING PROVEN WHAT IS PROVEN IS THAT IT IS DONE WITH THESE CHANGED LOOKS AND HAVE DIFFERENT WAY OF INTERACTING WITH THE MEMBRANE THAN THE WILDTYPE ONE THING JUST BEFORE I FINISH AND I HAVE TO SKIPâ SOME SLIDES WHAT DO WE KNOW ABOUT BINDING OF

INHIBITORS? SO THE ONE THAT WE TESTED WAS — [INDISCERNIBLE] IT’S A KINASE OF THIS TYPE INHIBITOR AND WHAT IT DOES IS MAKE COVALENT BOND IT IS THE BINDING OF THIS TYPE THAT HAS A COVALENT BOND TO A LYSINE RESIDENT THIS IS THE STRUCTURE AND THAT IS THE WAY WE KNOW WHERE THE ATP SITE IS — [INDISCERNIBLE] THIS IS YOU SEE HOW THE ACTIVE MEMBRANE TO THE CATALYTICp SO IF WE LOOK NOW AT TWO ISOFORMS AND LET’S LOOK FOR ISOFORM SPECIFIC INHIBITOR THIS IS THE ISOFORM GAMMA THIS IS PET FAR IN 55 — AS FAR AS CAN GET IF YOU LOOK AT THE BINDING, THIS STRUCTURE WAS DONEKóz BY THE GROUP OF — [INDISCERNIBLE] ALL THE RESIDUES — [INDISCERNIBLE] ALL RESIDUES INVOLVING ARE THE SAME SO IT IS CLEAR THAT — [INDISCERNIBLE]ö ISOFORM SPECIFIC INHIBITOR IT’S VERY GENERAL INHIBITOR HOWEVER, WHEN WE LOOKED AT THE STRUCTURE IN MORE DETAIL, WE SAW THAT THIS LOOK IN THE ALPHA EXPERIENCED BETWEEN UNBOUND AND BOUND, A LARGE CHANGE WHY THIS LOOKS HERE DOESN’T IN THE CASE OF GAMMA, IT’S THE OTHER WAY AROUND THIS LOOK DOESN’T EXPERIENCE ANY CHANGE THIS ONE EXPERIENCE A VERY LARGE CHANGE SO, EVEN THE STRUCTURES ARE EXTREMELY SIMILAR IN THE BINDING SITE, IT WOULD BE POSSIBLE TO DO ISOFORM-SPECIFIC INHIBITORS BY LOOKING AT THE CONFORMATIONAL AFFECT OF INHIBITOR BINDING I HAVE MORE THINGS LIKE THAT BUT I THINK I’M GOING TO SKIP THEM AND TO GIVE YOU AN IDEA WHAT OTHER THOUGHTS OCCUR WHEN WE LOOK AT THE BINDING SITES THIS IS THE A TP BINDING SITE IT’S LOCATED HERE AND I SHOW YOU WITH THE MEMBRANE THIS IS A VERY WELL STUDIED SITE AND ALL THE INHIBITORS TO DATE ARE GOING TO THE BINDING SITE BECAUSE THEY COME FROM THE BACKGROUND CHEMISTRY OF KINASES AND THAT IS WHAT THEY FOUND IN COMMON, NOT THE PHOSPHATE BUT THE ATP IT IS VERY DIFFICULT TO FIND BINDING TO THIS PROTEIN, IT’S VERY DIFFICULT TO SEE STRUCTURE.oz SO, WE LOOK FOR SOME EXPERIENCE LOOKING FOR PROTEIN SITES AND STRUCTURES AND WE CANNOT WITH THIS SITE, IN THE CORRECT POSITION TO BE PHOSPHORYLATED THIS IS THE POSITION TO BE PHOSPHORYLATED THE RELATION WITH THE MEMBRANE IS PERFECT SO, WE ARE NOW EXPLORING THIS SIDE AS IT IS POSSIBLE THAT WILL GIVE THE IDEA TO OPEN A NEW SITE FOR THE DEVELOPING OF INHIBITORS AND ALSO FOR SUBSTRATE88hj INHIBITORS, INHIBITOR THAT IS COVER THE ATP AND THE — [INDISCERNIBLE] THAT IS SOMETHING WE ARE EXPLORING JUST FROM THE BIOPHYSICAL POINT OF VIEW SO, JUST TO GET AN IDEA OF WHAT I TRIED TO TELL YOU IN 50 MINUTES, THERE SEEMS TO BE TWO GENERAL MECHANISM OF ACTIVATION ONE IS WHICH WE NEED NSH2 CLEARLY RELATED TO RECEPTOR TYROSINE KINASE BINDING AND SOME ONCOGENIC MUTANTS AND THEN CHANGING OF THE ENZYME WITH THE CELL, MEMBRANE AND BOTH OF THEM ARE PRESENT IN MUTATIONS BUT BOTH OF THEM ARE PROVIDING IMPORTANT — [INDISCERNIBLE] SO, HOW IS THE RELEASE OF ACTIVATION? THIS DOMAIN COMPACTS THREE DOMAINS AND POSSIBLY MODIFIES THE COMMUNICATION BETWEEN THOSE THREE(R ACTIVATING PHOSPHOTYROSINE PEPTIDES RELIEVES INHIBITION BYW 9Uz THE NSH2 DOMAIN WHICH IS THE DOMAIN THAT IS MODIFYING INTERACTIONS HOWEVER, ACCESS TO THIS SITE BY THE RECEPTOR TYROSINE KINASE REQUIRES THAT THIS DOMAIN IS AT

LEAST PART OF THE TIME REMOVED FROM THE POSITIONS SO THERE ARE THREE IN THE NEED FOR DYNAMIC EFFECTS FOR THE MUTATIONS, MANY OF THE MUTATIONS OCCUR AT INTERFACES BETWEEN DOMAINS AND SOME OF THESE MUTATIONS USE THE SAME MECANISM AND ACTIVATION AND THE MUTATIONS CAN OCCUR DIRECTLY IN THE INTERFACE AND WHEN THEY OCCUR VERY FAR, THEY ARE COMMUNICATED BY THIS COIL, WHICH IS A DROP OF THE MOLECULE THE AFFECTS OF WEAKENING THIS INTERACTION IS A CHANGING THE FACE OF DYNAMICALLY UPSET THERE ARE PROBABLY NOT VERY WELL DEFINED CONFORMATIONAL CHANGES IN THE CASE OF INHIBITOR BINDING, IT IS POSSIBLE THAT ISOFORM-SPECIFIC REAGENTS CAN BE PAID BY LOOKING AT THE CONFORMATIONAL CHANGE THAT IS OCCUR AFTER BINDING THE KNOWN CD4 AND THEN THE OTHER MECHANISMS INVOLVED INTERACTIONS WITH THE CELL MEMBRANE AND INTERACTIONS WITH THE CELL MEMBRANE SENSITIVITY OF THE INTERACTION WITH THE CELL MEMBRANE CORRELATE WELL WITH THE STRUCTURAL AND THE ACTIVATION BY THIS PARTICULAR ENZYME SO, I WANT TO THANK THE PEOPLE PEOPLE — [INDISCERNIBLE] HERE FROM THE BEGINNING AND DIRECTED MOST OF THE DAY-TO-DAY OPERATIONS OF THIS WORK THE STUDENT THAT DID THE STRUCTURE OF THE WILDTYPE [INDISCERNIBLE] [READING] A GRADUATE STUDENT GRADUATING IN A FEW WEEKS AND SHE LEAD ALL THE CALCULATIONS I SHOWED YOU SHE DETECTED FOR SOME TO THE LABORATORY [READING NAMES] AND THE OTHER COLLABORATORS FROM THE LABORATORIES AND WE DID MANY OF THESE EXPERIMENTS IN THE NATIONAL LABORATORY, FUNDING OF THEE’ç NCI AND THE NIH AND WE STARTED THIS PROJECT ON AN UNPHOSPHOR LATED TRANSFER PROJECT AND IT EVOLVED INTO THIS PROJECT AND WE HAD FUNDING FROM NICMH AND THESE ARE PEOPLE IN MY LAB THIS IS NOT A SLIDE PHOTOGRAPH BUT HERE ARE SOME OF THE PEOPLE I MENTIONED [INDISCERNIBLE] THANK YOU VERY MUCH FOR YOUR ATTENTION [APPLAUSE] I WOULD BE FLOOD TO ANSWER QUESTIONS I’M SURE NOT EVERYBODY BELIEVED EVERYTHING I SAID >> THERE IS A RECEPTION AT THE LIBRARY AFTER THIS >> YES? >> [OFF MIC] >> WHO HAS A MIC? >> THE MUTANTS WHO ALSO DIFFERENT DOMAINS SUPPOSED TO WORK ON THE SAME MECHANISMS, THE ACTIVATIONS OF THE KINASE IS SYNERGISTICS OR — YOU GET THE SAME EFFECT? >> IN THAT CASES, IT WAS — WHEN TUMORS ARE SEQUENCE, USUALLY YOU DON’T KNOW IF THE MUTATIONS OCCURRED MORE THAN ONE IN THE SAME SET TOOTS CONGLOMERATE OF CELL SO, WE DID NOT TRY MUTANT, DOUBLE MUTANT THAT SEPARATED IN SEQUENCE WE TRY THE HOTSPOT OR THIS HOTSPOT AND AS FAR AS I CAN

TELL, DOUBLE MUTATIONS OF DISTANT MUTANTS CHECK TO SEE — [INDISCERNIBLE] BUT WE WALK WITH THE MUTANT SPECIFIC WHO WOULD LIKE TO HAVE MUTANT THAT WALKS ON DIFFERENT DOMAIN BUT WALKS LIKE THE SAME MECHANISM NOT TO BE ADDED >> THAT IS CORRECT YES ONLY NONACTIVITY WAS SHOWN WITH RESPECT TO PHOSPHOTYROSINE BINDING BUTDbE NOT TO THE — NOT TOO MANY OF THEM CORRECT THAT’S A GOOD EXPERIMENT, >> SO YOU TALKED ABOUT THE DYNAMICS WHAT DO YOU THINK WHICH TIME SCALE DOES THE PROTEIN BEHAVE DYNAMIC? DO YOU THINK IT’S FAST ORN’?eñ SLOW? >> YOU REALIZE THAT SOME OF THE CALCULATIONS WERE NOT — [INDISCERNIBLE] ONE WAS DYNAMIC NETWORKS AND THEN — [INDISCERNIBLE] SO ALL THE SEQUENCE THAT IS WE ARE LOOKING AT ARE Aá FREQUENCY THE TIME SCALE IS DIFFICULT TO BELIEVE THIS WILL BE THE FREQUENCY AS O SUGGESTED — [INDISCERNIBLE] SO THEN 10 TO THE 7s OR SOMETHING LIKE THAT, WHICH IS I THINK DIFFICULT TO BELIEVE SO THE TIME SCALE IS DIFFICULT TO IT’S POSSIBLE — [INDISCERNIBLE] IT DOES REFLECT THE MOMENT FOR THOSE TO OCCUR, A STABLE STRUCTURE, IT HAS TO BE EASY TO MOVE IN BOTH DIRECTIONS THAT IS WHAT THIS CALCULATIONS REFLECT IF YOU’RE A STABLE STRUCTURE, THOSE DIRECTIONS THAT WE ARE SEEING ARE THE ONES WHERE IT’S EASIER TO MOVE ONCE YOU MOVE FROM THE STABLE STRUCTURE, HOW FAR OR HOW FAST IS MUCH MORE DIFFICULT TO DECIDE BUT IN A SYSTEMS OF THIS SIZE UNLESS NEW TECHNIQUES BECOME ACCESSIBLE, IT’S VERY DIFFICULT TO GET TIME SCALE >> JUST ONE — ACTIVITY NOT THAT MUCH HIGHER IN THE MUTATED — [INDISCERNIBLE] >> SAY IT AGAIN? >> THE ACTIVITY NOT THAT MUCH HIGHER, SO IT WAS NOT SOMETHING LIKE — [INDISCERNIBLE] >> NO, NO THAT WAS THE BIGGEST SURPRISE TO ME WHEN I GOT INTO THIS I WAS INTO — I COME FROM OTHER ENZYMES AND WE DO A MUTATION, AND WE MAKE DECISIONS WITH A FACTOR OF THREE AND THAT HAS NOTHING TO DO WITH THE ACTIVITY AND THE MECHANISM OF THE ENZYME THAT HAS BEEN MY STANDARD RESPONSE THEN I GET INTO SIMILAR AND VERY SMALL CHANGES WITH VERY LARGE AFFECTS I’M NOT COMPLETELY SURE HOW THAT IS ACCOMPLISHED IN THE SENSE THAT IF YOU DO HAVE THAT ACTIVITY, — [INDISCERNIBLE] YOU COULD SAY YOU HAD NO ACTIVITY BUT P10, THE PHOSPHATASE, IT IS ALWAYS JUST TAKING IT AWAY BUT SOMETHING$% [INDISCERNIBLE] WHY WOULD YOU PHOSPHORYLATE SOM% TO KEEP IT IN CONTROL? YOU CAN’T DO IT — [INDISCERNIBLE] SO I’M AS BAFFLED AS YOU ARE I SHOULDN’T BE BUT I AM, YES >> ANYMORE QUESTIONS? IF NOT, LET’S THANK THE SPEAKER AND PLEASE JOIN US AT THE RECEPTION

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G-protein signaling

many g-protein coupled receptors have a large extracellular ligand binding domain when an appropriate protein ligand binds to this domain the receptor undergoes a conformational change that is transmitted to its cytosolic regions which now activate a trimeric GTP binding protein or g-protein for short as the name implies a trimeric g-protein is composed of three protein subunits called alpha beta and gamma both the alpha and gamma subunits have covalently attached lipid tails that help anchor the g-protein in the plasma membrane in the absence of a signal the alpha subunit has a gdp bound and the g protein is inactive in some cases the inactive g-protein is associated with the inactive receptor while in other cases as shown here it only binds after the receptor is activated in either case an activated receptor induces a conformational change in the alpha subunit causing the gdp to dissociate GTP which is abundant in the cytosol can now readily bind in place of the gdp GTP binding causes a further conformational change in the g-protein activating both the alpha subunit and beta gamma complex in some cases as shown here the activated alpha subunit dissociates from the activated beta gamma complex whereas in other cases the two activated components stay together in either case both of the activated components can now regulate the activity of target proteins in the plasma membrane as shown here for a GTP bound alpha subunit the activated target proteins then relay the signal to other components in the signaling cascade eventually the alpha subunit hydrolyzes its bound GTP to gdp which in activates the subunit this step is often accelerated by the binding of another protein called a regulator of g-protein signaling or RGS the inactivated gdp-bound alpha subunit now reforms an inactive g-protein with a beta gamma complex turning off other downstream events as long as the signaling receptor remains stimulated it can continue to activate g-proteins upon prolonged stimulation however the receptors eventually inactivate even if they’re activating ligands remain bound in this case a receptor kinase phosphorylates the cytosolic portions of the activated receptor once a receptor has been phosphorylated in this way it binds with high affinity to an arrest in protein which in activates the receptor by preventing its interaction with G proteins arrest ins also act as adapter proteins and recruit the phosphorylated receptors to clathrin-coated pits from where the receptors are endocytosed and afterwards they can either be degraded in lysosomes or activate new signaling pathways

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I have something new to share! Plus of course lots of tips and tricks

welcome to Hedgehog Hollow today I’m super excited because I get to share with you the entire blue blossom trend front on ik studios and of course it’s my favorite color combination of pink of course and blue and here I have absolutely everything in their release I’ve got a little bit of everything to share with you I’ve got some tips some tricks samples all sorts of bits and pieces and I’m going to be sharing some top tips and I have a coupon to share with you as well so let’s dive in and take a look I’m going to start off with the paper site to put those to the side before we start making a mess and so the basic paper in this collection is ballet pink as with all of the crop perfect papers they have that canvas linen Buzzle texture however you like to describe it on one side and on the reverse side is almost smooth which I really like because depending on our project we can then decide which side we want face-up and which side we’d like to put on the bottom and they come in 8 and a half by 11 here in the US or a 4 in Europe and then they also come in 12 by 12 so if you like to scrapbook when you want to do larger projects you have that option too now all of the papers come in 5 packs and that also includes the 12 by 12 if you buy the a for eight-and-a-half by eleven it comes in a ten pack so they’re your options with regards to paper packs then of course we have all of our speciality papers too so the first one we have is the glitter card stock and I know lots of you love this glitter card stock because whilst I do lots of things with glitter and I have a concrete floor and to me it doesn’t matter if I get glitter everywhere lots of you have that glitter phobia of mister being everywhere and having to do all of that clear up well with glitter card stock you don’t have that issue it is no shared so I can do this nothing comes off I’ve always frightened of doing that and some glitter coming off but it really doesn’t and you can die-cut this so you can make words or shapes or ornaments memory books you don’t really absolutely anything with her at all you can cut it out you make card bases really really great is the perfect thickness for die-cutting so really really love this and it’s that blue and silver so you can make icicles for Christmas you can do snick you can do all sorts of amazing things and of course this collection is great because we can use it the whole year round where you can use it for blue babies pink babies we can use it this Christmas in particular because if you’ve been through those big-box stores you’ll see that these are really trendy Christmas colors too and they also happen to be my favorite colors you’ll notice I have pink hair a lot and I wear a lot of pink too so I am loving this collection we also have the navy navy 12 paper I think it looks more black than Navy particularly against some of the others but they say it is Navy and it has these like damask flowers and you see how it catches the light this is actually embossed in there it’s a very tactile paper and absolutely love this paper it comes in an a4 size for those in the US a4 is slightly longer but a little bit narrower than eight-and-a-half by 11 I actually have a course coming very soon on different paper sizes different paper weights so stay tuned if you are interested in that we’ll add a link to the blog below you can sign up for our newsletter if you want to find out when that is coming up because it’s probably one of the most frequently asked questions I get about gsm versus pound and different paper sizes so that will be coming very soon but this isn’t a four size and it’s a lovely lovely paper a really elegant – then we have the satin cardstock I mean how pretty is this this one is called blue obsidian orb sidon love this paper and you all know how much I love my black velvet I feeling this is going to be one of my Christmas papers they released a few in the Christmas trend release I’m definitely gonna be adding this one in because this is just so so pretty look at that almost like a sapphire e-type color as well or tanzanite color and the thing about satin papers is whilst I love a glossy cardstock it’s sometimes or Amira cardstock if sometimes can distract from your project or I think it can anyway whereas a mirrored cardstock still has a little bit of bling to it it doesn’t reflect which is one thing I really like about it which makes it easier to photograph too so I think the guys here like that but it also just whilst it brings your eye into the project and it has a little bit of shimmer it’s not too much shimmer and I think it’s just a little bit more elegant than a boss tee cardstock too so that’s why I really really like the satins we have two handmade cotton papers here as well and I know I’ve gone on and on about how many cotton papers if you’ve seen in my trend unboxings before you’d have heard this tip but you can always turn these how many papers

over and they have a reverse side and they always have the same texture on the reverse side and you can see where I was trying this out earlier you can take a door bar with maybe something like embellishment loose this is the new one out the trend and you can take someone little daughter like this and you can change the color or maybe you want to take the same paper but you just want to add an accent you see how here I can just add a little bit of an accent here with that pink and it just changes the paper slightly so they’re great for being able to alter them excuse me or create them in any color you’d like so that’s another option that you have with those handmade papers then another one we have is this rose gold posies so these craft foiled card stocks are amazing we’ve had some already earlier in the year there’s now a whole collection of them again everything I’m going to show you is linked in the video description plus some exclusive coupon codes and I’m also going to mention at the Hedgehog Hollow members area where you can get an extra temps enough so if you join our community tonic are giving all of our members an extra 10% off the entire store so if you want to grab things out of this trend and I’m sure you’re going to see things you love you can save even more but this is great because this is really pretty just on its own I mean how pretty is that but you can take things like your mica myths your maybe your distress oxide sprays any dye based ink water-based ink your aqua flows you can use you can pop them over the top or even take you alcohol markers and color in areas but the great thing about anything die based is the foiling resists that color like an embossing resist and I’m what I do is I pop an embossing resist video that Greg and I recently did in that top right hand corner if you want to click on it but you can go in put it over the top and then you come wipe away the excess and it won’t sit on the foil whereas if you use alcohol markers it would color over the foil but it’s a really fun technique to do and you can just alter this and make it your own unique colors really really fun and then finally we have a pearlescent pink here as well really really pretty it’s called a princess pink just a little bit of shimmer in there again just adds that little bit of interest to your projects one final a lot of people we have is the blue blossom designer paper here you have 24 sheets in your pad you can see all of them are double-sided there’s eight patterns in total and I have them down the front here so this is one sheet and this is one seat so you have two sheets of blue and then you have two in the pink and I just took them out so you could see the different patterns and you can see them here on the front as well so you have the checks and the dots you got stripes and darts you’ve got the gingham and hearts and then you’ve got stripes and the grid on the other side so you have eight designs of total six sheets of each of the eight different designs making the 24 in total assam delinked lien free so if you are a scrapbooker they’re not going to affect any of your photos in there as well so that’s the blue blossom panelist in one of those for each of the trends this year too so lots and lots of fun things there now we get to dive into all of those beautiful mediums we’ve got embossing powders here when one is the pink popsicle and the other is Dutchess blue and here I have these little samples they can also give you a top tip with so when you open up your embossing powders you’re going to see these little pieces on top and they’ll have embossing powder stuck to them it’s part of the manufacturing process and what I do is I just hold them with my tweezers like this and then I take out my heat tool I hate it so that the embossing powder sets and that’s why I put in my swatch book or I did want to use to swatch just with a piece of cardboard I’d stick that on my swatch cardboard with my label above and then you don’t have to do anything you don’t have to stamp it out and reimburse it you just put that straight in and you’re watch is done for you really really easy no extra work for you so top tip in there we have a new glitter marker and do go and watch our video on glitter markers again I’ll link it in that top right hand corner for you because you can use the glitter markers as watercolor you can use them as an ink pad which is super super cool you can just paint draw it onto your stamp and then stamp out and it works as a glittery ink pad like truly truly glittery ink pad this is how it looks on your white cardstock you can see really pretty there it’s called slinky any press peony this one but it also works on your dark card stocks too look how pretty it is even on your darker and there’s not many water-based products this is water-based you can use on both hence you can watercolor with it as well we also have three new ink cubes so in here you have raspberry smoothies siren blue and midnight surf and I pre swatch them out for you so you can see here that gorgeous raspberry pink that’s gonna coordinate siren blue look at that pretty sapphire I’d really call it like a sapphire blue there and the midnight surf is that beautiful steely blue too so lots and lots of things we can do

with that and you’ll be seeing projects coming up using these supplies very soon but I’ve priest watched everything so I could show you of course we have a new glitter paste or glimmer paste this is strawberry champagne and again we have lots of videos on the channel if you want to go and check out the inspiration we have tonic playlists will make sure they’re all linked up for you but this is what the glimmer face looks like so it’s a more gritty effect versus the glacier paste that I showed you a few weeks ago but here is how it looks dry and I always think things look different dry which is like why I like to have it priests watched as you see here you can use it thin or you can use it thicker and it looks very different of how dependent or whether it is thick or thin so that’s why I have the two different options available to you so they’re your options that we have new alcohol markers in flamingo pinks and the nouveau alcohol markers are a little bit different because their price point is great and yet they still blend beautifully Copic markers are now around $8 a pen which is insane I mean I’m lucky I’ve had mine a long time when I’ve got my collection but Nouveau alcohol markers are around $6 for three so for less than one Copic using up all three of these they come with a chisel and a bullet nib and these are the Flamingo Pink’s these three here and they blend together perfectly so you can get a seamless blend with each pack I take my pee touch and I label all three of these so I know that these three and my Flamingo pinks and it is a tonic download so you can swatch it around a color wheel I know that those three go together because they’re numbering system is actually a product numbering system it’s not like the Copic system that we are used to so just to let you know on that but really really love these Alva markers we put them in the Hedgehog hollow box numerous times and then also on this chart here because I know some of you are more to colorize some of your mock alcohol marker colorist you know you have preferences we do have the aqua flow so the colors this time are pink lemonade which is the bright pink down the bottom here we have cameo pink and we have blue velvet and again I have done six ways in a video to use liquid watercolor pens because there are really versatile medium you can do things with rice you can use salt I mean all sorts of different kitchen things that you can use in your projects and then use them with dyes you can use with stamps and so go check that video out too lots of fun things you can do with them you will extend their life a lot just by actually using your glass mat or your easy clean matte scribble down and then grab a paintbrush add water to them mix your own custom colors as well they’ll last a lot lot longer so some more top tips in there for you when you’re using those mediums and I think you’ll find you have a lot more control if you put them down on a separate surface and then use a paintbrush with them but I think you’ll really love watching that video of six different ways to use a liquid watercolor brushes it’s really great video lots and lots of people have watched that one okay let’s go back to that noose I showed you earlier too which is called poppy pink again i swatched it on some Navy cardstock and then also on some white car you can see it’s a really really light pink it does add just a nice shimmer to your white card stock but if really poppy when you put it on too dark cardstock and if you’ve never played with embellishment minutes of course you know I’m gonna say there’s a video but you can use it as a metallic watercolor you can add it into your lightness spray bottles and use it as a spray you can use it as a paste you can use it as just a medium on a door bar like this you can use it in so many different ways really versatile again Nouveau products are great because there’s always more than one way that you can use them and of course that leaves me really nicely in to some of the confetti zand things that we’ve got so we have the Rose blossoms rose shell blossoms I should say so look at that pretty rose gold color but the really great thing about these is look at that it’s actually a little flower shape how cute is that so again the other thing is when you’re doing shaker cards and things so if you’re doing things like the tonic blisters which again we have videos of a two-minute shaker card and you literally can make a shaker in two minutes with those blisters you don’t necessarily these are actually tea light holders from Ikea and I like to make my own custom mixers in these and then just pop them into my carts this is the new Bluebell glitter which matches perfectly with that glitter card stock it has a rich blue with a little bit of a silver twinkle so you can see that in there too we have these peachy pink tutu with iridescent sequins and there’s multiple sizes in here so you don’t just have one size if you want to stick them on your cards rather than use them as a confetti mix you can do that I mean look how

pretty you can just see that your adjustment shimmer in there too and finally we have our Bluebell hearts so again this is a heart-shaped confetti and see it on my finger there but it has the blue color but also beautiful iridescent as I move this around you can see how they are iridescent absolutely really really pretty lots of fun things we do can do so if I wanted to I could pour some of these in there and then you can see how I can mix them together so now I can make my own custom mixes and then if you wanted to you can just pop it in a ziploc or little box the little sauce packets you get five guys you know when you go and get the ketchup if you ask them they’ll always let you take some home because done that a lot and you can take the lids home and you can also mix in a little bit of glitter and then grab your craft pick or your craft spoon mix it up and now you have your own custom shaker mix so you don’t necessarily just have to use them straight out the packet to make your own little custom mix and that one now that’s really pretty so I can keep that one for a future shaker cup so there’s another option for you so finally we have all of our mediums so we have two new mica myths we have midnight horizon and pink carnation and I have already spritz them out for you so you can see this is how they’re going to look when you’re finished you’ve got that mica shimmer in them here you can put them through a stencil you can spray them directly on your project or spray them onto your mat and then paint them on if you wanted to as well again we do have videos of maintenance from mica sprays because there are a couple of things that are really really important one is to store them on their sides the other is to make sure you shake them around and around not up and down because you’ll clog that nozzle and then when you’re done put it upside down spray until your nozzle is empty and then give it a wipe and finally then just run the nozzle under the tap and that will make sure that your nozzle works next time otherwise you’re gonna have issues with clogged nozzles and mica being in there but that just that general maintenance works really well so store it on its side shake it around it around and I didn’t tighten my lid beforehand shake it around and around and then when you’re done turn it upside down spray run it under the tap so they’re my top tips for that one this one is from where I cleaned it for when I did yesterday’s video then we have what we have next Oh new Nuvo drops to share with you so I have a swatch all of the new Nuvo drops out for you so we have a new glitter accents called a ballroom blue so that’s this one here so I did again do a and there is a download available at the Hedgehog hollow about the difference between bitter accents nouveau drops crystal accents dream drops on all the different drops available but this one dries more gritty so it has more texture to it and you’ll see with the glitter accents compared to a glitter drop the glitter drop dries more translucent and more smooth and this is your new glitter drop it is called enchanting pink so it’s got like that pink color and a little bit of pinky gold glitter inside of it they’re really really pretty again then we have a jewel drop which is called steel blue so it’s more translucent you also have an iridescent pink drop here which is your crystal drop called shimmering rose and then finally you have just a regular nouveau drop it’s not shimmery it’s a more matte finish and that’s called berry blue and they’re all going to dry opaque your regular nouveau drops whereas your jewel drop dries opaque translucent rather and again as I say we have a download telling you the difference and for those of you always say well like it Peaks in my nouveau drops and it doesn’t dry properly we do have videos to tell you how to always get the perfect nouveau drop we have a whole playlist on nouveau drops different shapes different sizes how to get the perfect one how to never get peaks on your nouveau drops how to make embellishments how to make hearts how to do all those different things so if you want all of those tips go check out all those different playlist on the channel hang around for a while bookmark them add them to your favorites come back later check those out lots and lots of different tips and tricks yet honestly you can find out lots of different information and we keep adding to them to make sure you hit that subscribe button and ring the bell because lots of tips and if you always have a request you can drop them in the comments we do compile this and we add those to our next videos that we’re going to make we always want to know what you would like to see here on the channel so if you’re struggling with something leave us a comment let us know and we’ll make sure we add it to our future videos list so the final thing I have for you is a new shimmer powder it’s called meteorite shower these again are one of my favorites because you can paint with them you can create backgrounds with them you can make sprays with them you can do all sorts of really cool stuff with them and this is how this one comes out here I was playing around with it this is lots of water this is just little bits of water there’s a color bus and it’s the only

one I know of that contains mica and mica means shimmer so you can see here it just has that like metallic shimmery finish to it super super cool again you’ve got blue you’ve got pink you’ve got black you’ve got purple lots and lots of fun colors you just kind of tap it out I always use the analogy of think of it like chili powder does start with a little oil you might want a lot by the end but start with little because you can’t take it back afterwards and then spray it with your light mist buffle I could even go in now reactivate it if I wanted to and put more water in and if you want to seal it you can absolutely do that the de-stress micro glaze is a really great way to seal it through the mail and once you’re happy with your finish if you want to do that so that’s what the shimmer powder comes out with if you want top tips you know the deal go check out the videos you can search the channel for those too even a nice to get myself a little blossom on there but doesn’t it look pretty it coordinates as always perfectly with the rest of that trend so that’s the entire blue blossom trend as I say check out the links in the video description below for the latest coupon if you’re not watching this in September 2019 you can’t go and check out one of our latest videos for the latest coupon codes we have them in every single month Ratanak but do join our community as well for even more savings and even better savings as well our favorite companies give us coupon codes every single month and of course we’ll be back at creative ation in january for next year’s trends tours of all the booths so make sure you hit that subscribe button and ring the bell for all of the latest videos we’ll be on the show floor first as always giving you the latest and best coverage and last year the official media partners so we might have some news on that very very soon or – thanks for joining me as always don’t forget to hit subscribe ring the bell hit that joint buns we parked the community and get all those amazing savings and receive your birthday card of course give us a thumbs up if you enjoyed this review of the latest trend and some of those tips and tricks and I’ll see you again tomorrow happy crafting everyone bye

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TUTORIAL: AliDropship Woocommerce Store (Create a Woocommerce Dropshipping Store) UPDATED 2018

Hey, guys It’s me, Sarah, here from Wholesale Ted and in today’s video, I’m gonna be doing something that has been highly requested I’m gonna be giving an updated, step-by-step tutorial on how to create an AliExpress dropshipping store with WooCommerce and AliDropship So yes, if you are one of those people that prefers to use WordPress and WooCommerce to build your dropshipping empire, then this tutorial is for you This is an updated version of my 2017 WooCommerce tutorial In that one, I used WooDropship but these days, AliDropship is easily the best plugin on the market for AliExpress dropshipping This is going to be a 17 step tutorial and for each step, I’m gonna switch over to my computer so that you can see exactly how to complete each step and follow along with my screen Here are the 17 steps that we will be following If you want to skip ahead to a particular step, then I will have timestamps so that you can jump straight to it in the video description below All right, the fun begins I’m gonna switch over to my computer now and give you a quick preview of the store that we are about to build So here is the store that we are going to build It features a custom logo and a nice, big splash image to give the store character and style Scrolling down, you can see that we’ve created a simple yet professional design by previewing different products listed in our store So coming back up, let’s take a look at the menu We’ve got a working store menu including a submenu under the products tab, showing off the different categories of items that we sell So here are the coffee mugs we’ve listed in our store Let’s open one up so that you can see a product page Here is a product page On the right, we’ve got bullet points promoting and selling our item, which have been proven by Amazon to be effective for conversions We’ve also got a working image gallery on the left and customers can check out the different photos And this product has multiple variations Customers can purchase either a black mug, a blue mug, a green mug, or a red mug Scrolling down the page, you can see we’ve got a bigger product description, great for SEO, and related products at the bottom Scrolling back up, let’s finish this preview off by taking a look at how customers can purchase items I’ve added one of these self-stir mugs to my cart And here is our nice, simple, yet professional checkout page As you can see, we’ve set it up so that customers can pay for their items with PayPal and we’ve set it so that each item has free shipping All right So that is the store that we are going to build Let’s get started Step 1, set up our hosting and domain name with SiteGround To set up our hosting and our domain, we are going to be using a low-cost yet high-quality service called SiteGround and this has gotten a four out of five-star rating from TechRadar Now in my 2016 and 2017 tutorials, I recommended that you used iPage because it was the cheapest form of hosting but now, it no longer is And the reason for that is because as of February this year, Google now requires that websites like e-commerce stores that collect data from customers have what is called an SSL certificate If you don’t have one, they will flag your site as being unsafe, massively hurting your sales and conversions And an SSL certificate is unfortunately not included with an iPage subscription, however, it is included for free with a SiteGround subscription A one-year subscription to SiteGround will cost you $4.95 a month paid upfront, however, sometimes you can catch it for $3.95 a month and if you find it for that price, I would recommend that you snap it up fast Another option is to instead choose to purchase a one-month trial with SiteGround, however, this is not something I recommend that you do unless you are really strapped for cash The reason for that is they will charge you $4.95 for that one month plus a huge $14.95 fee so just one month is going to cost you $20 And after that, if you want to keep your site active, then you’re going to need to purchase at least a one-year subscription plan so it’s much better to just go ahead and do it upfront If you would like to follow along with this tutorial, you will find the link to how you can get a SiteGound subscription in the video description below Please be aware, that is an affiliate link Here at Wholesale Ted, all of our YouTube tutorial videos are free and affiliate links help us keep our videos free Come and click on the Web Hosting button in the top menu Click the button under Shared Hosting Come to the StartUp plan and select Get Started Now, let’s register our domain name Type in the domain name that you want Make sure that you have .com selected I don’t recommend using any other type of URL Then, click the Proceed button and just let the page load All right Now you need to create an account For this, you’re going to need to choose an email address to use as your username and

a password Make sure you choose an email address that you have easy access to and that you don’t mind receiving emails in I’m gonna be blurring out all of my personal details in this section to keep them private And of course, select a strong password as you will be using this site to make sales and collect customer’s private data so having a strong password is crucial All right Once you’ve created your account, you are going to need to fill out your personal details You will need to give your full name, your address, and your phone number Other information is optional After that, fill out your credit card details This will be the credit card that you will be using to purchase your hosting and domain name from SiteGround Again, I’ve blurred out my details for this video for obvious reasons If your billing address is different from the address you gave above, you can deselect this checkbox and fill in the correct details Under Hosting Services, select the period that you want For most, it’ll be either the trial or the one-year plan As this is just a tutorial and I don’t plan on keeping this store, I’m selecting the trial plan And after that, just scroll down to the end of the page, click the confirm checkbox, and select the Pay Now button Then, just let SiteGround load and create your account And, done Now just click the Proceed to Customer Area button Now, if you are really strapped for cash, then you can choose to purchase your domain name through another site like GoDaddy or NameCheap to save a few dollars but if you do do that, then you are gonna have to go through the technical process of updating the nameservers and then pointing that domain name at your store which is why I recommend that most beginners just purchase their domain name through SiteGround They will connect your domain name for you, which means that you will have less technical hurdles to have to overcome, meaning that you get set up faster and that you can start making money faster Step 2, install WordPress WooCommerce is a plugin for the content management system called WordPress Before we can install WooCommerce, we need to install WordPress Luckily, WordPress is free and SiteGround makes it super easy for us to install it in just a few clicks Let me switch over to my computer and show you just how easy it is to do this Come to the Members Area inside SiteGround Make sure you are in My Accounts Then, come and click the Launch Setup Wizard button From the menu, click the Start a new website checkbox Once you’ve done that, choose to install WordPress, not WooCommerce You will then need to create your WordPress login details Choose an email address that you have easy log-in access to You will also need to select a username and a password Don’t create a weak password It is very important to keep your account secure And once you’ve done that, click the Confirm button On the next page, they’re just trying to sell you unnecessary extras you don’t need, click the Confirm button again On the next page, tick this checkbox and then click the Complete Setup button and then wait for the page to load This may take a few minutes This is absolutely normal Don’t exit Awesome Now just select the Proceed to Customers Area button to return to your SiteGround admin area and you are done Step 3, install WooCommerce Now that we’ve installed WordPress, we can now install WooCommerce and that is really easy to do Let me switch back to my computer and show you how to complete this step Once again, start out in your SiteGround account Make sure that you are in the My Accounts section of it Then come and click the Go to Admin Panel button Now you’re at the log-in page for your new WordPress site Log in using the login details that we created in step 2 And now you’re in your new WordPress site On the sidebar menu, click Plugins Now, click the Add New button All right Now we just need to come to the plugins search bar and type in WooCommerce Once you’ve typed it in, the WooCommerce plugin should show up and then you just need to click the Install Now button It may take a few minutes to install Don’t click away, just be patient Awesome Once it’s installed, just click this button Now we need to complete our WooCommerce setup You’ll need to select what country you live in and fill out your address I recommend keeping the currency to USD and selecting that you plan to just sell physical products Once you’ve done that, click the Let’s go! button Now again, we just need to let it load This can take a few minutes That’s absolutely normal Don’t click away, just be patient

And that’s it We’ve successfully installed WooCommerce Let’s move on to the next step Step 4, install and activate the AliDropship plugin AliDropship is the best AliExpress dropshipping plugin for WordPress It is very similar to Oberlo Not only does it greatly speed up the process of adding items to our store, but it is going to semi-automate the fulfillment process, making it much easier to process our customers’ orders AliDropship costs $89 to purchase Keep in mind though that this is a one-off purchase, unlike Oberlo which charges you fees once you sell 50 or more items a month Once you purchase AliDropship, that is it I will have a link to where you can get AliDropship in the video description below Please be aware, that is an affiliate link If you would choose it, we would greatly appreciate it All right So to begin with, we need to start out by purchasing it and installing it Let me switch over to my computer and show you how to do this Come to the AliDropship site Again, I’ll have the link to it in the video description below Click Buy Plugin button On the next page, click the Buy Now button On the checkout page, enter your email address You don’t need to add any comments Then just scroll down and enter your credit card details If you’d prefer, you can choose to buy the plugin with PayPal instead of a credit card After that, click the Complete Order button Now just let the page load It may take a few minutes, that’s normal Don’t click away, just be patient On this page, just click the Continue button This is an upsell page and we don’t need what they’re selling us Awesome We’ve purchased AliDropship Scroll down and find the AliDropship WooCommerce plugin and click the Download button When you click it, the plugin should download to your computer Okay, so what we’re gonna do is switch back to our WordPress Dashboard while keeping this page open We’ll need our license key later On the left menu, click the Plugins button On the Plugins page, click the Add New button After that, just click the Upload Plugin button On the next page, you’ll need to select Choose File and find the AliDropship file you just downloaded and select it Then, click Install Now The installation process make take a few moments and again, this is absolutely normal Don’t click away Awesome Now just click the Activate Plugin button and you’ve successfully installed AliDropship All right Now that we have purchased AliDropship and installed it, we need to add our license key to it so that we can activate it Earlier, when we purchased AliDropship, I said to keep that purchase page open and the reason for that was because it had our license key on it So go back to that page now and I will show you how to add the license key to your plugin and activate it Come back to the AliDropship purchase confirmation page Find your license key and copy it And then switch back to your WordPress Admin area On the left sidebar menu, click the AliDropship Woo button Then come and select the License button Then all you need to do is paste in your license key and click the Activate button After that, it’s time to move on to the next step Step 5, install the AliDropship Chrome Extension Later on in this video, in step 9, we are going to automatically add AliExpress products to our store using the AliDropship Chrome Extension That means if you haven’t already, you are going to need to get the Chome browser and install it So if you haven’t done that already, go do so now To download the AliDropship Chrome Extension, you will need to go to the Chrome web store and I will have a link to how you can get there in the video description below All right Let me switch back to my computer and show you how to do this Come to the Chrome web store extensions Again, I’ll have a link to it in the video description below Go to the search bar and type in AliDropship When the extension appears, click the Add to Chrome button On the pop-up, click Add Extension When it successfully installs, you’ll see this success message in the top right of your browser Close this message Then click the AliDropship Chrome button and type in the URL of your new store and click Add And then that’s it You’ve successfully added the AliDropship Chrome Extension It’s time to move on to the next step Step 6, install the AliDropship Theme – Shopper One of the quirks of working with the AliDropship plugin is that it is not compatible with many WordPress themes You need to make sure that you are choosing a theme that is compatible with AliDropship There are multiple themes to choose from and you only need to use one of their free ones You don’t need to pay for any of their premium themes I think that the easiest theme for beginners to use to create a simple yet professional

store is the Shopper theme so that is what we are going to be using in this tutorial To download and install it, you will need to go to the AliDropship Themes page and again, I’ll have a link to that in the video description below All right So let me switch back to my computer and show you just how simple it is to do this Start out by coming to the AliDropship Theme page Again, I’ll have a link to it in the video description below So scroll down until you find the Shopper theme We’ll be using this one so click it On the Theme page, click the Download button When you do this, it will start downloading to your computer When it’s finished downloading, switch back to your WordPress dashboard On the side menu, put your mouse over the Appearance button and click the Themes button from the submenu On the Themes page, come and click the Add New button Then on the Add Themes page, click the Upload Theme button Click the Choose File button then find the Shopper theme file that we downloaded to your computer earlier and open it And then just click the Install Now button and wait for it to install Awesome All that is left for you to do is to click the Activate button and you are done It is time to move on to the next step Step 7, edit the AliDropship settings In this step, we’re gonna be doing a couple of things Firstly, we’re gonna be setting it so that AliDropship will override the customer’s phone number with our phone number on the checkout page That means the supplier, if they need to get in contact with us, will use our phone number and not the customer’s Secondly, we’re gonna be adding in an automatic note to each order that will tell the AliExpress suppliers to not include any invoices or marketing materials with our order This is called “blind dropshipping” and it means that the customer will not find out how much that we originally paid for the item We’re also gonna be setting it so that AliDropship will check if any of our suppliers have run out of stock and if they have, it’s gonna set our inventory to zero So I’m gonna switch back now and show you how to edit AliDropship settings Come to your WordPress Dashboard and click the AliDropship Woo button on the left menu When the page loads, come and click on Settings in the left menu Now we’re gonna update the Checkout settings First we’re gonna set it so that when customers order an item, the AliExpress supplier will be given our phone number not the customer’s So come to the Default phone number and type in your phone number Don’t worry, the customer won’t see this Then click this button so that your phone number will override the customer’s number And after that, type in this custom note This will mean that no promo materials or invoices will be included in the package In this industry, we call that blind dropshipping Then just click Save Changes All right Now we’re gonna change the Order Update settings Come to the left menu and click the Updates button Now, come to this button and click it Change it so that when a variation disappears or is out of stock, that the quantity for it will automatically set to zero Keep the rest of the settings like they are You can change these if you like though It’s entirely up to you Now just come to Auto Update and click the Enable button Keep it set to update once daily and then just click Save Changes Step 8, add a custom logo and update our store’s color scheme Adding a custom logo and giving it a custom color scheme is gonna help give our store a brand and a unique feel Now, you can create a logo for free using a logo maker and I will have a link to one in the video description below For most of you though, you’re probably gonna get a much better logo made if you just head on over to Fiverr and get a graphic designer to make one for you and it’s gonna cost you just $5 plus a $0.50 transaction fee And something that you will want to do before you go ahead to complete this step is to pick a color that matches your store’s niche For me, I’m selling coffee accessories, so I’m going to choose the colour brown since coffee beans are brown So once you’ve gone ahead and chosen your colour, follow along with me on my computer Start out on the Dashboard On the left sidebar, click the Appearance button Now click the Customize button after putting your mouse over the Shopper Theme Okay Start out by clicking Site Identity Then, click Select logo Click the Select Files button Now, find where you saved your logo on your computer and open it Once loaded, click Select On the next page, you’ll have an option to crop the image I don’t need to crop my image so I’m just gonna click Open Image Fantastic We’ve now added a custom logo to our store Let’s modify the color scheme

Come and click the back button Now, select the Color button in the left menu Come to the Link color and click Select Color Choose the color that you’ve already chosen for your store Because I’m selling coffee accessories, I’m gonna be selecting a brown All right So when you’ve chosen your color, come and click the Publish button to save it Next, click the back button On the left side menu, select Buttons Now come to Background color and choose the same color that you chose before Almost there Now just click Publish And that’s it We’ve added a custom logo and color theme to our store Step 9, add products to your store For this step, we’re gonna be going over to AliExpress and then importing products directly in using the AliDropship Chrome Extension So, of course, that means that to complete this step, you’re going to need to open up your Chrome browser So go ahead and do that now and then head on over to AliExpress Come to AliExpress and do a search for the product that you want to add Let the results load up Once the results have loaded, scroll through the list and find the product that you want to add Put your mouse over it and click the AliDropship button Wait a few moments while the product is being added to your store When you get the tick, it means it was successfully added Okay So I’m just gonna scroll through the listings and add one randomly All right So let’s choose this one I’m gonna click the AliDropship button and give it a few moments to add it to my store Success Now I’m gonna switch back to my WordPress Dashboard to show you it’s been added I’m gonna come and click on the Products button and look, there are the products that we selected So go through now and add each of the products you plan to sell from AliExpress Step 10, how to add product categories Come to the left sidebar menu and hover your mouse over the Products button and select Categories from the submenu All right So I’m gonna add in one item category, coffee mugs So I’m gonna come to name and type in coffee mugs Next, I’m gonna add in a Slug If you don’t know what this means then don’t worry about it It is optional Now I’m just gonna scroll down the page and click the Add new category button And then that’s it Coming here, we can see that my coffee mug category has been successfully added I’m now gonna pause in the video and add in two extra categories Done I’ve gone and added two new categories, coffee spoons and travel mugs So go on and add your product categories now Step 11, how to create an About Us page On the left WordPress menu, click the Pages button Then come and click the Add New button Come and enter a title A simple title like About Us works great Now you just need to enter your content I’ve already written up some filler content for this video I’m also going to use an image as part of my About Us page I’m going to set it so that the picture is on the left side of the page with the text I’ve prepared in advance written around it Don’t stress too much over your About Us page It’s important to have one as it creates trust and legitimizes your store but a simple page with three to four paragraphs is plenty in the beginning I find that a lot of beginners overthink things like the About Us page and put too much emphasis on it You can make a better page later, once you are making sales Once you’ve finished adding content, click the Publish button And then that’s it We’ve successfully created our About Us page You can preview it by clicking the Preview Changes button It’s time to move on to the next step Step 12, how to add a Contact Us page On the left sidebar, click the Plugins button Now, click Add New In the search bar, type contact form Click Install Now next to Contact Form 7 and let it install Once install, click the Activate button Come back and click the button titled Plugins Click the Settings button under Contact Form 7 Come and copy the shortcode on this page Then click Pages Click Add New Type in a title Contact Us works great Now paste in the shortcode that we copied earlier Then just click Publish and you are done You’ve successfully created a contact form Messages from it will be sent to the email address you created with your WordPress account Step 13, how to add free shipping For this tutorial, I’m gonna keep it nice and simple I’m gonna set up one shipping option and that is free shipping It means that all of the products in your store will be eligible for free shipping

This means that you’re going to need to be keeping this in mind when you’re pricing your products since you need to absorb the cost of the shipping into the price that you are charging Luckily though, shipping costs on AliExpress are very, very cheap Something else that is very important to note is that for this tutorial, I didn’t specify any particular countries so my shipping was applied worldwide but I don’t suggest this for most people As I explained in my video “10 Things to DO BEFORE Dropshipping,” I recommend that beginners stick to countries that have fast, efficient shipping options And the absolute easiest country to begin with is dropshipping to the USA I recommend that beginners just start out focusing on the USA market because they have a very fast, efficient shipping option called ePacket All right Let me switch over to my computer and show you just how easy it is to do this On the left menu, hover your mouse over WooCommerce and then click the Settings button Then come and click on the Shipping tab Now come and click Add shipping zone Name it Free Shipping You can also select which countries you want to ship to in the Zone regions If you don’t select any countries, it will be a worldwide shipping option Once you’ve done that, click Add shipping method A pop-up box will appear From the dropdown menu, select Free shipping and then click Add shipping method And then that is it Something important is that if you selected particular countries, this shipping option will only apply to those countries and people from other countries won’t be able to order from you unless you create a shipping option for them This is a good way to limit which countries you ship to Step 14, how to set up PayPal To keep this tutorial and accessible for people worldwide, I’m gonna show you how to set up your checkout with PayPal To accept payments, you’re going to need a business PayPal account Luckily, turning your personal PayPal account into a business one is free Another good payment option is Stripe Now, if you are eligible to use Stripe while having your currency set to USD, I highly recommend that you add this to your store But for this video, we’re just gonna be adding in PayPal Let me switch back to my computer and show you just how easy it is to do this On the left menu, hover your mouse over WooCommerce and then click the Settings button On the Settings page, click the Checkout tab at the top of the page Now just scroll all the way down to the bottom of this page At the bottom, click the PayPal button All righty So enabling it is simple Just click the Enable checkbox This will enable PayPal Then, add your PayPal email address in the PayPal email box After that, just scroll down to the end of the page and click the Save changes button And once you’ve clicked that, you are done Now customers will be able to pay for their items using PayPal There are other options on the Checkout settings like Direct bank transfer and Cash on delivery but because we haven’t checked to enable them, they won’t show up for the customer Step 15, how to customize your homepage It’s a myth that you need a super fancy store design to make sales Having a simple, clean design with a bit of a unique feel is all you need for a profitable store So we’re gonna keep this tutorial nice and easy We’re gonna, first of all, add a big splash image to our homepage and then we’re gonna set it so that we’ve got a title and a subtitle overlaid on top of it And next, we’re gonna set it up so that we’ve got different products that we’re selling in our store previewed on our homepage It’s a really nice, easy way to keep our store looking professional Okay, come to the left menu and click on the Pages button Then come and click on the Add New button All right So what we need to do is create some headline text for our page and some subtext that will go under the headline The headline text should be typed into the Title box and the subtext should be placed in the big text box I prepared some text in advance Just a quick note While I’m writing worldwide free shipping, that is a more advanced tactic I recommend beginners start out by just dropshipping to the USA Once you’ve added some text, come to Page Attributes From the dropdown menu, select Homepage Next, we need to add a splash image This image should be at least 1080 pixels long and 500 pixels high A good place to find a splash image for your site is Google Images Be sure to search for images that are free to use and edit, even for commercial purposes If you don’t use images that have a license that lets you do this, then you will risk being sued For more information, watch my video, “How to Not Get Sued When Dropshipping,” which I will include a link to in the video description below Being sued may sound scary but don’t worry It’s very easy to avoid this happening to you as long as you follow my advice Once the image has finished loading, click the Set featured image button

Awesome We have now added our homepage splash image Now we just need to click the Publish button Now we’ve got to go and set this page as our homepage so on the left menu, come and hover your mouse over the Settings button and then click the Reading button Next, to your homepage displays, click the static page checkbox And then from the dropdown menu, select the page we just created After that, click Save Changes Almost done Now just come and click on the Appearance button in the left menu Then click on this Customize button Now click on Home Page Here, we can choose which product categories we will preview on our store homepage If you have all of these showing, the page will get cluttered Remove two different previews and remove the product categories and blog preview from the homepage And that’s it We have successfully customized and updated our homepage design As you can see, it’s got different product previews that we chose to keep and we no longer have the categories or blog posts listed on the homepage To finish, just click the Publish button Fantastic I’m now gonna open up the homepage on my full screen so that you can take a better look at the design All right So here is the homepage As you can see, our splash image is at the top and it has the title text that we wrote and the subtest that we created is on here too And, of course, scrolling down, we can see the product previews we chose are here as well We’ve created a nice, simple, clean homepage design It’s time to move on to the next step Step 16, update the product pages For this step, I’m gonna be editing two different products I’m gonna be editing the self-stir mug, which comes in multiple color variations The second product that I’m going to be updating is this camera lens travel mug and it comes in a single variation and that is black I’ll also show you how to set a product on sale using the self-stir mug and I’m gonna show you how to set a product to not be on sale using the camera lens travel mug Now, this part is easily going to be the most time-consuming part of setting up your store Creating unique product descriptions can certainly be quite time-consuming but it’s well worth it So let’s go ahead and edit our product pages All right So come to the left menu and click on the Products button So the first product that we’re going to edit is the self-stir mug It’s a product with multiple variations so I’ll show you how to delete the ones you don’t want and edit the ones you want to keep First things first though Delete the product title and enter your own Next, delete the description and enter your own I’m gonna be pasting in a description I have prepared in advance Now scroll down the page until you come to the short description box In this box, write some bullet points that promote and sell the item I’m gonna be pasting in the ones that I’ve prepared in advance Okay So next we need to edit the product data Don’t worry about changing any of this in the Inventory tab and you don’t need to change anything in the Shipping tab either And you can optionally choose to link products for upsells and cross-sells Something we do want to edit though is the Attributes For the Color Attribute, I’m deleting the colors I plan to remove, the sky blue mug and the yellow mug And then after I’ve done this, I’m going to be going through and hiding some attributes that I think look clunky and unprofessional to include I’m gonna be hiding these because they’ll show up on our product page This data has been pulled directly from AliExpress and a lot of suppliers on there have awkward English, which leads to these clunky attributes And in addition to hiding them, you can permanently remove them or you can even edit them But all of these are optional You may disagree with me and want to include some attributes but hide others and that is absolutely okay These attributes won’t be in the main section of your product description They’ll be in their own separate tab Once I’ve finished editing this self-stir mug, I’ll show you where they appear And I’ll be honest, setting up product listings is one of my least favorite things to do but you need to make sure that you don’t take shortcuts It’s important that you edit them to make them look professional and engaging and it’s at this point that a lot of people get bored and they give up Don’t be one of those people Dropshipping requires more than just throwing money at ads We need to do our best to convince the customer that not only should they buy this item, but that they should trust us as well And having professional-looking product pages helps to go a long way in getting them to trust us Fantastic We’ve updated our attributes Now we’re gonna click on the Variations tab This sky blue mug is a variation that I don’t want so I’m going to remove it Cool

It’s been successfully removed I’ll now show you how you can update the prices of the variations that you do want to keep I’m gonna be putting this mug on sale from $19.99 down to $14.99 All right So as this is quite a long process to watch, I’m now gonna pause this video and remove the variations of the mug that I don’t want and update the prices on the variants that I want to keep Done Okay, after you’ve edited the variants, click the Advanced tab Untick Enable reviews and then that is it We don’t need to update the Supplier info or the Review tab Then come and click the category that the item fits into And then just click Publish Perfect, we’ve updated a product with variations I’m gonna preview this page now so you can see what it looks like So on the left is the image gallery and on the right are the bullet points from our short product description Down here, you can see the main description that I added And if we click on Additional Information, you’ll see the product attributes that we chose to have are visible All right So let’s switch back and update the camera lens travel mug On the left menu, come back to the Products button and click it Now, I’m just going to find the camera lens travel mug and open it This item will be different to edit as it has no variations Again, the first thing we want to do is delete the title and put our own one in Next, just like before, we want to delete the description and write our own And then come to the Product short description box and write some bullet points promoting the item Coming back to Product data, just like before, we don’t need to make any changes to the Inventory or Shipping tabs but you can optionally add upsells or cross-sells if you’d liked to in the Linked Products tab Once again though, we need to go through the attributes and hide or remove any that we don’t want While I’m doing this, I want to take the opportunity to talk a little bit about my product description I wrote that product description as an example of one that is optimized for search engine optimization traffic, that is why the description is very long However, if you’re planning on running paid ads to it, then don’t feel you need a long product description A short two to three paragraph description along with stuff like a shipping disclaimer and trust lines like Secure Checkout with Mastercard and Visa should be more than enough That product description was just filler Don’t copy it I just wrote some text to help people visualize what theirs could look like Once you’ve updated the product attributes, click Save attributes Now, if we come to the Variations tab, you’ll see that it looks very different because there are not multiple variations of it but we still need to come to this page to update the item price I’m not going to put this mug on sale so that you can see what an item looks like when it’s not on sale All right And just like before, we’re gonna come to the Advanced tab and turn reviews off And we don’t need to make changes to the Supplier info or Review tabs Tick the correct category for the item I accidentally ticked coffee mug instead of travel mug, whoops And then click the Update button And that’s it We’ve updated the page I’m gonna preview the product listing now so you can see what it looks like So we’ve got the image gallery and the bullet points at the top and there is no sale price this time And scrolling down, you can see that the description I added in is here along with related products at the bottom And if we click the Additional Information tab, the attributes we chose are visible Looking at these closer now, I think that I would edit some of these since the values in them are wrong but luckily, as I’ve shown in this section of the tutorial, updating them is easy So go on, edit your product pages and we can then move on to the next step There are a couple of things that I want to note Firstly, I accidentally said that my items would arrive within one to two weeks If you are doing AliExpress dropshipping with ePacket as your shipping option, I would recommend telling the customer that it’s gonna take two to four weeks to arrive And again, I really don’t recommend that beginners start out by dropshipping worldwide If you are new to this, then I recommend just focusing on USA and only selling items that have ePacket as a shipping option I also forgot to show you how to edit images in the image gallery It’s pretty simple though You can delete images and reorder them in this gallery section here All right We are almost done Congratulations on making it this far Just one final step to go Step 17, create a store menu All right Now we just need to create our store menu and on it, it’s gonna have a link to our homepage, our About Us page, our Contact Us page, and a link to all of our products And we will also be creating a submenu that contains links to our different product categories All right So let’s switch over to my computer and complete this final step

On our left menu, hover your mouse on Appearance and then click the Menus button We’re only gonna be creating one menu, the main menu Come and type in a name for it The name Primary Menu works well After that, just click Create Menu Awesome When the page loads, click Primary Menu as the display location After that, click the Save Menu button All right Now come and tick the pages that I am The first page on the list should be your homepage Don’t worry, we will be changing the name of it for our menu Once you’ve selected all of these, click to Add to Menu Perfect Let’s change the funny name of our homepage to to say Home I’m typing in caps because as part of the theme design, all menu items get capitalized anyway I also like changing Shop to the name Products since I think it’s more descriptive People will know that if they click it, they’ll see a list of our products After that, it’s time to add in categories Unfortunately, we have to add to add these URLs in manually so in another window, open up your Product Categories page Now you’ve got two options You can either right-click the View button under the category you want to add and click the Copy Link Address button to get the URL or you can open up the page and you can copy the URL in the browser screen Some people might find that easier to do Once you’ve got the URL, come back to your Menu page, under Custom Links, paste in the URL And then in Link Text, type in the name of your category Then just click Add to Menu Perfect Now come and click the category and drag it so that it is both under the Products tab and indented like I’ve done here This turns it into a submenu item for that Products button Okay So what I’m gonna do is I’m gonna pause this video and then add in my two other categories Awesome So you can see, I’ve added in my two other categories to create the submenu So what I’m gonna do is show you a preview of the site so that you can see how the submenu looks All right So if we come to the Home button, you can see it takes us to the homepage And if we hover over the Products button, you can see that our submenu pops up And by clicking on the different items in the submenu, we can go to their respective categories And if we click About Us, you can see that that works And finally, if we click Contact Us, it takes to that page as well Well, everyone, that’s it We’ve completed the final step Congratulations, you’ve completed your very own AliExpress dropshipping store using WooCommerce and AliDropship And that is it We are done Congratulations on creating your new store Now there are still several things that you can add to it to make it even better For example, I recommend that you add links to your different social media accounts in the footer and I also recommend adding a refund policy page And I would highly recommend going in and updating the metadata of your store, in particular, the title But for now, if you follow this tutorial, you can set up a nice, basic, semi-automated dropshipping store that works Now, if you would like to get even more free dropshipping training, then be sure to subscribe to Wholesale Ted and click that little notification bell next to it so that you can be notified anytime that we post a new video And we’ve actually got another freebie for you Here at Wholesale Ted, we have a free eBook, “How to Make $10,000 a Month Online with Dropshipping.” You can find a link on how to download this eBook in the video description below

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ZODIAC STYLE SWAP: Dressing like EACH OTHER&#39;S Signs!

(laughing) – What? (beep) – What’s your numerology number? – Who? (beep) – What? It’s so Aquarius, what are you talking about? (beep) (seventies music) – Hey guys, welcome back to Clevver Style We are sitting here on a couch which is kind of a change up for us We wanted to be super comfy because today is all about exploring (ethereal music) our personal souls – Wow – Our s-, is that the video I signed up for? – [Loryn] I didn’t sign up for that – So what Sinead is talking about is today we’re gonna be doing an astrology exploration where we’re gonna dress like our signs but with a twist – Right, so we’re not gonna dress like our signs, we’re gonna dress like each other’s signs – [Erin] Yeah – And make it a competition We have been encouraged to use props in our outfits which is very interesting You guys might need to go to Trader Joe’s and get some wine – You’re a Pisces, what’s your sign? – I’m an Aquarius – I’m a Aries – We are doing three looks total and each one of us will be judging our sign You will judge the Aries street style, I will judge the Aquarius formal wear, and you’ll be judging the Pisces lounge wear – And to be clear, I will be dressing as your signs – Right – And you’re gonna be dressing as my sign And then I get to judge what you guys picked out – Right – For myself – We’re gonna judge the outfits on an emoji – I’m gonna do fire – I’m gonna do the emoji with straight tears (baby crying) – I’m gonna do – Black heart? – Yeah let’s do the black heart because that’s rebellious and apparently everyone in this entire office has thought it’s so weird and nobody’s ever said anything – At the end whoever has the most points, apparently they’re telling me right now, oh yes, yes, that we will be winning a prize at the end but none of us know what the prize is But the only hint that I have is that it’s really, really good – That it’s actually a prize this time – [Erin] Science stuff – I’m prize motivated – I’m free-stuff motivated for sure – I am, too – Well I don’t know much about my sign at all At all – We know more about your sign than you do – Should we start with Loryn’s birth chart? – No, why? – What’s your numerology number? – Who? – You don’t know what time you were born? – Do you? – Yes, 4:16 p.m – 8:35 in the morning (crickets chirping) – To be fair the reason I think I don’t think I know much about my sign is because Pisces are the least interesting sign – The Pisces are really sensitive Sorry, babe, Neals Is a Pisces – How are we friends? – What do you mean? Neals is a Pisces – I know and you’re so blunt – I love him, I love you, but you guys both drive me nuts (laughing) – I usually don’t like Pisces for a fact (crickets chirping) – Crickets! – So as an Aries you are outgoing – Ding – Passionate – Ding – Independent – Ding – Competitive – You are competitive in these videos – You’re competitive (bell chiming) Keep telling me more about myself – Okay, okay, okay – Alright, sorry Courageous – I think so, moderate – Have you seen your outfit? (record scratching) Hey-o! – Keep going, next one – Energetic – Yep, sometimes – Life of the party, no (buzzer sounding) (laughing) – Well that was real quick – I think Might have focused or manic energy – Yeah, I don’t need Adderall as much as some people do – Rude – I don’t know what that says about my style but – Speaking of your style, Erin, these are the style characteristics of Aries Love attracting attention with your outfit – I wear weird stuff but not because I think other people Like I don’t dress for other people (buzzer sounding) – Do you love the color red and black and white? Literally what you’re wearing – She’s not a natural red head, you guys That was a choice – And you’re wearing black and white right now – Oh lots of hats and hat accessories and a giant head – Oh, giant head’s in there? – No – Yeah – Yeah I love hats – Such an Aries thing – You do wear a lot of questionable hats – [Loryn] Doesn’t your husband hate your hats? – [Erin] Yeah my husband hates my hats – Sporty and masculine looks, that couldn’t be further from the truth – Yeah you’re wearing, but she’s wearing biker shorts right now – This is not normal And I also haven’t shaved my legs in like three days so that’s pretty masculine – Is that on here? – Think French girl meets grunge – Not me (buzzer sounding) – This is very confusing – I don’t think this is you at all – No – So next up is Sinead’s characteristics as an Aquarius Rational and analytical – Dude you analyze (beep) to death – Yes and no (giggling) Because rational, I think rational analytical are two different things I’m gonna analyze exactly what this is right now (sped up talking) I guess for the most part I’m pretty rational, definitely analytical – [Loryn] Yeah – That’s why we’re still talking about this – Okay, independent – Yeah – Easy going – No (buzzer sounding) – The OCD is not an easy going trait – Definitely not easy going – Okay, doesn’t get stressed very easily – No (buzzer sounding) – No Curious – Yes I ask so many questions all the time – Yep – [Loryn] Okay takes time to make decisions – What do you mean, yep? Why are you saying it like it annoys you? – Well we, it’s all known that you talk a lot – Yeah that’s true – Okay takes time to make decisions – What do you think? – Takes time to do anything – Time is a, yeah – Takes time to drive, to do our makeup – Doesn’t display emotions very often but is very internally emotional – I don’t think I display emotions on the outside a lot – You do if they’re like, anger (growling) – Okay let’s go through your style – Okay – Artistic meets chic – [Erin] Yes – What are you talking about? – You wear stuff that like, is so cool – I wish I was chic though I don’t ever feel chic – I don’t really know what that means – Chic would be like, “I work at a magazine”, which we do, oh – I feel like on Instagram she’s chic – Maybe we don’t know what chic means – Experimental, rebellious, unique and unusual looks I totally see that – 100%

I think unique 100% Everything you wear I’ve never seen anybody wear or how you wear it or the things you choose Like that’s why whenever we do these fashion episodes we never pick out the same things – That makes me feel so nice ’cause I literally, I never think I have a style, ever – Oh my gosh, your style is more stylish than anybody here – Yeah, 100% – Yeah, for sure So your colors are violet, turquoise, electric blues, anything vibrant and red – For jewelry I have tones of like, turquoise, teals, purples, things like that But clothing, you guys know, I wear black and I wear white and mostly black – Oh, accessories are a statement That is so you – That is so you – The category for you that we’re shopping for is formal wear – I would say channel Harry Styles He gender bends a lot with his style and I think that is part of being an Aquarian Well ’cause it could be dresses, or it could be like pantsuits, or it could be two pieces – So that is chic, then ‘Cause you said you’re not chic but that feels very chic – We don’t know what chic means (crosstalk) Moving on to Pisces, which is what Loryn is It’s a water sign – She knows nothing about her sign – I know of my sign and I don’t feel like it at all – Creative – Yes – Great intuition – Yes – Sensitive, you cry all the friggin’ time – Yes – You didn’t know that until like five minutes ago You just said that like, you literally have it written down in a book Love to daydream – Daydreaming sounds boring (buzzer sounding) – Can find beauty in every situation – 100% no (buzzer sounding) – When she doesn’t like something she is done – And she will tell you – Love nature – No (buzzer sounding) – Wait I feel like you’re always snowboarding – So here’s the thing I love snow but it covers up all of nature So when I was a kid and when I was growing up, I’d go play in the woods and there’d be bugs everywhere and I’d get gross But then when it snows, I will go outside, I will climb the trees because everything’s clean and the bugs are gone – That is so funny – That is so deep That’s so deep So for style characteristics – Yeah – Romantic and flowy, yes – [Sinead] Absolutely – But she only likes big flower prints (laughing) – [Sinead] Or hearts, she loves hearts – And not small flower prints Very feminine, I would say yes – [Sinead] Yes – You think I’m feminine? – Very, very – I absolutely think you’re feminine – Oh – [Sinead] You have a very delicate style and delicate fashion, you know what I’m saying? – Okay – Delicate styles – Oh wow Did you read that before you said that? – No I didn’t I really didn’t – That’s weird – [Erin] Wow, that’s weird Whimsical, ethereal – No (buzzer sounding) – I wouldn’t say ethereal Soft fabrics? Who doesn’t love soft fabric – I mean, yeah, that doesn’t – A Pisces thing? – Like polar fleece, ’cause you know I’m all about that – So true, if you guys are doing lounge wear – But what if I did like a satin, like silky thing – No, no – She doesn’t like that – Nothing lingerie, nothing sexy Am I gonna vote for it? No – She’s not wearing it, we’re wearing it – Oh that’s true – I won’t vote for either of you if you’re wearing lingerie – Just the idea of it upsets you? – Yeah. (giggling) – Alright, your colors: sea green, violet, indigo and gem tones I could definitely say gem tones – [Erin] Gem tones – ‘Cause you don’t like pastels – For sure Accessories – Sea green, as opposed to what, regular green? – Have you watched “The Little Mermaid”? – [Sinead] Yeah – [Erin] It’s that green – [Sinead And Loryn] Oh – That’s a nice green – Some other famous Pisces: Justin Bieber, Millie Bobby Brown, Camilla Cabello (gasps) – I love Millie Bobby Brown’s style and what I was learning about modern interpretations of Pisces is that they’re young at heart and they tend to gravitate towards younger things So that is so funny that I love Millie Brown, Bobby Brown so much and she’s a Pisces – I don’t really know what to do with this information I’m gonna have to dress like Loryn in order for her to vote for me – You guys can do whatever you want, just make sure you follow all the rules – Alright to the mall we go – Alright, so we have our plan, we have our strategy, we’re shopping for three categories, two looks each – Yeah – I’m doing Aries street we-, Aries formal wear – No – No (beep) – I’m doing Aries formal wear – No – No You just said it the second time the exact same way – Oh my God (beep) – Aquarius formal wear – Yes – Aries street wear – And I’m doing Aquarius formal wear and Pisces lounge – Yes – But not lingerie, not lingerie – Not lingerie And I’m doing Aries street wear and Pisces lounge wear, but not lingerie, not lingerie – Not lingerie – Okay, and I’m just gonna buy some lingerie (laughing) What would an Aries do to get us off on our way? – Huddle – Okay, that’s not a huddle – Wait, what do you mean? – What are we doing? Oh my God this is so embarrassing, what are we doing? – One, two, three, let’s shop – Let’s shop? – Astrology? – Shop ’til you drop – One, two, three astrology? One, two, three – [In Tandem] Astrology – Wow that was so sad Why did we all whisper? – None of us wanted to actually draw attention – ‘Cause Aries are uncomfortable in public – Astrology! (laughing) – I’m walking behind Sinead and Erin and they look so confident There they are, not a care in the world Aries and Aquarius, just killing life Meanwhile, Pisces back here stressing Oh we’re passing ’em I feel like I’m in Mario Kart I just passed Erin – So I’m headed to Windsor I’m looking for my Aquarius former, formal wear I’m looking for a pant suit mostly because Sinead said pant suit And then also I’ve never owned a pant suit and I want to so, it’s a win-win for both of us I think so, let’s go – Alright you guys, my (fumbles over word), my strategy is pretty straight forward I’m just going to try to embody Loryn and Erin as much as possible and then put the rules as my next priority, even though I love following rules I think I know that they don’t pay as much attention to rules so

I think I need to try to channel them but I mean, I’ll probably still end up following the rules because I really can’t help myself I need help – Do you like this? Is this street-y? – No, I would never wear this is in the street, only in the sheets (laughing) – Ouch, poor Matt Turquoise? I keep getting distracted by things that I want Is that a Pisces trait? – Does this say like, “I’m ready for the job, I’m qualified I have lots of special skills, one of which includes Microsoft Word”? – Alright I’m wearing my sunglasses because I have to wear this 24-hour makeup for a separate video and it looks awful I am in H&M right now and I feel like for the Aries street style I really like the idea of doing maybe like a more feminine skirt and then like putting the grunge on the top Surprisingly enough I’m not mad at this skirt Really I’m just thinking about what I want to add to my wardrobe – I’m so conflicted emotionally What else is new? – Ooh, you know what I was thinking would be a good accessory? Come here Yeah? It’s not her birthday but it’s love jewelry that has a meaning – I think if I wear an actual face mask with the outfit maybe I’ll get points for creativity Holographic, maybe? It’s a shame that they don’t like, have like eye patches that say, “give me wine” on them ’cause I would definitely win if I could find that (upbeat pop music) – Well it said bold unique accessories, that’s so Sinead I’m gonna hold on to these two things I think Sinead would kill me if I went this formal I might go back to my original plan of like, electric blue It feels like more of the Aries colors, vibrant There’s violet, there’s electric blue and she likes black Ugh, Sinead you are so hard Do you think? – I like this one obetter – You do? Okay – I just think it’s more – More interesting You’re such an Aries – There are too many things to think about And it’s already hard enough when you shop for yourself thinking about the things you like for yourself But to try to then find things that other people would like for yourself is just so many layers of inception that is really is mind blowing I think I might need a hard adult beverage after this – Okay so my idea for Loryn ’cause hers is lounge wear and since she has very specific taste when it comes to pajamas She doesn’t want anything lingerie, nothing sexy, so I’m like what if I get like a cool tie-dye set as well but like different, a little bit different from the one she had Maybe like, a little off the shoulder On Amazon you can get iron-on patches And I’m gonna get an iron-on, wait, hold on It’s my competition I was about to share my like, my awesome plan for my Pisces outfit but Anyways, so then on Amazon they have these iron-on patches so I’m gonna get a white, white claw iron-on patch and make it look like a white claw set Right? That’s amazing I’m super stoked about it, too Thank you – Okay this is sort of along the line of what I was looking for but the wrong color Is this a skirt? Oh my gosh, what are kids wearing these days? – Okay so being the intuitive Pisces that I am, on our way out of that last store Erin like was touching a couple jackets and looking at them and I know she was not looking at them for what she needed to get for me or Sinead, so I think she just looked at it ’cause she liked it So now I’m gonna back here and I’m gonna buy it I’m thinking denim, these denim sparkles are very unique but comfy So I found this really cute little camo dress that would look really good with her hair but it’s not her, it’s me who has to wear it So what I’m gonna do is I’m gonna buy it smaller than I might ordinarily, squeeze into it, hope I don’t stretch it out, and then if she does actually like it I can give it to her ’cause I don’t like camo, but she, it looks so good on her, no wonder it’s like part of her style, hopefully I hope, I hope Erin That’s cute, too – I got some stuff from Forever, like a black choker, a hair bow and this belt bag Just because I know for a fact Erin likes stuff like this and I felt like that was a good way to appeal to her as well but also keep it like grungy And then for the top I’ll go a little bit more feminine and then like a bright red lip and then I’m done I just don’t have any props for that one, I’m gonna need some help Oh, headphones, I do! Oh my God, I should just accept my prize now Do you wanna tell me what it is? – [Erin] Okay – Oh darn I thought this said “driven” – Our shopping is complete, well at least at the mall – Mostly – Mostly, we all have to do some online shopping – Yeah – I feel like – You guys got a lot of stuff – A lot of stuff – I don’t have to do as much online shopping as I thought I was gonna Just maybe prop, accessory, shoes – Shoes – And that’s it I got all my main pieces for you guys – Oh I definitely don’t have all my main pieces but I do think I nailed a lot of the accessories – I’m excited, I have a really great accessory for you – I wonder – I have a surprise accessory for you – I hope it has like a 14% alcohol content

– See you back at the studio – We’re not actually walking away because – Oh, you’re supposed to cut already – Our producer’s holding the camera so we have to all leave together – We can go We can go – Don’t let her fool you (beep) Alright, we have completed our shopping endeavors – It’s time to finally assemble our outfits that we have put together I don’t know what accent that is – So we’ve compiled outfits, props, lots of surprises – I know there’s like food being delivered or something I can literally smell the box from here – What? – Yeah, I’m cool – Guys! – I don’t know what it is – You know my donut sensors go off – Stop looking at things – Okay, it’s fine – Loryn we need to get you – I didn’t think that anybody was gonna get me donuts – Do we have any Fireball? Oh, okay – Alright and we are of course going to judge these ’cause it is a challenge that we have named “I styled the sign” ♪ I styled the sign ♪ ♪ And it opened up my eyes ♪ (crosstalk singing) – We have to vote based on the emojis that we chose – Are Pisces forgetful because I don’t remember what emoji I chose – I think it was a fish – Or was it a cry face? – Maybe it can be a crying fish – I don’t think you can just invent emojis – Our editor can – And there will be a winner – And there’s a prize (burping) That’s not it It smells so bad – Like salmon, like fish? – You just had salmon – Ew – It smells like Pisces – So we will be rewarding each other with emojis in three separate categories: Would I wear it? Does it match my sign’s style? – And can I eat it? – The last one is actually creativity – Okay I’m gonna judge the first round so you guys go get on your lounge wear – Okay – And I will wait here as the Pisces that I am Actually we’re really impatient, so hurry up (pop music) Ladies in lounge wear (panting) Oh my God, oh my God, hi! – Do we look just like you? – We actually look like very similar – Like we’re going to the same pajama party – Oh my God – [Erin] Did you see? – Oh ’cause Pisces are extremely creative so I made sure to add a DIY element – I will upsell her because I got one of these bags for you as well – [Loryn] It’s really cute – So you get to take one of these home with you – Oh! – Well you also get this Harry Potter water bottle – Aww – And, and this lightning bolt necklace – Oh my God! – To match the Harry Potter water bottle – Guys, I feel popular I feel like, I feel so loved – So I think my biggest thing was I wanted to at least incorporate some of the sign’s, like the whimsical feeling The blue actually really worked really well into that And then, like, I was like oh gold, and then like maybe like, some sort of like hair clippy thing – Yeah – Like things like that – The details – Like the details – I feel like I’ve seen you wear something like just like this like on stories with your cauliflower escapades I feel like the bow – I had one on earlier – I know I like saw it as I was checking out, I was like this is something Loryn would like I just thought this like felt very like, underwater, aquatic. (record scratching) Is that a word? – Aquatic – Aquatic? – It’s like you combined the sound a duck makes (duck quacking) – This is so hard You guys did a really good job I am honestly impressed and surprised Okay, so, I’m going to rate you for the first category, would I wear it? Five emojis! – Yay! – I knew she was psyching I knew it – I was so nervous – [Erin] I knew it – Do I think it fits my sign style? This is tough ’cause I know that you were totally basing this off of me I feel like it doesn’t match as a whole So I’m gonna give you – I’m sorry, what? – A four – That’s fine – Creativity is where you get all the points The fact that you DIY’ed that yourself So I’m gonna give you five for creativity You know what? I’m gonna give you two extra points for all the Harry Potter stuff – Yeah – Which has nothing to do with Pisces but it is very personalized to me and my love language Okay Erin, would I wear it? Clearly, this is a five I really like the matchiness of it all, it looks super cozy, very comfortable Next one, do I think it matches my sign’s style? I really do I think this is a five for match my sign’s style I’m gonna give you a four for creativity – If there was a fireball in this bag things would be different – I thought there was going to be ’cause you said that – I know I thought about it once it was too late – It’s still creative, I like everything you put together You just didn’t DIY anything – [Erin] I know I didn’t DIY, I didn’t – I’m gonna give you two bonus points ’cause I get to take a bag home and it’s really cute – Yay! – So who wants to go next? Oh God you’re soft Holy moly – Okay I hate to do this to you guys but I will judge the next one which means you guys both have to take off like your really comfy cozy lounge wear and stuff and put on formal wear

It’s okay – Well we knew that It’s fine, I mean (pop music) – I’m gonna get that camera pregnant It’s on birth control but it don’t matter – Three, two, uno! Oh my God you guys look great – Oh Sinead, oh wow – First of all, I feel extremely under dressed So everyone’s aware – Oh, you didn’t get the memo? – And donuts – Yes – Teacher’s pet over here – Cosmic donuts – I didn’t really wanna go too far Aquarius ’cause I know your style is so really not that You wear a lot of black, you wear a lot of white – You look great Like this is a really nice sleek chic outfit I like it, you look super cute – Thanks, thanks – Alright so I know that Sinead is a stickler for the rules so I really went hard on the Aquarius side The electric blues, the like deeper bright colors, I definitely leaned into that And then I know she loves accessories and she usually picks like out of the box accessories I know that you don’t like to show your toes – Nobody will ever see my toes – Have we never seen her toes? – [Sinead] Not like actual heel heels – Oh my God, what do your toes look like? – Ever – Okay, I don’t know that Aquariuses are all about their accessories but I know that you are so I threw some hair clips in, I got the sunglasses, I got the bag Of course you’re obsessed with donuts so got ’em to go – Those look so good – And this isn’t for me, this is for you You get to have these, I mean I’ll take one – Get a room (laughing) – I’m so impressed, honestly Like you really did a lot So, would I wear it? I mean I would wear something very similar to the style, so like personally I’d give you like a three What?! It’s a c-bell accessory – Yeah and this is the worst part – [Loryn] I knew you would never – Of the whole outfit (laughing) – [Loryn] What? – What?! it’s so Aquarius what are you talking about? – Everything I think I know, I don’t know – It is safety! – You’re taking away from something that’s so sleek and like nice and like (phew) – If I took it off I would be nude! – I’m okay with that I’m like way too much of a mom, I’m gonna cry myself – You’re fine, it’s okay – Is it my star sign’s style? No But there are elements definitely of it that I think really do work more so with my style Like the sleekness and the edginess of it for sure I think I’m gonna have to give you another three, please don’t be mad at me – Oh I’ve been mad at you, it’s fine – Okay, so – Can I have one of those? Does it have booze in it? – She’s taking a long time to judge – Yeah She talks a lot, remember? – Okay I do, I do talk a lot – Dang, these are dense – Creativity, creativity Formal wear is hard so I think that in order to make it look as formal as you did, is like still a really good, a really great feat So all together for creative I’ll give you a (laughing) – What? – [Loryn] What a heart – I’ll give you a five for creativity – What? – I’ll give you two bonus points for incorporating my two favorite colors, black and white Alright Kesha, let’s go – Hi, Kesha? – So, would I wear this? There are a lot of things I would wear about this I would wear every single one of these accessories, those shoes are amazing actually – Thanks – And the style, this is my style Yeah the colors are a little – Aquarius? – Predictable, in a way Like the rhinestones match the rhinestones match the rhinestones, you know what I’m saying? Would I wear this? I’d give you like a four, honestly Is this my sign’s style? I mean I think it’s pretty obvious that you are a five because Aquarians are obnoxiously obnoxious when it comes to colors – Yay – Yeah, creativity, I mean your creativity is like off the charts – You’re holding my creativity – It’s delicious as well – Yeah, creativity is like off the charts So for creativity I would give you five Obviously I have to give you a couple bonus points for the donuts – Well, as an Aries, I’ve been waiting for my moment to shine, slash judge, the heck out of you two And I have already taken notes based on how you’ve judged me – What? I gave you a donut – It’s time for me to judge my street wear – [Sinead] Oh no I’m in trouble, she’s mad – Uh-oh, you did it – Don’t poke the bear (pop music) I don’t wanna look – C’mon, just do it – What the (beep) is going on? What is happening? – We look so totally different – I don’t know what the Taco Bell is – Bitch loves tacos – It’s dope – But doesn’t it look like a purse? (laughing) – I feel like both of these outfits are from like, I don’t know, Lizzie McGuire or something – Are you kidding? I look like you! So I know you have a big thing for hats, so I got one – It’s a head accessory as an Aries, yep – Yep and red is the fire sign color so that’s why I picked red – I saw you touch this jacket when we were shopping I don’t know if that was you touching it ’cause you thought it was God awful

or if you liked it, and I also know that you always wear your jackets like this Green looks really good on you and you like camo but this felt like a cute camo But also, fun fact, this dress is your size Doesn’t fit me, it’s not zipped up You get to take it home And then I got you tacos ’cause I know you like ’em – Very interesting, okay Let me just clarify In the Billie Eilish video there is a point in that video where I say I don’t like everything that I touch but I just can’t stop touching everything – Crap! Hold – True, true – Hold, turn that way (devious plucky music) – Okay – Well what about now? (laughing) I couldn’t decide – I actually like this better – Oh, okay – Believe it or not I feel like it looks more intentional – Yeah this is all very interesting – I don’t really eat Taco Bell very much – Oh, but you tweet about tacos – I do tweet about tacos – I do though, so So for my look I tried to channel the Aries that was like French girl meets grunge But then I also wanted to appeal just to things that I know you kinda like So I know that you really like plaid, and like booties And like even though I’ve never seen you wear anything this edgy I do feel like these are something you would actually enjoy – I like those – The plaid like school girl skirt, I feel like that’s so you Another thing I know about you is that you love like, studs – Yep – And like details like embellishments in a way, you know, like – Oh like rhinestones on a denim coat? Apparently not – So this is probably like the least you but I wanted to just really try to tie in like, that French vibe so I got a button up shirt and a sweater like they do in the movies, and then of course the matching burgundy headphones – Anyway, okay Gosh I have to start rating now? This is really hard because I’m gonna be real I hate both of these outfits I would like literally never wear them However – Then why did I get this in your size? – I would wear it if it was here and I had nothing else to wear, I just threw up on myself – Y’all I wanna do my round again – Out of five I would give this, would I wear it? I would give it like a three I do think it fits my sign’s style pretty well with the red hat and like kinda just being bold and like making a bold choice So I’m gonna give it a four for that For creativity I really – Tacos, you’re holding my creativity again – That’s true So I’m gonna give it a three ’cause there was effort there and I appreciate that I have to give bonus points to Loryn because food is the gateway to my soul and spirit I’m gonna give three because there’s enough tacos here to feed a stadium of people – Dude, you would never wear that outfit in your whole entire life – That’s not what bonus points are for, Sinead – The bonus points are here in this box and I’m gonna give it a three – Alright maybe I’m taking this bag and these headphones home with me – So Sinead, would I wear this? On a scale of one to five I’m gonna give it a two I like plaid but it’s kind of like a bland color But I do feel like it does fit the French vibe of like the Aries like look so I think I’ll give you like a three for hitting it on the head for the Aries look You did like accessorize and really like think of the red – [Loryn] That was pretty good – I played soccer for 10 years – So did I and I can’t do that (laughing) – It’s my round! – Sorry, keep going – Okay – I’m not even mad just keep going – Creativity, I’m gonna give it – C’mon now, let’s call a spade a spade I’m creative AF right now – You do have a lot of accessories so I think I’m gonna give you a four just because there are multiple accessories So those are my final scores I don’t know what our final final numbers are, though Okay, the votes are in The source of the tally is in my bosom (intense violin music) This is shocking news Breaking news everyone, there is a tie – What? – So who’s the runner up? – The runner up is Sinead – Oh whatever, your guys’ all sucked, so (laughing) (beep) Listen, your styles are so similar that this makes sense I was targeted (laughing) – But you didn’t lose by much You had a score of 25 emojis Loryn and I were tied with a score of 29 emojis, which is weird because I felt like I did not do a good job in this video – So you guys can open your eyes So you have won presents and prizes – What is it? – Spill it already – So we have a tarot deck, a Mystic Monday’s deck, a deck for the modern mystic – You can have it! – I can’t take that home – It’s for you – It’s too scary – No, no – I think it’s too scary – Some of these cards in the deck, oh my God! You want me to take this? I guess if the winner can’t fulfill their duties as being grateful for the presents then I will take it – Is that wine? – No – Is it close? – No – Can I drink it? – It’s for water, tea and for water – What? – I want that one – Oh I love that – No I want that one (crosstalk) – It is a crystal infusion bottle,

this is cute – Oh that’s really cool – And you put crystals in it – Is there something in there? – It’s got gemstones in it – Ooh, thank you – Gemstones, wait! But that’s a Pisces thing And they’re purple – But I’m an Aries and I make bold – Aries are thieves – Accessory statements – Thieves

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