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it will called calcein and this is a

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relatively impermeable molecule with 363

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Dalton the molecular weight is similar

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to typical chemotherapy and I'll just

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direct you through this these are

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microscopic images 10x looking at for

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tumor cells here GBM tumor cells and

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these mrs. control condition with no

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room for sound and just by looking why

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light microscopy and if we go across

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this is the looking at the fluorescent

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image of these same cells so you can see

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these four there's a little bit of the

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fluorescent dye tech passes into these

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cells under control conditions but when

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we apply infrasound the intensity of

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this fluorescence increases and so you

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can see that it's increasing the uptake

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of this fluorescent dye unity cells

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indicating the membrane is being more

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permanent so this is one experiment this

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is a histogram using a flow cytometers

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to be to get more quantitative measure

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of that so basically this is looking at

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10,000 events of measuring these

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increases so here is 10,000 events with

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no in person in the meeting of

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fluorescence is here so you see a shift

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towards more fluorescence in these cells

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so this is very exciting we made it

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halfway if we were seeing that we are

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seeing an impact on the membrane

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permeability with it for some so before

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moving to the second hypothesis we

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wanted to get a little bit better

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on what was coming out of this G machine

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because of this to this stage we were

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just pressing a button and there was a

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little bit light saying that something

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was coming out there but we couldn't

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hear it and we really just for trusting

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that this was making the Chico like

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stuff so we we got an engineer to come

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in and basically to a spectral analysis

45
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and and so we were pleased that we did

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indeed see infrasound that looking

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coming out the machine was was in this

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usually again the 20 Hertz cut off so we

49
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saw it here between seven and 15 with

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some peaks in between so we we forged

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ahead oh wait before we first add then

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there's 172 that to the chico machine

53
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and let me go back and you can see some

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of these peaks and there was this

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difference is it was basically get

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dynamic frequency and also the richard

57
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lee the scientific director at the

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Institute that produces the machine has

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spent some time talking to him they feel

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very strongly the dynamic component of

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this is very important to to protect

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against biological adaptation so we

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thought well this is really fascinating

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and then you know what before we move

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forward we wanted to be sure that we

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were going to be applying there the best

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kind of genome a half site that's kind

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of a infrasound so we wouldn't test that

69
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so we made our own g gold generator our

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infrasound generator

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and I am going to just get you to focus

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just on this cluster this this is a

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box-and-whisker plot and just to orient

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you to it we'll just stick with this

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cluster right here it's going to be you

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know we have data from four different

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human cell lines that are derived from

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four different GBM tumors to look for

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the generalizability of the results but

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just if we look at just one tumor here

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u87 the results of this box are from the

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dynamic output of the Chico machine and

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here are the results from our infrasonic

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generator at 8.5 just a single frequency

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11.6 and 15 now if you're not familiar

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with box and whisker plots there they're

87
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meant to give a depiction of all the

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data and basically that the mean is

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shown I know that it's convergence of

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the hourglass shape and the top of the

91
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box will be the 70 75th percentile than

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twenty fifty percent on the bottom the

93
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top of the whiskers are ninety-eight

94
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percent I on the bottom is that two

95
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percent and any outliers any and every

96
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outlier are shown with a cross and and

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just to take away from minutes you want

98
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to look for the boxes that don't overlap

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and and we really found that most of our

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boxes did overlap there were occasions

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where where we would get it looks like

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an effect but the overall message from

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this that we found is that for this

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outcome for membrane permeability with

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the infrasonic exposure we could pretty

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much say that we get we get this

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increase in in the fluorescent uptake

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whether we use the dynamic frequency or

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the single frequency another thing to

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note about in some cases the box is not

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overlapping is that for the different

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cell types there's a look there can be

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some differences so some of the reaction

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is it's not completely generalizable so

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now we move to hypothesis too and we're

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looking we're going

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the response to a chemotherapy so take

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you through the title slowly first we

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chose cisplatin very common chemotherapy

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it's a DNA damaging agent and our

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outcome measure with a pitocin and this

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is a its program cell death it's a very

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common outcome measure in cancer

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research and it's important because it's

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an important outcome for cancer because

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you said cell basically undergoing cell

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suicide rather than necrosis which can

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lead to inflammation and be more

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problematic so a good chemotherapy will

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induce apoptosis so that catch ourselves

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were just nice you know packaged up in

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and go away so to look at a pitocin we

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we stained we had two different stains

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and these the results again of a flow

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cytometer there are four panels for four

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different conditions so we'll get you

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know we can focus up here just to orient

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you to the craft therefore this is a

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control condition with no infrasound and

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no cisplatin so you can see that the

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healthy cells this is where they live on

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this kind of a plot and this lower left

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quadrant the end of you because they

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don't stain for annexin 5 and this is a

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stain that will pick up whenever a

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particular lipid will flip its

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orientation on the on the membrane and

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it's an indicator of the early stages of

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a pitocin or programmed cell death so

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these healthy cells do not move along

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the x-axis because they're not they

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don't have this flipping of a membrane

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that indicates they've kind of the early

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stages of AP ptosis this axis measures

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propidium iodide which is a vital dye

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which will be allowed into the cell at a

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late stage of of cell death regardless

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of whether it's a puto sasur not so if

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we just look at these quadrants what

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you'd see is healthy cells here and then

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when we treat them with the key

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therapy you see many of the population

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this is 24 hours later moving over and

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being stained as early a pathetic and

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then also moving up into this late stage

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cell death so you can see that you know

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at 24 hours there were some that were in

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the beginning of their program of death

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some had already progressed in till late

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stage and here's where debris is left

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there these are you know completely dead

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so this was control and as I've showed

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that this is where cisplatin treatment

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alone and you can see this death and

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then down here this is Empress sound

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treatment alone is pretty much

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indistinguishable from the the control

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condition so there we have the

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demonstration of non toxic nature of the

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infrasound in this model and we did see

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that you can see there are some more

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dots over here indicating that on top of

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the cisplatin effect we were seeing more

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so these are results and again these are

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10,000 events these are results from one

186
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experiment comparing the four conditions

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and so to look at again four different

188
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cell lines and this represents the data

189
00:08:27,730 --> 00:08:25,730
from three independent experiments for

190
00:08:29,680 --> 00:08:27,740
each of the lines compared to the

191
00:08:32,350 --> 00:08:29,690
control condition so again with

192
00:08:34,719 --> 00:08:32,360
infrasound alone you just saw this kind

193
00:08:36,820 --> 00:08:34,729
of noise not real not a significant

194
00:08:39,780 --> 00:08:36,830
difference in terms of programmed cell

195
00:08:42,370 --> 00:08:39,790
death rate mitosis this is platinum

196
00:08:44,260 --> 00:08:42,380
cause of death and in each case we saw

197
00:08:48,370 --> 00:08:44,270
an increase in the amount of death

198
00:08:50,020 --> 00:08:48,380
caused by the combination so this is

199
00:08:51,370 --> 00:08:50,030
this was a very exciting for us this is

200
00:08:54,430 --> 00:08:51,380
what we were looking for that we could

201
00:08:57,640 --> 00:08:54,440
see an impact in this non toxic agent to

202
00:09:02,560 --> 00:08:57,650
make that chemotherapy more effective so

203
00:09:05,350 --> 00:09:02,570
one more thing we wanted to ask was how

204
00:09:07,240 --> 00:09:05,360
much of the membrane effect how much

205
00:09:11,350 --> 00:09:07,250
could that explain of our cisplatin

206
00:09:13,630 --> 00:09:11,360
effect and this many regression gave us

207
00:09:16,860 --> 00:09:13,640
as you know admittedly it's only three

208
00:09:19,900 --> 00:09:16,870
points but gave us the confidence that

209
00:09:22,870 --> 00:09:19,910
we could talk about a mechanism to

210
00:09:25,060 --> 00:09:22,880
to please the NIH funders and say that

211
00:09:28,210 --> 00:09:25,070
indeed there was a tight correlation

212
00:09:30,280 --> 00:09:28,220
between this and and in addition to

213
00:09:32,950 --> 00:09:30,290
pleasing the funders that also perhaps a

214
00:09:35,080 --> 00:09:32,960
clinical application is that because we

215
00:09:37,720 --> 00:09:35,090
saw some variation and the cells derived

216
00:09:40,750 --> 00:09:37,730
from different tumors this this

217
00:09:42,700 --> 00:09:40,760
indicates it just a simple die just just

218
00:09:44,830 --> 00:09:42,710
looking at the ability of a simple die

219
00:09:46,810 --> 00:09:44,840
to be pushed in the South Amazon might

220
00:09:48,760 --> 00:09:46,820
predict for patient whether this would

221
00:09:49,980 --> 00:09:48,770
be appropriate for them in terms of

222
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assisting in their chemotherapy