<div dir="ltr"><div>Amy, can you please make sure Bruno has the necessary customs pre-clearance info?</div><div>Thanks very much, Bruno!</div><div><br></div><div>Also, with the remaining material let's run the SDS titration. To minimize protein requirement, perhaps run the tests at lower protein concentration. I think we should run at 0.5 OD 280 nm (~25 uM) with 0.001, 0.002, 0.005, 0.01, 0.05, and 0.1 % SDS, 6 <u>samples, 3 cells</u>. Run at 50,000 rpm.</div><div>Thanks! -Borries</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Mon, Mar 22, 2021 at 2:22 PM Bruno Lomonte <<a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
<div>
<p>Sure! 11.0 mg to be shipped by courier tomorrow...</p>
<p>thanks to all</p>
<p>Bruno</p>
<p><br>
</p>
<p>+++</p>
<p><br>
</p>
<div>On 22/03/2021 01:33 p. m., Montina,
Tony wrote:<br>
</div>
<blockquote type="cite">
<div>
<p class="MsoNormal"><span>Hello
Amy,<u></u><u></u></span></p>
<p class="MsoNormal"><span>I
guess we should use as much myotoxin as possible this time
around for the NMR measurements.<u></u><u></u></span></p>
<p class="MsoNormal"><span>We
can use this sample for the NMR time booked this weekend.<u></u><u></u></span></p>
<p class="MsoNormal"><span>Once
more arrives, we should try to further concentrate the NMR
sample to the 20mg/mL.<u></u><u></u></span></p>
<p class="MsoNormal"><span>Bruno,
this works out to almost 10mg of myotoxin, plus what Amy
needs for AUC.<u></u><u></u></span></p>
<p class="MsoNormal"><span>Are
you able to send this large of a quantity?<u></u><u></u></span></p>
<p class="MsoNormal"><span>Cheers<br>
Tony<u></u><u></u></span></p>
<p class="MsoNormal"><span><u></u> <u></u></span></p>
<div>
<p class="MsoNormal"><span style="font-size:9pt">_______________________________________________<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Tony
Montina <u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Director |
Science Operations<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Director |
Magnetic Resonance Facility<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Instructor
| Department of Chemistry and Biochemistry<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Faculty of
Arts and Science<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">The
University of Lethbridge<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Lethbridge,
Alberta, Canada, T1K 3M4<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Office:
1-403-394-3927<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:9pt">Lab:
1-403-329-2230<u></u><u></u></span></p>
</div>
<p class="MsoNormal"><span><u></u> <u></u></span></p>
<div>
<div style="border-color:rgb(225,225,225) currentcolor currentcolor;border-style:solid none none;border-width:1pt medium medium;padding:3pt 0cm 0cm">
<p class="MsoNormal"><b><span lang="EN-US">From:</span></b><span lang="EN-US"> Myotox
<a href="mailto:myotox-bounces@biophysics.uleth.ca" target="_blank"><myotox-bounces@biophysics.uleth.ca></a>
<b>On Behalf Of </b>Henrickson, Amy<br>
<b>Sent:</b> March 22, 2021 12:33 PM<br>
<b>To:</b> Myotoxin-II discussion
<a href="mailto:myotox@biophysics.uleth.ca" target="_blank"><myotox@biophysics.uleth.ca></a><br>
<b>Subject:</b> Re: [Myotox] myotoxin2-SDS titration<u></u><u></u></span></p>
</div>
</div>
<p class="MsoNormal"><u></u> <u></u></p>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">Hello All, <u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">There is only about
3-4mg total. So we could possibly get ~6mg/ml by diluting
into 500uL, but this would leave every little if any for
the further AUC tests.
<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">Therefore, Bruno if
you do have some that you could ship that would be greatly
appreciated. <u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">The address that it
can be shipped to is:
<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">Attn: Amy Henrickson
<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">4401 University Drive
W<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">TIK 3M4, Lethbridge,
AB<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">Canada<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">Thanks, <u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:12pt;color:black">Amy Henrickson<u></u><u></u></span></p>
</div>
<div class="MsoNormal" style="text-align:center" align="center">
<hr width="98%" size="2" align="center">
</div>
<div id="gmail-m_-7833723596732541401gmail-m_3250609032166108197gmail-m_-4066707762680686423gmail-m_-1567372153100094991divRplyFwdMsg">
<p class="MsoNormal"><b><span style="color:black">From:</span></b><span style="color:black"> Myotox <<a href="mailto:myotox-bounces@biophysics.uleth.ca" target="_blank">myotox-bounces@biophysics.uleth.ca</a>>
on behalf of Opyr, Michael <<a href="mailto:michael.opyr@uleth.ca" target="_blank">michael.opyr@uleth.ca</a>><br>
<b>Sent:</b> Monday, March 22, 2021 9:08 AM<br>
<b>To:</b> Myotoxin-II discussion <<a href="mailto:myotox@biophysics.uleth.ca" target="_blank">myotox@biophysics.uleth.ca</a>><br>
<b>Subject:</b> Re: [Myotox] myotoxin2-SDS titration</span>
<u></u><u></u></p>
<div>
<p class="MsoNormal"> <u></u><u></u></p>
</div>
</div>
<div>
<div>
<p class="MsoNormal">Hi Everyone<br>
<br>
Looking at my notes from last time I prepared for the
Shigemi<br>
I used 450 - 500ul to fill the tube, so I would say plan
for 500ul.<br>
<br>
This means 10mg per nmr sample.<br>
--Mike<br>
<br>
*************************************************************************<br>
Michael Opyr<br>
Magnetic Resonance Facility Technician <br>
Faculty of Arts and Science<br>
The University of Lethbridge <br>
4401 University Drive<br>
Lethbridge, Alberta, Canada T1K 3M4<br>
Phone: 1-403-332-4526<br>
<br>
-----Original Message-----<br>
From: Myotox <<a href="mailto:myotox-bounces@biophysics.uleth.ca" target="_blank">myotox-bounces@biophysics.uleth.ca</a>>
On Behalf Of Borries Demeler<br>
Sent: March 21, 2021 5:50 PM<br>
To: Myotoxin-II discussion <<a href="mailto:myotox@biophysics.uleth.ca" target="_blank">myotox@biophysics.uleth.ca</a>><br>
Subject: Re: [Myotox] myotoxin2-SDS titration<br>
<br>
Caution: This email was sent from someone outside of the
University of Lethbridge. Do not click on links or open
attachments unless you know they are safe. Suspicious
emails should be forwarded to
<a href="mailto:phishing@uleth.ca" target="_blank">phishing@uleth.ca</a>.<br>
<br>
<br>
Thanks, Tony! We'll shoot for 20 mg/ml.<br>
<br>
Amy:<br>
please confirm if we have the required amounts available
to do both the AUC and the NMR experiments. If not, please
alert Bruno so he can send some more this week.<br>
<br>
Thanks! -Borries<br>
<br>
On Sun, Mar 21, 2021 at 10:37:32PM +0000, Montina, Tony
wrote:<br>
> Hi Borries<br>
> For standard NMR tubes we typically make up 600-650
uL of the solution.<br>
> Then we put 550uL in the NMR tube<br>
> But this work is being done in a Shegimi tube, which
uses far less volume, as it restricts the sample to the
active coil area only. This increases the sensitivity and
the glass in the shegimi tube is also susceptibility
matched for d2o/h2o which allows for better shimming.<br>
> For these tubes we need around 280-320uL of sample.
Maybe plan for 350uL just to be safe.<br>
> Mike, can you please confirm if I am correct on the
shegimi tube volumes ?<br>
> Cheers<br>
> Tony<br>
><br>
> Tony Montina<br>
> Director, Science Operations<br>
> Director, Magnetic Resonance Facility<br>
> Faculty of Arts and Science<br>
> Instructor, Department of Chemistry and Biochemistry
University of <br>
> Lethbridge SA6214, Science Commons<br>
> 4401 University Drive West<br>
> Lethbridge, Alberta, Canada, T1K3M4<br>
> Office: 1-403-394-3927<br>
> Lab: 1-403-329-2230<br>
><br>
> This message has been sent from my mobile device so
please ignore any autocorrect or typing errors.<br>
><br>
> On Mar 21, 2021, at 4:08 PM, Borries Demeler <<a href="mailto:demeler@gmail.com" target="_blank">demeler@gmail.com</a>>
wrote:<br>
><br>
> Caution: This email was sent from someone outside of
the University of Lethbridge. Do not click on links or
open attachments unless you know they are safe. Suspicious
emails should be forwarded to
<a href="mailto:phishing@uleth.ca" target="_blank">phishing@uleth.ca</a>.<br>
><br>
><br>
> Thank you, Tony!<br>
> Can you remind me of the volume needed to do the
experiment you planned?<br>
> This will determine if we need Bruno to send more
protein.<br>
><br>
> Thanks, -Borries<br>
><br>
> On Sun, Mar 21, 2021 at 06:43:18PM +0000, Montina,
Tony wrote:<br>
> Hello All,<br>
> I agree with both Paul and Borries.<br>
> The higher we can get the myotoxin concentration in
SDS the better for signal to noise and overall NMR
experiment time in our room temperature probe. Also, as
Paul and Borries both mentioned, using a 5mM concentration
of the buffer and also a significantly lower SDS
concentration (at least one order of magnitude but
potentially two) will greatly improve the dynamic range of
the ADC convertor and hence should result in better signal
to noise (and possibly resolution).<br>
> The last experiments we used ~2.5mg/mL concentration
and our first experiments used 4 mg/ml.<br>
> This was because things started precipitating out at
any higher of concentration. Hopefully this was due to the
micelle formation and once this problem is gone - now that
we are working with SDS below the CMC - we can get
myotoxin into solution at much higher concentrations.<br>
> First, Borries and Amy can determine the minimum
amount of SDS required that is below the 0.2% CMC of SDS.<br>
> I would then suggest that Amy and Mike use this SDS
concentration to determine the max solubility of the
myotoxin in that SDS concentration and 5mM buffer (but not
exceed 20mg/mL of myotoxin as suggested by Borries).<br>
> Once this is done, we can re-run the NMR experiments
on the SDS (at lower concentration) + Myotoxin (hopefully
at a higher concentration) in 5mM buffer. Hopefully we can
get the concentration of myotoxin up much higher than
4mg/mL so that we can run these experiments quickly.<br>
> Amy, do we have enough myotoxin on hand in Lethbridge
to go up to 20mg/mL?<br>
> Lastly, I agree that a NOESY experiment will be
useful, but that would be more useful for the later paper
and depending on the final concentration of myotoxin we
are able to use in SDS, may or may not be possible.<br>
> For example, if this is a 2 week long NOESY
experiment we may not be able to run this on our room
temperature probe. Increasing the concentration will help
greatly with this problem.<br>
> I hope that helps clear up the sample prep questions
for NMR.<br>
> Amy, please reach out to Mike and I when you are
ready next week and we can get the new sample prepared.<br>
> This is really good timing, as I believe Mike has
booked the NMR instrument for the entirety of next
weekend.<br>
> Cheers<br>
> Tony<br>
> ________________________________________<br>
> Tony Montina<br>
> Director | Science Operations<br>
> Director | Magnetic Resonance Facility Instructor |
Department of <br>
> Chemistry and Biochemistry Faculty of Arts and
Science The University <br>
> of Lethbridge Lethbridge, Alberta, Canada, T1K 3M4<br>
> Office: 1-403-394-3927<br>
> Lab: 1-403-329-2230<br>
><br>
> -----Original Message-----<br>
> From: Myotox <<a href="mailto:myotox-bounces@biophysics.uleth.ca" target="_blank">myotox-bounces@biophysics.uleth.ca</a>>
On Behalf Of
<br>
> Hazendonk, Paul<br>
> Sent: March 20, 2021 10:41 PM<br>
> To: Myotoxin-II discussion <<a href="mailto:myotox@biophysics.uleth.ca" target="_blank">myotox@biophysics.uleth.ca</a>><br>
> Subject: Re: [Myotox] myotoxin2-SDS titration<br>
><br>
> As high a conc of protein as is possible, considering
aggregation issues etc, etc. It will really speed things
up especially the 2D experiments.<br>
><br>
> -----Original Message-----<br>
> From: Myotox <<a href="mailto:myotox-bounces@biophysics.uleth.ca" target="_blank">myotox-bounces@biophysics.uleth.ca</a>>
On Behalf Of Borries
<br>
> Demeler<br>
> Sent: March 20, 2021 9:38 PM<br>
> To: Myotoxin-II discussion <<a href="mailto:myotox@biophysics.uleth.ca" target="_blank">myotox@biophysics.uleth.ca</a>><br>
> Subject: Re: [Myotox] myotoxin2-SDS titration<br>
><br>
> Caution: This email was sent from someone outside of
the University of Lethbridge. Do not click on links or
open attachments unless you know they are safe. Suspicious
emails should be forwarded to
<a href="mailto:phishing@uleth.ca" target="_blank">phishing@uleth.ca</a>.<br>
><br>
><br>
> If this is possible that would be very exciting!
Paul, can you please indicate the concentration and volume
of protein needed to perform these experiments. We will
then test by AUC how low we can go with SDS and still see
the shift in s.<br>
><br>
> -Borries<br>
> On Sun, Mar 21, 2021 at 02:36:30AM +0000, Paul
Hazendonk wrote:<br>
> This is very exciting. May I suggest we also try a
NOESY experiment one we have done the analysis on the low
sds conc. Sample since if the binding is very specific we
may be able to identify the amino acids with which it is
interacting.<br>
><br>
> From: Myotox <<a href="mailto:myotox-bounces@biophysics.uleth.ca" target="_blank">myotox-bounces@biophysics.uleth.ca</a>>
On Behalf Of Bruno
<br>
> Lomonte<br>
> Sent: March 20, 2021 4:40 PM<br>
> To: <a href="mailto:myotox@biophysics.uleth.ca" target="_blank">myotox@biophysics.uleth.ca</a><br>
> Subject: Re: [Myotox] myotoxin2-SDS titration<br>
><br>
> Caution: This email was sent from someone outside of
the University of Lethbridge. Do not click on links or
open attachments unless you know they are safe. Suspicious
emails should be forwarded to <a href="mailto:phishing@uleth.ca" target="_blank">phishing@uleth.ca</a><<a href="mailto:phishing@uleth.ca" target="_blank">mailto:phishing@uleth.ca</a>>.<br>
><br>
> Interesting as always, Borries and all ! the
combination of your biophysical analyses + NMR are
providing lots of exciting new information, as you
explained. I will send more protein this week to Amy's
address. Thanks a lot for all the interest and hard work.<br>
><br>
> Best regards,<br>
><br>
> Bruno<br>
><br>
><br>
> +++++++<br>
> On 3/20/2021 11:20 AM, Borries Demeler wrote:<br>
><br>
> As promised, here is an update on the SDS titration
experiment.<br>
><br>
> Amy found plenty of Myotoxin for doing these
experiments in our<br>
><br>
> freezer and performed an AUC experiment doing a
titration with very <br>
> low<br>
><br>
> concentration of SDS (0.01 and 0.02 percent of SDS,
which should be <br>
> well<br>
><br>
> below the CMC of SDS). This clearly shows that there
is a significant<br>
><br>
> change in the sedimentation profile (see attached).<br>
><br>
><br>
><br>
> A few notes:<br>
><br>
> 1. The monomer control was actually run at 300 nm
with ~21 mg/ml<br>
><br>
> concentration - so very high, and no evidence of
dimerization there.<br>
><br>
> The protein concentration in the SDS titration
experiments was much,<br>
><br>
> much lower at only about 10 uM.<br>
><br>
><br>
><br>
> 2. even the 0.01% SDS sample exhibits significant
shift in the <br>
> s-values<br>
><br>
> which can only be explained by dimerization (as seen
in the SDS-PAGE),<br>
><br>
> not by micelle formation with multiple myotoxin
molecules being<br>
><br>
> incorporated into the micelle.<br>
><br>
><br>
><br>
> 3. The 0.02% SDS concentration sample sedimented
*slower* than the<br>
><br>
> 0.01% SDS sample. If I think about this it makes
sense if we<br>
><br>
> assume that instead of additional oligomerization we
simply bind<br>
><br>
> more SDS to the complex, because SDS could
significantly affect<br>
><br>
> friction. It also affects the partial specific
volume. It is less<br>
><br>
> dense than protein so the overall partial specific
volume of the<br>
><br>
> complex becomes less dense as more SDS is bound.<br>
><br>
><br>
><br>
> 4. There isn't just one peak visible, consistent with
either monomer <br>
> or<br>
><br>
> dimer, but there are at least 2 major peaks in both
samples, <br>
> suggesting<br>
><br>
> possible even high order structures than dimer are
present, or at <br>
> least<br>
><br>
> some equilibrium between at least two oligomeric
structures. It is not<br>
><br>
> clear to me what oligomers are present, but with a
little additional<br>
><br>
> AUC work we could probably figure this out.<br>
><br>
><br>
><br>
> I think this brings up more questions, but also
answers one important<br>
><br>
> question for Paul and Tony:<br>
><br>
> We can definitely significantly reduce the SDS
concentration to avoid<br>
><br>
> the strong overlay signals from SDS in the NMR
experiments. Also, we<br>
><br>
> can keep the very low phosphate concentration (5mM).<br>
><br>
><br>
><br>
> I think before we go to the next NMR experiments I
want to do one<br>
><br>
> more AUC experiment: Amy, can you please find out the
lowest possible<br>
><br>
> SDS concentration that causes a shift for a given
protein concentration?<br>
><br>
><br>
><br>
> What I found surprising is that the 0.02% solution
sedimented slower<br>
><br>
> than the 0.01%, this suggests the formation of
discrete species, with<br>
><br>
> tight SDS binding, and no change in oligomerization
between the two <br>
> SDS<br>
><br>
> concentrations. That's important. I would like to
know if we can find<br>
><br>
> out the lowest SDS concentration that causes this
behavior. I think<br>
><br>
> the SDS binds very tightly, so do we see the same
behavior at 0.001%<br>
><br>
> SDS? I think we should do a titration one order of
magnitude lower,<br>
><br>
> and at the same time increase the protein
concentration. THis would<br>
><br>
> tell us two things: 1. what is the best ratio of high
concentration<br>
><br>
> protein and low concentration SDS that clearly starts
showing this<br>
><br>
> oligomerization? That ratio would be best for NMR,
maximum protein <br>
> signal,<br>
><br>
> minimum SDS signal. Let's find the critical ratios
with AUC. We may <br>
> have<br>
><br>
> to run the SDS titration with more points and also
repeat it at<br>
><br>
> multiple protein concentrations. I would also like to
know the maximum<br>
><br>
> s-value shift that can be observed, and whether the
2-peak pattern<br>
><br>
> persists.<br>
><br>
><br>
><br>
> Paul/Tony: What protein concentration would you like
to run ideally?<br>
><br>
> 1 mg/ml, 10 mg/ml, 20 mg/ml? Since Bruno is willing
to send more <br>
> sample,<br>
><br>
> I think we can play with this ratio a bit. Amy, how
much is left?<br>
><br>
> For AUC I would prefer not to go much above 20 mg/ml
to avoid<br>
><br>
> concentration dependent non-ideality effects.<br>
><br>
><br>
><br>
> Let me know what y'all think about this approach.<br>
><br>
><br>
><br>
> -Borries<br>
><br>
><br>
><br>
> _______________________________________________<br>
><br>
> Myotox mailing list<br>
><br>
> <a href="mailto:Myotox@biophysics.uleth.ca%3cmailto:Myotox@biophysics.uleth.ca" target="_blank">
Myotox@biophysics.uleth.ca<mailto:Myotox@biophysics.uleth.ca</a>><br>
><br>
> <a href="https://demeler7.uleth.ca/mailman/listinfo/myotox" target="_blank">https://demeler7.uleth.ca/mailman/listinfo/myotox</a><br>
><br>
><br>
><br>
> --<br>
><br>
> Bruno Lomonte, Ph.D.<br>
><br>
> Instituto Clodomiro Picado<br>
><br>
> Facultad de Microbiología<br>
><br>
> Universidad de Costa Rica<br>
><br>
> San José, COSTA RICA<br>
><br>
><br>
><br>
> tel.of. (+506) 2511 7888<br>
><br>
> <a href="mailto:bruno.lomonte@ucr.ac.cr%3cmailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr<mailto:bruno.lomonte@ucr.ac.cr</a>><br>
><br>
> _______________________________________________<br>
> Myotox mailing list<br>
> <a href="mailto:Myotox@biophysics.uleth.ca" target="_blank">Myotox@biophysics.uleth.ca</a><br>
> <a href="https://demeler7.uleth.ca/mailman/listinfo/myotox" target="_blank">https://demeler7.uleth.ca/mailman/listinfo/myotox</a><br>
><br>
> _______________________________________________<br>
> Myotox mailing list<br>
> <a href="mailto:Myotox@biophysics.uleth.ca" target="_blank">Myotox@biophysics.uleth.ca</a><br>
> <a href="https://demeler7.uleth.ca/mailman/listinfo/myotox" target="_blank">https://demeler7.uleth.ca/mailman/listinfo/myotox</a><br>
> _______________________________________________<br>
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<pre>_______________________________________________
Myotox mailing list
<a href="mailto:Myotox@biophysics.uleth.ca" target="_blank">Myotox@biophysics.uleth.ca</a>
<a href="https://demeler7.uleth.ca/mailman/listinfo/myotox" target="_blank">https://demeler7.uleth.ca/mailman/listinfo/myotox</a>
</pre>
</blockquote>
<pre cols="72">--
Bruno Lomonte, Ph.D.
Instituto Clodomiro Picado
Universidad de Costa Rica
San José, 11501
COSTA RICA
<a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a>
tel. +506 2511 7888
cel. +506 8392 0012
</pre>
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</blockquote></div>