- Good afternoon everyone
and thank you, Dr. Scottgale.
Welcome everyone to our final talk
of the 43rd Darwin festival.
And just to remind you today is alumni day.
Traditionally, over the last 43 years,
the final day of the talk is alumni day.
Some of you in the audience may not be aware
why do we always have this in February.
It's to time the talk with Darwin's birthday,
which this year didn't quite overlap
because his birthday's tomorrow
and most of us perhaps
would be doing something else on a Saturday.
Just a quick reminder, before I introduce an alumnus,
Mr. Peter Shearstone.
That if you're a student, a bio student
in the, Mya Hall building.
After this talk, if you wanted to come down to the lab,
Mya Hall 414,
I've got some little surprises that I'll be giving away.
With that let me introduce Mr. Peter Shearstone.
As I said, Peter is an alumnus.
He graduated in 1989 through our biology department.
He joined Thermo Fisher Scientific in 2018
as vice-president for global quality and regulatory affairs,
and is responsible for leading
the company's global QARA team,
which consists of over 8,000 employees.
Thermo Fisher Scientific is headquartered
in Waltham, Massachusetts,
and is the world leader in serving science.
Their mission being to enable customers
to make the world a healthier, cleaner, and safer place.
As I said, Peter graduated from bio,
our biology department in 1989 with a BS in biology
and through his company, Thermo Fisher Scientific,
they are providing support
for the Darwin festival over the next five years,
enabling us to provide these talks
to the broader north shore of Massachusetts.
Peter, thank you very much for joining us today.
- Dr. Fisher, thank you so much.
It's an honor.
And I had to do some quick math
to realize that when I was at Salem,
I was attending the sixth and seventh and eighth and ninth
annual Darwin festival.
So obviously completely thrilled and honored
to be here today and to provide support for the festival.
Really honored to introduce you all
to Dr. Laranjo and to Sydney.
And let me just give you a quick,
some quick comments on both of them.
Dr. Laranjo is an assistant professor
at Salem State University biology department.
She's in her third year with the university.
She obtained her undergraduate biology degree
from UMass Lowell.
So another state college grad.
And joined Salem state from Brandis
where she completed her work on her PhD.
Born and raised in Brazil,
she came to the us when she was 15
and whilst working on her bachelor's degree at Lowell,
worked in a neurobiology lab,
was part of a national science foundation research
experience for undergraduates program REU,
and internship, rather, excuse me.
And that's what inspired her really to pursue her PhD.
Whilst at Brandis, she did quite a bit of work
and research in a genetics lab,
studying a specific class of mutation created
during DNA replication.
And in her lab at Salem State,
she's very fortunate to work with a team of incredible
students who are just as curious as she is.
And I will say personally, curiosity is what you know,
gets many of us started, I think on the science track.
Her goal and the team's goal is to continue their work,
to test FDA approved drugs for their ability to cause
specific types of DNA mutation
using bacteria as a model organism.
And they're also collaborating
with Framingham State University,
which is also very good to see for see.
For Sydney, she's a 2021 Salem State graduate in biology.
She's also a graduate of Malden High School
and was a research assistant at Tufts University,
actually during her senior year of high school,
where I suppose Sydney,
that's probably where you got the bug,
like many of us did.
You've been a resident assistant at Salem State
as well as a supplemental instructor in organic chemistry.
I wish I had known you back in 1985,
when I took organic chem,
you could have probably helped me a bit there.
And she's worked with Dr. Laranjo on her honors project
and is co-presenting our topic today,
which is investigating FDA approved anti-tumor drugs
for effects on template-switch mutagenesis or TSM in E coli.
So please join me in welcoming Dr. Laranjo and Sydney.
Thank you.
- Thank you so much for such a nice introduction.
And start over here.
And good afternoon everyone.
I am very happy, very excited to be here,
sharing with you a bit about our work.
And as was mentioned,
we're gonna talk a little bit out FDA drugs and the effect
on a specific class of mutation.
But maybe just, maybe you don't think about cells
and DNAs every day.
So I wanna give you a little bit of a background reminder.
So maybe you've seen a picture like this in your fifth
or sixth, seventh grade biology class,
or even in a intro bio class.
We know here we have a cell,
the smallest structural and function unit of an organism.
We learn in school that inside of the cell,
we have the nucleus and in the nucleus, we have chromosomes,
which are lots of DNA wrapped around proteins
in a way that will fit inside of the cell.
When we look into DNA,
we know that DNA is composed of two chains that coiled
around each other.
They form what we call a double helix,
and that carries the genetic instructions
that is used for growth, development, functioning,
reproduction of all known organisms.
But sometimes they make mistakes and those mistakes,
we call DNA mutations and any of these mistakes
have the potential to change the genetic code.
So you can see here that a lot can happen,
and there's so much going on in the DNA
that it opens up good amount of field for us to study,
and a lot of questions for us to ask.
I have here simplified what we call a replication fork.
So if you look it over here,
we have our DNA and our DNA needs to be replicated
because if it doesn't replicate the cell will die.
And it needs to replicate.
And it opens, the two strands opens up.
And at that point it's called replication fork.
It doesn't really look like any fork I have at home,
to be honest, but this is what people named it.
And you can see that there is some specific characteristics
of this replication and fork.
We know that it has directions,
and this is very important because it allows us to track,
which strand is which.
And we give names to them,
one, the leading strand and the other, the lagging strand.
So the leading strand is also known
as the continuous strand.
The replication process here is gonna be smoother.
The lagging strand is still going to be replicated,
but other players are gonna have to come and join us.
But when you look at the DNA and you look
at the replication process,
here is a very simplified cartoon,
but you can see that there is a lot of players here.
So all of this players here brings the question,
how does all work?
How do they all work together?
And how do they work to be doing what they're supposed to do
and not have that many mutations?
So the first time that I got interested in the topic,
I was in high school,
and I just kept thinking things have a place to go,
they have a function to do,
and why are some things going out of control?
So you also call that a perfectionist mentality,
which is bad and good.
But I took the good part of it,
and I decided to use to investigate how the DNA uses
its resources to be perfect,
to do what is supposed to do,
to replicate in the way it's supposed to do.
But it also brought up the question,
what if you make a mistake?
We all make mistakes our DNA is gonna make mistakes.
How is it going to get fixed?
And that is where my passion for DNA mutations is started.
And it's interesting because as Peter mentioned,
I worked in a neurobiology lab.
I truly enjoy my time there,
but it was a totally different project.
And I just wanted to bring this up because sometimes you are
not going to get the opportunity to work on the thing you
are passionate about right away.
But take, take any opportunity and make the best of it,
use those skills.
So then when it comes the time that you can study
what you want, you are ready for it.
So that's what I did. But let's talk about mutations.
I started out wondering how often these mutations happen.
Is it something that the DNA has to do at it
on a daily basis or every so often?
And that's not something
we think about it all the time, right.
But actually they happen much more often than we think.
For example, the human genome is estimated
to go through 10 to the 16 power DNA mutations
in a single day.
Of course, a lot of them are going to be corrected.
Most of them otherwise, death and diseases
will happen much more often.
But even though they have great mechanisms to fix,
it is still estimated that we have around 8,000 human
diseases caused because of DNA mutations.
So think about it like this.
If you are an undergrad and you are working
for or five years in your degree,
that estimates you to have about two times 10 to the 19th
power of mutational events during your college career.
So that is a lot of mutations that can happen.
And this number can be increased by lack of nutrients.
If you don't have health,
food habits by stress and so on, lack of sleep.
And if you are professor,
I wanted to think about every 10 years of you teaching.
Every 10 years of you teaching is estimated
that in that timeframe,
you're gonna go through 3.7 times 10
to the 19th power of mutation.
And also this number can be increased
by stress, lack of sleep, and so on.
Now maybe you'll like, yeah,
those are a lot of zeros afterwards, a lot of numbers,
but that's a lot of years and a lot of days.
Well, just during this talk that I'm giving to you, our DNA,
mine and yours can go through up
to 4.2 times 10 to the 14th power mutations.
My point is mutations happen very often
and that number just keep going up,
but there are different sources of DNA mutations,
and that is why it's difficult to study them.
If there was one source,
we could all study that together
and we could make that work.
But a lot of things can cause DNA mutations.
Some of those sources are external and others are indulgent.
So for example, a viral infection, radiation, chemotherapy,
things from the outside that will affect your DNA.
And when we talk about radiation,
we talk about daily radiation too.
For example, if you are flying a lot in airplanes,
you are exposed a lot more to radiation
than if you're don't, if you were here.
But there are other sources that are inside of the cell,
such as replication, stress,
and other things that happen in a regular basis.
But the source that I was interested,
it's actually not really talked about.
And people are studying more and more now,
but it's not in the diagrams as you can see here.
And it, we don't know much about it.
So I fixed this diagram. There you go.
We're gonna talk about secondary structures
as a source of DNA mutation.
So secondary structures are shapes that the DNA takes
and they can make funny figures like this.
We are gonna focus on this type of structure here.
It's called a hairpin loop, 'cause it looks like a hairpin.
And as you can see, we are always gonna look at directions,
but this is a shape that the DNA can make
based on how it is formed.
So I'm going to explain to you how these shapes are created
by giving you an example of something that you'll probably
know more about it than DNA, and which is tacos.
Taco is a very, very delicious, it's great
and what does that have to do with DNA?
Well, if you look at this taco cat,
the spell backward is also taco cat.
That's a palindrome that we talk about here, right.
But in DNA, the basis of DNA,
we use letters for those to identify each phase and they can
also be in a setting that they are going
to look like palindromes or quasi, semi-palindromes.
So the letters can be organized in the same way
that it's going to be the same letters backwards or not.
And those structures have been linked to forming hairpins.
So regions of palindromes in the DNA
form the structures that we call hairpin.
And if you think about it,
there's different forms of hairpin, there's different sizes.
But if you have one in the cell,
you wanted to replicate your DNA.
And as I mentioned, you want to replicate the DNA
exactly like it was before.
You don't wanna add any mutations.
You don't wanna delete anything.
You don't wanna change the genetic configuration.
You wanted to have this hairpin be duplicated and so on.
You want all the same thing.
And in order for you to do that,
you're going to look at one of them as a template.
So this is going to be my hairpin template
and the cell is gonna wanna do over and over the same thing.
Sometimes if you change the template
you can have problems.
I'll give you an example.
Imagine you are in an art class and you receive
an assignment and your assignment is to draw a horse.
You receive from the professor, a template of a horse.
And you know, you're gonna draw this horse.
You know, you can see everything there.
You're like, okay, I have the template.
I can just trace it.
This is what I'm going to do.
And then you leave your assignment at a table and maybe you
have a toddler at home like me
and you forget that you leave your assignment there.
The toddler comes and it changes
that template to something similar.
A horse is nice, but how about unicorn?
Very similar, very similar to a horse,
but not the initial one.
So now instead of using the horse as the template,
you ended up using the unicorn.
You submit this assignment and it doesn't matter how nice
this your unicorn looks. It is not what you was asked for.
Same thing goes with the hairpins.
These hairpins have been associated with a mutation that it
changes from a quasi palindrome
to a perfect palindrome.
What does that mean?
It means a region of the DNA that formed this hairpin
had an imperfect hairpin, a quasi almost palindrome.
Something happened, a mutation happens
and it makes it a perfect.
And just because it's perfect and it's nice,
like the unicorn does not mean it's what we wanted to.
So even though it's a perfect palindrome,
that is a mutation.
And this mutations correlated with the structure
can have very implications,
many implications in human health and so on.
So I'm gonna tell you,
how do we believe that this mutation happens
going from a quasi palindrome to a perfect palindrome.
So this model was first proposed by Ripley in 1982.
So not that long ago.
Here we have a DNA strand and this shapes,
they pretend that each one of them is a base of the DNA.
And we have a region of an imperfect hairpin here.
Our DNA opens up,
and DNA polymerase is gonna come to replicate
that strand of the DNA.
It's going through and then for some reason,
when you reach the region of an imperfect hairpin,
the polymerase can momentarily disassociate from the fork.
It doesn't run away and go to other places,
but it stays nearby.
But it's no longer attached to this new strand
that it was just making it out.
And it's no longer attached to the template
that it was using it to make this new strand,
because this is a region of a hairpin,
because of the configuration of how the basis work.
This strand can now fold on itself
because of its nature of the basis.
When it folds on the top and on the bottom,
we also call this a cruciform structure,
but you can see here, it starts to fold on itself.
And at this point, or at some point,
the polymerase has to come back.
It has to finish replication.
But at some frequency instead of the polymerase,
returning to your original template
and continue to making this new DNA.
At some frequency, it can bind to the new strand instead.
And now it's going to use the new strand as the template.
So as if the bottom is the horse, the top is the unicorn.
You're trying to make the horse,
but now you went to the wrong one.
And now it's gonna use the new template as much as it can.
Because this is a new strand,
you can see it's we don't have any more bases here.
So it's going to go and replicate and then, okay.
What do I do now?
Polymerase will have to jump back and switch template again
in order to proceed with the replication.
So this is the second template switch.
This forms where we know this, the cruciform is structure.
That repair mechanisms are gonna come here and help resolve
this structure and allow the DNA to proceed the replication.
But once all of that happens,
we will ended up with template switch mutations.
We started with an imperfect, a quasi palindrome
and we ended up having a perfect palindrome.
So even though it's perfect, it is a mutation
and that is not great.
These mutations and clusters of these mutations
have been seen from bacterias to humans,
which suggests that it's a conserved mechanism.
So in bacteria, in some regions of the DNA,
that more mutations happen more than others.
We call those hotspots.
We can see 500 times more template switch mutations
than in other locations.
We also looked at in yeast and in yeast,
we see that it's induced during double strand break repair.
And if you break your double strand DNA, you have to fix it.
If you don't fix it, the cell's going to die.
And in humans,
a few diseases have already been associated with them.
53 related cancer edemas,
and the most known one is the osteogenesis imperfecta.
So we are still understanding exactly what cascade
of events is set.
But we do know the template switch mechanisms,
do play a role into all of these mutations.
So at this,
at the time I was at Brandeis University
doing my graduate work
and I had the honor to work on creating
and testing quasi palindrome mutation reporters.
So reporters is something that it's gonna tell you
what it does, right.
So we tested many and we were able to find reporters.
They are very specific.
We created them high accuracy,
and we made reporters that will tell us how often,
what is the rate of quasi palindrome
temple switch mutations.
So this is how Dr. Lovett designed these reporters.
She chose her favorite gene.
So that stands for your favorite gene.
So she chose her favorite gene and she found a region
that already had a perfect palindrome there.
So we added four base pairs into each side of the hairpin.
So in this case here, I only have a one side,
but we did a reporter for the leading
and the lagging strand.
So because it has directions,
we could make a reporter for each side.
So once we put this four base pair insertion here,
this gene is no longer active.
So the cell, the colony,
the culture that we're gonna played,,
can only survive at specific conditions,
if something happens and make this gene good again.
And the only way for us to remove these four base pairs here
in this gene, in E coli,
is if it goes through a template switch mutation.
So we did that in E coli and in yeast.
In yeast we use directory,
and today I'm not gonna be talking about this data.
We stay with E coli and we did this research
where we could see that the reporter is very,
very accurate in such a way that all of the times
that we've lost this four base pairs,
the only way possible
is actually going through a template switch mutation.
So the assay works like this.
We could take, in both systems we're very similar.
So that's why I wanna just show it here.
But we inoculate the colonies
with appropriate media for each organisms.
And then we either didn't do any treatment in each of them,
but we also later on treated with some drugs, the culture,
and then we played it in a specific plates.
So we played it in plates
that would give us the total population count.
So for bacteria, it's the lb
and for yeast, we had a selective marker LEU2.
So in this plates here, everything would grow.
We had all the nutrients,
everything is gonna be happy and grow.
So we know how much we have it.
And we also played in specifically plate,
and this place are called selective plate
because they selected for the mutation.
Because only the cells that went through a template,
switch mutation and activated that gene that we deleted
is now going to, the gene that we made it inactive.
It's now going to grow.
So here will be a LacMin.
So there is no lactose here unless lacZ gene
is activated again, the cells are not gonna grow.
And the same here was for the URA.
So URA was inactivated,
as soon as mutation happen in now activates back in.
We did this multiple, multiple, multiple times,
and we got some good data.
We published few papers on this, and we looked at it,
how things inside of the cell
actually prevent this class of mutation.
So we looked at single strand binding proteins.
We looked at polymerase,
their mutants and saw that the cell itself tries
to fix this problem.
As I mentioned, many mutations happen,
and we do have things in the cell that says,
hold on, let's fix it.
But we also noticed that without some abilities of the cell,
and without some proteins of the cell working well,
this type of mutation happens much more often.
And this case here would be the polymerase.
So we notice that if we mess up with the polymerase,
now we have more time for this event to happen
and we see more mutations.
Here in the bottom are some of the yeast genes
that I screen to see if we would increase
or decrease the frequency of mutation.
And we worked a lot of that.
And then that's when it came the idea.
What if we treat the cell with chemicals
that will make DNA replication be stalled,
or that will interfere with DNA replication.
Since that our hypothesis is that if replication stops,
the polymerase leaves the fork,
there is more time for this event to happen.
Then we could be onto something here.
So we decided to look at DNA, protein crosslinks
as how they are barriers to the replication fork.
So DNA proteins crosslinks,
they can be produced by natural metabolisms,
and they haven't seen in all organisms.
They are a barrier to the fork
because they stop replication.
And if they stop the replication,
now we have a higher chance
of the polymerase leaving the fork.
So then the hypothesis will be that DNA protein crosslinks
will promote this class of mutation.
There are many normal cellular enzymes.
I have here cytosine methyltransferase and topoisomerase.
That they are known to produce covalent complexes
as intermediates in the reaction mechanism.
So the trapping of these intermediates
would promote covalent DNA, protein crosslinks.
So I try with five-azacytidine,
which is a anti-cancer drug
and ciprofloxacin which is an antibiotic.
But we also look at aldehydes,
the formaldehydes knows to fix and trap protein and DNA.
When we look at the data for this,
we saw that five-aza as well as the other,
but I'm show you first five-aza,
induces this class of mutation.
So we can see here,
the leading strand and the lagging strand.
In the leading strand,
we saw a 44 fold increase in mutations
after treating the cells with a sub lethal dose
of five-azacytidine.
On the lagging strand, we saw a nine fold increase,
which is still very significant.
Here on the Y-axis, we have the rates of mutations.
The funny thing is that this difference between 44
and nine X was very expected,
because in previous work we've shown
that the lagging strand has a lower number.
Not because it happens less often,
but because of all their proteins
that are involved in the lagging process.
And I'll be happy to answer questions
if you have on that topic.
But we see that, we see a pattern
of five-aza induced mutations.
And from now on,
I'm only gonna show you data from the leading strand,
but the lagging strand also happens
with the leading strand being the strongest response.
When I look at formaldehyde and ciprofloxacin,
I also saw a very large increase.
If you look at this scale here, this is a log scale.
So 33 fold increase of mutations
after treating formaldehyde,
But with the antibiotics ciprofloxacin
we saw over 670 fold increase of mutations.
This was by far the highest mutation
that we have ever reported it.
So this, it is very interesting
because we need to more fully categorize the system
and know more about it and know
the pathways and how everything is happening.
So at this point I joined Salem State,
and that was awesome because I talked to my graduate advisor
and I said, I wanna continue doing this.
And I wanna look at FDA approved drugs.
And I wanna look at these drugs because
they are well studied, we know a lot about them.
I can buy a lot of them in bulk, in libraries.
And I wanna know, like,
are these drugs interfering with temple switch mutation?
So I joined Salem State and I had the best time.
I received a lot of support from the faculty,
from my supervisors, with the lab.
They say let's do this. I'm so excited.
And my first goal is to find motivated students.
So I need to find students,
because it's not really super exciting
to say, I'm starting from scratch.
So we're gonna have to read a lot.
We're gonna have to order materials.
We're gonna have to clean up lab.
We're gonna have to order, order many products
that we might not have it now.
At the same time, my colleague Dr. Brown
was so helpful in sharing his lab space and say,
I'm gonna make some space for you. Come here.
I'm gonna help you. It was great.
It was the best welcoming I ever received.
I got the students and I told them, look,
we're gonna have to work from the bottom.
And from now, from that time to now,
I had a 11 students going through my lab.
Out of those 11, eight graduated already.
And some told me I wanna help,
and I don't actually wanna do research.
I wanna learn how to prepare media.
I wanna learn how to write grants.
I wanna learn how to read these papers that you have to read
in order to propose experiment. So it was really nice.
So I was like, okay, find motivated students.
I got that. And you're gonna meet one of them today, Sydney.
Here Sydney, Becky, they just graduated from the lab.
I had that down.
Then I had some funding. I had department funding.
Very generously. I was very happy about it.
But I told my students, I wanna work over the summer too,
with just starting. I need a lot of work.
Let's work over the summer.
Myself, I knew that I could not afford
working over the summer in a lab unless I had funding.
So I told them, let's apply for funding over the summer.
And I am very happy to share that both of them got it each
in one summer, the Kathy Murphy summer research award,
which was essential into getting the work that they did.
So we started, we did all the controls.
We set up everything,
and all of this a few months have passed, right.
We set up the controls. We did all the testing.
We wrote all the proposals. Everything is good.
Now we are ready to do the research.
We are in lab, we are setting up, we have everything ready
and this is like March 2020.
I am so happy. Let's do it.
And, you know, what happen in March, 2020.
The pandemic started.
At that point,
we had to get back to the lab virtually and decide
we need to do something. We don't wanna stop the research.
How can we adapt?
How can we start working and get something done before,
going back to the lab. So it was a big challenge.
And I'm very, very happy
to introduce to you Sydney Addorisio,
that is going to be talking about
what she did during this time.
I'm happy to tell you that she did return to lab
once the lab was open, but she did a lot of work remote.
So Sydney did a lot of work and she'll tell you about it.
But she became a first author
in the paper we published this year.
She won the Kathy Murphy award.
She presented her work in multiple conferences,
not only Salem State,
but also at the Boston Bacteria Meeting, SACNAS,
Massachusetts Undergraduate Research Conference.
She's currently interviewing
for graduate school and she now works at Biogen.
So I'm gonna let you know now what she has done
and what is the next step into our project.
Sydney the floor is yours.
- Thank you, Dr. Laranjo.
So as Dr. Laranjo started to talk about.
You know, we got ready,
we were geared and ready to go in March of 2020,
and then COVID hit.
So as researchers, we said, you know,
we can't stop here. We need to adapt.
So first what we decided to do and what we knew we needed
to do was just read, read, read, read,
and learn all about these different drugs.
So this image is actually
an image of the drug library that Dr Laranjo purchased.
And these were the drugs that we could possibly choose
to investigate for this template switch mutation
that Dr. Laranjo just explained.
So we had to learn about all the different drugs
that we had available to us,
and what their mechanisms of actions were,
or how they worked.
What kind of concentrations
we wanted to possibly study them at.
And we had to take all of this information,
put it all together to formulate a hypothesis.
So I came up with a couple of different drugs
that I wanted to look at,
their concentration, studied their mechanisms of action.
And under the guidance of Dr. Laranjo,
I was able to take the hypothesis
and all of this information,
formulate it into a graduate level proposal
with specific aims.
And this is ultimately what became
my honors thesis as an honors graduate.
And on top of all of this,
we were also learning the lab techniques, the statistics,
about how to calculate mutation frequencies,
and also how to present those statistics.
You can do all the statistics in the world,
but if it doesn't make sense, what's the point.
But also just as a quick little plug,
there was a period of time that we couldn't be
in the lab at all and it was so far out
that we actually decided to take
advantage of the opportunity to not be in the lab.
To collaborate with Dr. Pena and her students
at Framingham State University.
And it was during this time that we actually wrote
a review article focusing on oxidative stress and bacteria.
I specifically was reading and learning
about microbial genome regulation under oxidative stress.
And this process was absolutely amazing.
I mean, I, as well as those other students
got to learn how to write scientific writing,
that is actually publishable.
Working with people, both within a lab, people I knew,
as well as people in a lab that I'd never seen before,
I'd never met them before.
And being able to give and receive feedback
from people I haven't met before.
And that was super important.
And knowing that we had to give feedback that was going
to present us as strong scientific writers
and present a really strong piece.
And I am happy to say that this article
has actually been accepted for publication
in the journal, Fine Focus as of the beginning
of this year.
And as the first author,
I also got the kind of inside scoop
on how the submission process works.
I was able to kind of be the liaison between
the editors and everybody else, fielding reviewer comments,
kind of seeing that sometimes daunting email
with the long list of comments
and having to deal with those as well.
So going back to being in the lab,
and once I was allowed to be back in the lab.
When I was looking at this large array of drugs,
I had to think about where do I wanna start?
There is a lot that goes into DNA replication,
a lot of proteins and components.
And I had known that Dr. Laranjo already had some work
out relating to SSBs as well as polymerase.
But where did I want to include my research?
What did I want to investigate?
And for me, that answer was topoisomerase.
So topoisomerase are proteins that actually relieve tension
in the DNA, and there are two types of topoisomerase.
Topoisomerase one relieves tension
by cutting one strand of the DNA,
whereas topoisomerase two relieves tension
by cutting both strands.
So the first drug I decided to look at from this drug
library is CPT-11.
And CPT-11 is a topoisomerase one inhibitor
that is also a chemotherapeutic.
So it kills cells that are dividing a lot
via this inhibition of topoisomerase one.
So it's slightly in more detailed mechanism of action
is that it traps the DNA
and induces protein-linked DNA breaks
that the cell cannot survive with, they're cytotoxic.
Now it wasn't just about picking a drug.
We also had to pick at what concentration
I wanted to study these drugs.
And I had picked 0.5 and 1.0 micromolar
as literature sited these concentrations
as being clinically relevant.
So we figured we would start there.
The second drug I decided to investigate
was doxorubicin hydrochloride,
which I will abbreviate from here on is DOXSO.
And this is a bacterial topoisomerase two inhibitor.
So cutting the two strands of the DNA.
And this particular drug causes cell death
because it stabilizes intermediates
from the complex between the topoisomerase two
and the DNA.
But overall, the main takeaway
is that it is stopping replication.
So for this drug,
I decided to investigate the concentrations
at 0.025 and 0.4 micromolar.
Now these two are quite far apart,
especially compared to CPT,
but it was found in the literature that 0.4
was a topoisomerase two poison concentration
without killing the cells that we were working with.
So just a reminder, Dr. Laranjo spoke
on these mutation assays already.
But just to remind you,
the work I did was specifically in E coli,
had nothing to do with yeast.
And that the lb media allows for all cells to grow,
so that provided me with a total count.
Whereas the LacMin media you see here
allowed me to count only the mutants to create a frequency.
So in doing this research,
there are three possible scenarios.
Now the first thing is that without any drug,
there is always going to be a baseline frequency
of this mutation type.
It is going to happen,
whether I expose the E coli to drug or not.
And if we find that the rate or the frequency,
my apologies of mutation is similar to the no drug,
or is very close to it.
This just means that we are gonna have more work to do
in the lab.
More questions need to be answered and investigated.
The second scenario is if we find that there is
a three times or greater increase
in the frequency of this mutation type.
And as Dr Laranjo mentioned,
there have been antibiotics and chemotherapeutics
that have been bound to express this particular scenario.
And if we found that the drugs I'm investigating did too.
This is a good thing, we're adding to the literature,
we're adding to that list.
Now the third scenario is if there was a three times
or greater decrease compared to
that baseline of mutations
that are going to occur, regardless.
Now this would be groundbreaking and super fascinating just
because there hasn't been drug or antibiotic
that has seen this pattern.
So this would be particularly groundbreaking.
So this is my data. We're gonna start with CPT-11.
So we see that baseline when there is no drug exposure
whatsoever, the mutations are happening.
And then once I expose the drug,
the E coli to 0.5 micromolar, we see a decrease,
and it is quite large.
We're seeing a 2.5 fold decrease.
However, that threefold threshold that I was talking about,
that is what Dr. Laranjo and I decided
we were going to stick to,
to determine whether a difference was significant.
And as you can see, 2.5 is not three.
And then when I expose the E coli to 1.0 micromolar
it decreased even a little bit more.
Now you can round numbers,
you can do all of that, but we were very firm with
our three time threshold and 2.8 while being very close
is not quite there yet.
So this means that we still have work to do
and there's still questions to be answered.
Now, looking at DOXSO, we again,
we see the baseline mutation.
It's going to happen regardless.
Then if I look first at 0.4, which is in the middle,
just so you know,
this is the larger concentration I'm showing first.
There was a decrease just like with CPT, but not as big.
We're seeing a 1.7 fold decrease.
But with 0.0, 0.025,
we still see a decrease slightly less
than with 0.4, 1.8 fold.
So just like with CPT-11 more work needs to be done.
There are more questions that need to be asked and answered.
So the conclusions from this study.
Drugs have different mechanisms of action.
And this is why I investigated
a couple of different concentrations,
because the concentration of a drug
that you use will affect one pathway.
So in a very abstract example, DOXSO at concentration one,
might yield mechanism one.
Whereas a separate concentration,
it might have a totally different mechanism of action.
So these different concentrations
are going to impact different pathways.
So a suggestion that we have following the study would be
to adjust the variables.
Obviously one at a time in isolation,
but changing the concentration perhaps.
Maybe if we increased that 1.0 CPT to 1.25,
maybe that drop in mutation frequency
would hit that three time threshold,
or perhaps we leave the concentrations the same,
but increase the exposure time of the drugs to E coli.
So overall, all of, out of all the drugs tested, not by me,
but including all of the research
that's been done out there, Dr. Laranjo.
These were the only two that showcased
a decrease in mutation frequency.
Keeping in mind,
it wasn't a significant in the sense that it didn't hit
that three time threshold, but there was potential there.
- And that was how we got to those two drugs
and then Sydney decided to graduate
and pass the Baton to the next one.
So I'm happy to tell you that we do have students
that will continue to work on them,
but they also have all the selection of drugs to keep going.
And all of the other ones that the other students
have tested had actually seen a slight increase or increase.
But as Sydney mentioned,
these are the only one that shows a potential decrease,
which that is gonna be very cool if this holds,
because it could be elucidating mechanisms
to prevent this class of mutation.
Because if the mutation is less,
we are preventing this mutation.
So we might understand more about the mechanisms
and how to prevent them.
- And it could just mean that the drugs I tested
simply had nothing to, their mechanism,
had nothing to do with topoisomerase
in relation to this mutation type.
- Absolutely.
But with that,
I do wanna acknowledge some people and places
that have given us support.
I wanna start with my lab members.
Here we have Sydney and Becky that just graduated,
but all of the past and current members
have been honestly a blessing.
I am so proud of all of you and I can't wait to keep seeing
you being amazing researchers.
I wanted to thank the collaborators,
people that I share lab with, my colleagues, my department,
my supportment system.
The biology department as a whole at Salem has been amazing.
I wanna thank the Dartmouth festival committee
for allowing us to share this good news.
And I wanna thank the Kathy Murphy award
for funding Sydney's research.
The biology research interest group,
it's a great new group that if you're not in it yet,
you should join.
Where we talk about research
and we talk about opportunities for research.
And one of them, big one, is by participating into SACNAS.
So I wanna leave my plugging here.
SACNAS stands for Society for Advancement of Chicanos
and Native American Science.
And we are opening a chapter at Salem,
as soon as we have enough students sign up for it.
But if you wanted to go to conferences, participate,
and you have any questions, feel free to reach me.
I wanna give a shout out to Dr. Lovett.
She was my graduate advisor,
and she's the one who taught me everything
from the beginning of this project.
And I wanna give a shout out to her and Dr. Warnick.
Dr. Warnick and Dr. Lovett
were my mentors as an undergraduate and a graduate career.
So I wanted to leave this here
because a lot of students are present.
And I wanna encourage you to find a mentor.
You might not find a mentor that agrees
with everything you wanna do.
You might not find a mentor that is perfect.
People make mistakes, mentors make mistakes,
but they make such a difference.
And because of these two mentors in my life,
my career changed completely.
They guided me, they taught me things I didn't know existed.
They told me all those secrets
that people don't talk about it,
on how to get internships, how to apply,
how to go out there.
And me coming from another country,
I didn't know the culture and I didn't know a lot of things
or not even how to start.
And these two professors have completely changed
the course of my life.
So if you haven't got a mentor yet,
and you are interested in doing research,
I wanted to encourage you to find one and to get as much
research experience as you are able to and enjoy this path
that science gives us.
And with that, I will take any questions.
- Thank you, Dr. Laranjo and Sydney Addorisio.
That was a wonderful talk. That was really, really cool.
And we've got lots of questions
so I'm gonna get straight to it.
Along those lines one of the questions is,
is there any way a student
could get involved with something like this?
- Absolutely. So I wanted to, if you,
if the person who asked the question is not involved yet,
I wanted to encourage you to join
the biology research interest group.
So this is a group of professors
who do research or are interested in research,
or are there to support research
that we meet a few Mondays throughout the semester.
And we talk about research opportunities,
and maybe we are not gonna have a spot
at that moment in our lab, but we know someone who might,
and you can get connections with other labs
and other opportunities as well.
So at Salem,
we do have the biology research interest group
and keep an eye out. Dr. Fischer sends an email
and he includes that on that email.
- Dr. Brown.
- Great. So I'll ask another question from the audience.
Are these mutations recognizable in humans easily?
Is it always a four based pair addition or mutation?
- That's a great question.
So in humans, in order, all of these mutations,
when they are first recognize,
the only way for us to see is by sequencing.
We need to sequence the whole DNA.
In E coli, this is very quick, it's $4 and it's quick.
We do that, we do it in an afternoon
and we have it by the other day in the morning.
Here is the catch.
In E coli by adding these four bases,
the only way the mutation happens that remove those bases
is by templates switch.
In yeast we had to do adjustments
because yeast has other mechanisms
that remove those four base pairs
that is not through temple switch.
So that was a challenge.
So that's an excellent question.
So these reporters,
we only have it in bacteria and in yeast,
but in order to recognize this mutation in any organism,
we can only do by sequencing the whole, the whole genome.
- Another question from the audience.
The person says
I think you're work is incredibly fascinating,
but I'm wondering,
do you think natural selection or evolution
has anything to do with the way the DNA mutates?
- Yes. (laughs)
Yes, I do think so. Especially because we know that,
we don't understand the whole mechanism,
but we know that anything that will affect the replication
for will increase the chances of mutations.
So we might not even be talking about hairpin specifically,
but other parts of the DNA can be affected
that will cause replication fork to be blocked.
And then coincidentally,
if that happens near these palindrome mutations
who have more template switch mutation, so absolutely.
And that is why it makes us so difficult to study,
'cause we don't know everything
and we cannot control any external environment yet, right.
So, excellent question.
- Can I add to that a little bit?
- [Laura] Yeah.
- Yeah. So that diagram too, that we showed a couple times
showing like the topoisomerases, the SSBs, the polymerase.
All of those are possible targets that could possibly lead
to this template switch mutagenesis.
So anything that goes kind of array
in any of those regions might play a role.
- [Laura] Absolutely.
- Yeah.
So I have a quick question about
kind of directed to Sydney, I guess.
How, what ideas do you have for how you would go about
trying to figure out whether the mechanism
of the effects that you saw for CPT-11 and the DOXSO,
were actually related to interfering with the topoisomerase
or if there's some other mechanism?
Like how would you, how would you address that?
- Oh, I love this question. Okay.
So, first I just wanna say too, with CPT, CPT is a pro drug.
So it requires a certain metabolite that will allow for it
to be converted to a more active form
that, than it is without that metabolite.
So that would be kind of my first step there is just to do
that first and see if the mutation frequency changes.
And then after that, and this would apply
for both DOXSO and CPT.
If there was a way that I could possibly track
the interactions with the drug
and the topoisomerase one or two,
whether that be through fluorescence or, you know,
some possible way that I could track that,
that would be ideal.
Because as I mentioned different, like DOXSO specifically,
has at least four different mechanisms of action, right.
So it's important as you say,
to make sure that the concentration I am using is inducing
that one specific mechanism of action so that somebody could
say it is that one mechanism of action that is causing it
and maybe not, you know, one of the other three.
- Yes. So in her specific aims,
that was exactly aim three.
So we first we'll test it and then once we see an effect,
we're gonna look at all the mechanisms
and modify specific things on those mechanisms
and see if this mechanism is affected,
we should see a response.
So that's an excellent question.
- Okay.
I think this is another question directed at, for Sydney.
Was it intimidating
to present your research at different conventions?
- Absolutely. (laughs)
At first, absolutely.
And that's okay.
You know, I think especially the first time, you know,
it is very nerve wracking.
But and I'm gonna plug SACNAS again in here a little bit.
SACNAS was my first conference that I presented at.
And that was a great format,
because even though it was virtual,
it was very collaborative. It was very informative for me.
And a lot of the people that gave me feedback,
it was all constructive criticism.
So there was nothing, you know, I didn't feel like judged.
I didn't feel like I was ever wrong.
You know, it was people that really just wanted to see
what came of my research.
They really wanted me to grow as a researcher.
And one of the people that talked to me
was actually a cardiologist.
And he approached me and he said,
I have no idea what any of this means.
I have zero experience with it.
However, I do know what topoisomerase is.
And have you thought about this?
You know, so it's always nice
to present at conferences and get some other perspectives.
- And Sydney said yes to all of them.
I kept sending it to her. She said yes to all of them.
And this one in particular,
one thing that she didn't know is that my previous advisor,
the ones that advised me in this project
were going to be there.
- Wait what? (all laughs)
- So they report great things.
So I was very proud of her.
And it's exactly what she said.
I told her like, we will never know everything
and that's okay.
And even now you might ask a question that I don't know
the answer and that's okay.
But that exchange of ideas is,
might be the solution to a problem
that we hadn't even considered.
So if you are, if you wanna present at a conference,
I'm gonna plug in the research interest group again.
You can come and practice with us.
We can take a look at your poster
or we can look at your presentation.
These are the things, we are here to help you.
And--
- Practice a talk with them too. It's great.
- Yeah, so you can always,
you can always count on us to help.
Because think about it like this.
It's a lot of people with PhD that if you go to a job
and you hire them as a consultant, it's a lot of money.
And we all wanna do it because we wanna help.
So you have all of this free resource to you.
So if you wanna present, go for it. It's okay.
You are going to get practice
and you're gonna make mistakes and it's good too.
- Absolutely.
I feel so much stronger in my presentation skills.
Now that I've done, you know, four or five of them.
- Great.
I bet you do know the answer to this question
since you sort of teed it up earlier.
But can you elaborate a little bit on the,
the strand bias mechanism and what's going on there?
- Yeah, absolutely.
So we were looking into it and everybody
who was studying this saw this exactly effect.
It was called the leading strand bias effect.
So they say are there more mutations happening
on the leading strand than on the lagging strand.
So my work, this was my first scientific publication.
What we did is that we looked at things that were present
in the lagging strand and actually preventing mutations
from happening or destroying them
before we were able to catch it.
So for example, there are exonucleases.
Exonucleases are able to degrade that three prime end
of the DNA that I told you that has to fold
in order to make the mutation.
And there are a lot more of exonucleases
in the lagging strand than on the leading strength.
So if the exonucleases come in the lagging and destroy that,
now the polymerase has a chance to go back
and go back to the original template.
The other things that besides having more exonucleases
on the lagging strand.
The lagging strand has single strand binding proteins,
which recruit more exonucleases.
So the leading strand, yeah.
Some exonucleases do go there and do help by degrading
and not letting the mutation happen.
But on the lagging strand, we have tons of exonucleases,
plus a strong wrong pull to the ones
that would go to the leading strand.
So it's just that the lagging strand is better
at correcting the mutations.
It's not better at preventing them.
So that's when we eliminated the exonucleases
and when we mutated the single-strand binding protein,
we saw that the disease trend bio was eliminated.
So if we do a playing fair field,
we have the same rate of mutation
in both leading and lagging.
- Cool. Thank you.
- Another sort of related questions
is it seems that the formation of the palindrome,
is it causative or is it an effect of
the polymerases falling off?
Or not falling off, but just stopping I guess.
But it may, there's single stranded binding proteins,
aren't they supposed to prevent the formation of all of this
and what's going on with palindromes?
Why are they forming in the first place?
- Yeah, that's a great question.
So they have physics experts,
they're actually calculating the pole
to make it a hairpin versus a cruciform.
And without going too much into a physics,
there is some force that because of the region
is a quasi palindromic region,
because it's or a palindromic region,
there is some force to make those structures.
So it is first, it's not that these structures
make the palindrome, is that the palindrome,
because of the sequence,
because of how the basis are the order that they are,
it makes this specific force that will cause this effect.
So single strand binding protein prevents
a lot of it from happening.
And that's why this mutation is not so common
as other mutations.
And when we've removed the single strand binding protein,
then we see very, very, very high rate.
So you are absolutely right.
So the palindrome is the,
is what cause, what forces,
but we have a bunch of stuff in the cell,
like single and binding protein, trying to prevent that.
But at some, at some portion,
it's not able to prevent everything.
- Okay. I see it's three o'clock.
And so we're supposed to try to stay on, on time here.
Thank you again Dr. Laranjo
and Sydney Addorisio. That was wonderful.
Really did a great job.
And I'm gonna pass it over to my,
our colleague, Dr. Ryan Fisher, for some closing words.
- [Laura] Thank you.
- Thank you, Dr. Scottgale.
And thank you, especially to Sydney and Dr. Laranjo
for such a interesting talk about the role of DNA
in all of our lives.
Just a few announcements before we close for,
close this 43rd festival, and they include these.
The recordings of all these webinars will be available
in about seven to 10 days.
So keep an eye on your emails for where you might find them.
They will be digitally lodged in our library.
In addition, if you're a student
and thinking about your assignment that you might
be doing for this webinar today.
The attendance sheets will be passed
on to your professors sometime next week,
that provide evidence that you were here.
I'd like to thank all our alumni that have been present.
So Sydney, who's a brand new alumni.
Thank you very much for joining us Sydney.
I can quite imagine seeing you in 10 years,
just like Dr. Pelatio this morning,
coming to speak to us as Dr. Adrishio.
Also for Peter Shearstone who dropped by and joined
and spent the day with us today after graduating
all those years ago.
Also to thank the funders.
The two primary funders of our annual event
is the Charles Albert Reid Trust,
which is administered by the Salem City Council.
And for this year in the next four years,
Peter's company Thermo Fisher Scientific.
Thank you very much.
Before i forget we've had a lot of fun behind the scenes
running these webinars,
and we are pretending that these are flights,
air travel flights.
And so thank you to Gail, who's our pilot today,
and to Derek Barr who couldn't be with us today.
He flew all the other webinars earlier in the week.
Finally, just to thank all my colleagues
in the Darwin festival team,
as well as all my fellow biologists here at Salem State.
Thank you very much.
We will be back next year with more Darwin festival talks
and I will quickly pass it back to Sydney and Dr. Laranjo,
just to say goodbye to everybody.
- Bye everyone. Thank you so much.
- [Sydney] Bye, thank you so much.