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Dear all, <br>
<br>
Ana and Borries just read my mind... I was just going to ask the
same today! :)<br>
<br>
Of course it would be totally understandable if it has been
difficult to run the assays, since obviously we are all facing the
health risks of this pandemy<br>
<br>
Sending you all my best and hoping that everyone is doing well,
science aside<br>
<br>
Bruno<br>
<br>
<br>
+++++<br>
<br>
<br>
<br>
<div class="moz-cite-prefix">On 12/4/2020 2:15 PM, Ana Gisele da
Costa Neves Ferreira wrote:<br>
</div>
<blockquote type="cite"
cite="mid:CAPgscZg6VMYdBaFi-jBg=oSKxghY4qsrdtYaHRTFDakhQ7iEKQ@mail.gmail.com">
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<div dir="ltr">Dear colleagues,
<div>I hope you and your families are in good health. We are
already at the end of the year and I think it would be great
if we could finish the manuscript on myotoxin II and submit it
for publication as soon as possible. Could you give us an
update on the latest NMR data progress ?</div>
<div>I wish you a joyful and peaceful holiday season, despite
these difficult times.<br>
</div>
<div>Warm regards,</div>
<div>Ana Gisele</div>
</div>
<br>
<div class="gmail_quote">
<div dir="ltr" class="gmail_attr">Em ter., 1 de set. de 2020 às
16:33, Montina, Tony <<a
href="mailto:tony.montina@uleth.ca" moz-do-not-send="true">tony.montina@uleth.ca</a>>
escreveu:<br>
</div>
<blockquote class="gmail_quote" style="margin:0px 0px 0px
0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">Hello
Borries,<br>
I think we can simply run a blank sample of the SDS in buffer
to determine where the peaks fall in the spectrum.<br>
Mike and Amy, can the two of you please work together to get
this blank sample prepared, as well as the sample with SDS
included with myotoxin?<br>
Cheers<br>
Tony<br>
<br>
_______________________________________________<br>
Tony Montina
<br>
Director, Science Operations<br>
Director, Magnetic Resonance Facility<br>
Instructor, Department of Chemistry and Biochemistry<br>
Faculty of Arts and Science<br>
The University of Lethbridge<br>
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" moz-do-not-send="true">myotox-bounces@biophysics.uleth.ca</a>>
On Behalf Of Borries Demeler<br>
Sent: September 1, 2020 1:24 PM<br>
To: <a href="mailto:myotox@biophysics.uleth.ca"
target="_blank" moz-do-not-send="true">myotox@biophysics.uleth.ca</a><br>
Subject: Re: [Myotox] high temperature NMR experiments<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. Please forward
suspicious emails to <a href="mailto:phishing@uleth.ca"
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<br>
<br>
Hi Tony,<br>
Thanks so much for the update! I think the plan is really
solid and will provide additional information. The linewidth
data gathered from the two temperature should tell us if there
are differences between +/- SDS, which will be highly useful
for the first paper, and tell us if SDS is either stabilizing,
destabilizing, or has no effect. I would expect<br>
*some* change, since SDS clearly causes dimerization. We may
not need residue assignments, just knowing the general trends
could already be useful.<br>
<br>
I am copying the entire list to see if anyone else has any
thoughts on the two-temperature experiments. The fluorescence
data Amy collected suggested temperature effects starting
around 40C, so staying below 40C is in my opinion a good idea,
since it would be non-physiological anyway.<br>
<br>
Once we get the cryo-probe there will be plenty of additional
structural information that we could access from 2D
experiments, and that should shed further light on this
interesting story, so for now the 1D experiments are fully
sufficient.<br>
<br>
One point I am not clear on: When we add SDS to the buffer,
how to you correct for the additional signal - is there some
sort of baseline subtraction you can make? Of course we don't
really need to characterize the SDS molecules, but we do care
about the effect it has on the protein structure, and any
details that we can obtain that would explain the dimerization
effect.<br>
<br>
Amy, can does Tony have enough sample from the lyophilized
stock that Bruno sent us?<br>
<br>
Thanks! -Borries<br>
<br>
<br>
<br>
<br>
On Tue, Sep 01, 2020 at 07:07:35PM +0000, Montina, Tony wrote:<br>
> Hello Borries,<br>
> I just met with Mike to go over the next steps of the
myotoxin work.<br>
> We have tried 2D NMR work for a while now and we have
come to the conclusion that most of the 2D work (if not all of
it) will require a cryoprobe to complete.<br>
> So this is what we have determined Mike will do next:<br>
><br>
> (1) Mike will take the normal myotoxin sample and run
this sample at both 10 degrees and 37 degrees. At each of
these temperatures he will obtain the 1H proton NMR and the T1
and T2 data for the proton NMR. This should allow Paul to
determine if there are relaxation or linewidth affects over
that temperature range. Mike will start these experiments as
soon as possible (this week probably).<br>
><br>
> (2) SDS experiments - we had decided to run the myotoxin
in SDS and obtain the 1H NMR spectra and the T1 and T2 values
in SDS. Do we need this for the first paper? If so, Amy, can
you please help Mike with determining how to prepare the
myotoxin sample in SDS with buffer.<br>
><br>
> So Mike will start on step one above as soon as possible.
Amy and Borries, please let us know about step 2 and Mike and
Amy can make arrangements to figure out the sample
preparation.<br>
><br>
> Let me know if you have any questions or concerns.<br>
> Tony<br>
><br>
> _______________________________________________<br>
> Tony Montina<br>
> Director, Science Operations<br>
> Director, Magnetic Resonance Facility<br>
> Instructor, Department of Chemistry and Biochemistry
Faculty of Arts <br>
> and Science The University of Lethbridge Lethbridge,
Alberta, Canada, <br>
> T1K 3M4<br>
> Office: 1-403-394-3927<br>
> Lab: 1-403-329-2230<br>
><br>
> -----Original Message-----<br>
> From: Borries Demeler <<a
href="mailto:demeler@gmail.com" target="_blank"
moz-do-not-send="true">demeler@gmail.com</a>><br>
> Sent: August 27, 2020 12:38 PM<br>
> To: Montina, Tony <<a
href="mailto:tony.montina@uleth.ca" target="_blank"
moz-do-not-send="true">tony.montina@uleth.ca</a>><br>
> Cc: Henrickson, Amy <<a
href="mailto:amy.henrickson@uleth.ca" target="_blank"
moz-do-not-send="true">amy.henrickson@uleth.ca</a>>;
Hazendonk, Paul <br>
> <<a href="mailto:paul.hazendonk@uleth.ca"
target="_blank" moz-do-not-send="true">paul.hazendonk@uleth.ca</a>>;
Opyr, Michael <<a href="mailto:michael.opyr@uleth.ca"
target="_blank" moz-do-not-send="true">michael.opyr@uleth.ca</a>><br>
> Subject: Re: high temperature NMR experiments<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. Please forward
suspicious emails to <a href="mailto:phishing@uleth.ca"
target="_blank" moz-do-not-send="true">phishing@uleth.ca</a>.<br>
><br>
><br>
> Thanks, guys! Also, Amy did collect the high temperature
unfolding data based on intrinsic trp fluorescence change, and
we did see changes in conformation starting at around 45C, is
this enough for the high temperature experiments you had
envisioned? If you need higher temperatures, since the
stability is not sufficient at higher temperatures, I think we
would have to skip the higher temperature experiments.<br>
><br>
> Amy, can you please coordinate with Mike? I believe we
have lyophilized sample so you can dissolve in whatever buffer
(D2O, H2O, pH, etc.) suitable for your experiments, though I
prefer to keep things in PO4 buffer when possible so we keep
things comparable to the AUC experiments.<br>
><br>
> Thanks, -Borries<br>
><br>
><br>
><br>
> On Thu, Aug 27, 2020 at 06:28:25PM +0000, Montina, Tony
wrote:<br>
> > Hello Borries and Amy,<br>
> > I was away for the second and third week of August
for holidays and Mike was away this past week.<br>
> > Mike and I will meet on Monday or Tuesday next week,
review our notes from the last meeting and discuss the next
best steps.<br>
> > In the meantime, Amy, can you please make
arrangements with Mike to drop off the sample to him?<br>
> > I believe he will be on campus tomorrow afternoon
(Friday).<br>
> > Talk soon<br>
> > Tony<br>
> ><br>
> > Tony Montina<br>
> > Director, Science Operations<br>
> > Director, Magnetic Resonance Facility Instructor,
Department of <br>
> > Chemistry and Biochemistry The University of
Lethbridge,<br>
> > 4401 University Drive West<br>
> > Lethbridge, Alberta, Canada, T1K 3M4<br>
> > Office: SA6214 Science Commons 1-403-394-3927<br>
> > Lab: SA6216 Science Commons 1-403-329-2230<br>
> ><br>
> > -----Original Message-----<br>
> > From: Borries Demeler <<a
href="mailto:demeler@gmail.com" target="_blank"
moz-do-not-send="true">demeler@gmail.com</a>><br>
> > Sent: August 27, 2020 9:28 AM<br>
> > To: Henrickson, Amy <<a
href="mailto:amy.henrickson@uleth.ca" target="_blank"
moz-do-not-send="true">amy.henrickson@uleth.ca</a>><br>
> > Cc: Hazendonk, Paul <<a
href="mailto:paul.hazendonk@uleth.ca" target="_blank"
moz-do-not-send="true">paul.hazendonk@uleth.ca</a>>;
Montina, Tony <br>
> > <<a href="mailto:tony.montina@uleth.ca"
target="_blank" moz-do-not-send="true">tony.montina@uleth.ca</a>>;
Opyr, Michael <<a href="mailto:michael.opyr@uleth.ca"
target="_blank" moz-do-not-send="true">michael.opyr@uleth.ca</a>><br>
> > Subject: Re: high temperature NMR experiments<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. Please forward
suspicious emails to <a href="mailto:phishing@uleth.ca"
target="_blank" moz-do-not-send="true">phishing@uleth.ca</a>.<br>
> ><br>
> ><br>
> > Paul/Tony,<br>
> > do you have any updates for us?<br>
> ><br>
> > Thanks, -Borries<br>
> ><br>
> ><br>
> > On Tue, Aug 25, 2020 at 09:30:15PM +0000,
Henrickson, Amy wrote:<br>
> > > Hello Everyone,<br>
> > ><br>
> > > Have you been able to book anytime to do the
next experiments?<br>
> > ><br>
> > > Best,<br>
> > ><br>
> > > Amy Henrickson<br>
> > ><br>
> > > ________________________________<br>
> > > From: Borries Demeler <<a
href="mailto:demeler@gmail.com" target="_blank"
moz-do-not-send="true">demeler@gmail.com</a>><br>
> > > Sent: Tuesday, August 4, 2020, 12:58 p.m.<br>
> > > To: Hazendonk, Paul; Montina, Tony; Henrickson,
Amy<br>
> > > Subject: high temperature NMR experiments<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. Please
forward suspicious emails to <a
href="mailto:phishing@uleth.ca" target="_blank"
moz-do-not-send="true">phishing@uleth.ca</a>.<br>
> > ><br>
> > ><br>
> > > Hey guys,<br>
> > > In our last meeting Paul mentioned that it
would be desirable to <br>
> > > measure myotoxin-II at higher temperature to
get additional <br>
> > > information. Amy performed a melting curve
using fluorescence <br>
> > > detection and determined a stability curve -
Amy, can you please <br>
> > > indicate the onset of the transition
temperature where the protein <br>
> > > changed fluorescence, and where the middle of
the transition is <br>
> > > for myotoxin in the presence and absence of
SDS?<br>
> > ><br>
> > > I would like to know what experiments you would
like to do next <br>
> > > and what information we could add to the paper
from them. As we <br>
> > > discussed, this could turn into a rabbit hole
of further <br>
> > > experiments, and some of them may be best
postponed until we get a <br>
> > > cryo probe. For now, we just want to pick the
low-hanging fruit, <br>
> > > and Amy obtained more sample at high purity
from Costa Rica we can <br>
> > > now give to you. Please contact Amy to arrange
for her to bring <br>
> > > them to you. The samples we have are
lyophilized and can be suspended by you in any buffer needed.<br>
> > ><br>
> > > Please let us know how you want to proceed.<br>
> > ><br>
> > > Thanks! -Borries<br>
> > ><br>
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-- <br>
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<div><span
style="color:rgb(11,83,148);font-family:tahoma,sans-serif;font-size:x-small">Ana
Gisele da C. Neves Ferreira</span><br>
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<div><font style="background-color:rgb(255,255,255)"
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<pre class="moz-quote-pre" wrap="">_______________________________________________
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<pre class="moz-signature" cols="72">--
Bruno Lomonte, Ph.D.
Instituto Clodomiro Picado
Facultad de Microbiología
Universidad de Costa Rica
San José, COSTA RICA
tel.of. (+506) 2511 7888
<a class="moz-txt-link-abbreviated" href="mailto:bruno.lomonte@ucr.ac.cr">bruno.lomonte@ucr.ac.cr</a></pre>
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