# Talk Birdy To Me

Monitoring bird populations is extremely important to gain an understanding of the overall ecosystem. Additionally, many species distributions are currently moving north, which is a sign of climate change. In order to measure the bird populations, trained ornithologists use the “point count” methodology, in which the observer stands in an area for a given amount of time and tallies the birds that can be heard or seen. The “point count” methodology has a few limitations. Since bird identification is an imprecise science, two very experienced ornithologists can survey the same area yet identify different birds. To account for this, most surveys increase their sample size. In order to reach the high number of survey areas in the given amount of time, ornithologists must start to cut corners, such as finding survey areas that are close to roads. However, birds are very sensitive to noise pollution, so the roadside surveys don’t accurately represent a population within the inner forest.

A new methodology to study bird populations and reduce the limitations associated with “point count” includes the usage of drones. Many ecologists use drones in their research, since they provide a unique and developing technique for studying wildlife. By using drones, we can save and refer back to an audio recording, thereby allowing ornithologists to agree on the birds present. Additionally, drones are fast and easy to maneuver, and will be able to travel to more remote survey areas.

Dr. Andy Wilson recently determined that counting birds from audio recorders on drones yields similar results to the “point count” method (Wilson 2017). Due to these benefits, drones may have a future in bird population monitoring. However, this methodology is still very new, and Dr. Wilson is skeptical about the impacts of drone usage on bird behavior. Because birds are sensitive to noise pollution, could noisy drones affect bird singing behavior? If so, there are ethical issues with using this methodology. Therefore, Dr. Wilson’s lab is currently testing the effects of drones on bird song. Check out video footage from our drone!

We travel to State Game Lands 249, where we have a permit to conduct research in the surrounding fields. We placed a 200m grid on the land, so that all survey points are equally spaced out (Figure 1). At each survey point, we create a 50m square array of SM3 wildlife audio recorders (Figure 2). After placing and turning on the SM3 recorders, we leave the area at least 10 minutes before the experimental data collection starts. This allows the birds to settle and resume normal activity. Next, we allow the recorders to continue gathering audio data for 4 minutes. This period is used as the “before drone” data. After this period, we fly the DJI Mavic Pro drone to the middle of the survey point at 48m high. The drone hovers at this location for 3 minutes. A portable audio recorder is hung from the drone by 8m of fishing line, placing the recorder at 40m from the ground. After the drone returns from the hovering point, we allow the recorders to continue for four minutes, as the “after” period. In this way, we have data from four SM3 recorders before, during, and after the drone flight, as well as data from the recorder suspended from the drone. The recordings are taken back to the lab for further analysis.

Figure 2. Google Imagery for survey point 17. The drone hovers at the yellow point and the four recorders are placed at the surrounding black points.

Figure 1. Thirty-six survey points in the State Game Lands 249.

Each individual bird has its own distinct call pattern that can be used by a skilled birder or new lab workers to identify the species and location of the bird. After clipping our audio files to the 11-minute segment of interest, we use two high pass filters to eliminate as much of the drone’s noise without sacrificing the birdcalls. The final step in the editing process includes clipping every other minute for analysis. This allows us to identify which birds are singing throughout the experiment. In this way, we can compare the number of birds singing in the before (1st and 3rd minute), during (5th and 7th minute), and after (9th and 11th minute) segments. Raven Pro version 1.5 allows us to simultaneously look at the four SM3 recording spectrograms for a single minute and identify the birds calling (Figure 3). However, there can be many individuals of the same species singing at the same time. Plus, birds may move further away from the drone throughout the experiment, which would be an important result. Therefore, we seek to determine each bird’s exact location. In order for a bird to be localized, the song must be identified on at least three of the four recordings. By using the correlator tool in Raven, we can determine the time of arrival differences to millisecond, in order to determine the location of the bird (Figure 4).

Figure 4. A correlation completed in Raven, comparing Field Sparrow song at recorder A and B. The correlation coefficient is 0.03 (the blue number in the middle). That number is entered into Soundfinder.

Figure 3. Spectrograms from the four recorders in Raven. For example, this is Point 10 Minute 0. There is a Field Sparrow at 2.5-5 seconds.

Now that we have three separate correlations, we can enter the data into a downloadable program called Soundfinder. The only information that we need to supply is the temperature of the air when we took the recordings, the three correlation numbers from Raven, and the GPS coordinates of the ground-based recording array. Soundfinder is then able to localize the bird to within 3 meters of the birds’ approximate locations. The UTM (universal transverse Mercator) coordinates provided by Soundfinder can be plugged into Google imagery to double-check the birds’ locations. For example, you would know that you had incorrectly correlated a bird’s call if a woodland bird, such as the White-eyed vireo, was localized in the middle of a field. Therefore, there is some trial by error involved and so it takes a practiced eye to properly correlate a single birdcall.

Raven and Soundfinder allow us to track individual birds throughout the 11-minute long experiment, and determine if the birds are moving away from the drone or if they stop singing. We hypothesize that the drone will not have an effect of the bird behavior, but further analysis of our data are needed to determine our results. If there is no effect, then this methodology can be used in the future. An ornithologist would only need to use the drone and the hanging recorder to gather audio data, thereby quickening and simplifying the bird population monitoring process.

Works Cited

Wilson, A.M., Barr, J. and M. Zagorski (2017). The feasibility of counting songbirds using unmanned aerial vehicles. American Ornithological Society, 134, 350-362.

# BioacTiVity

Here’s Alex making titanium ascorbate!

# Out with the Mold, in with the New

## In a fungus, cell division meets mRNA transport

Why would anyone choose a common bread mold fungus to study the way cells work? –  in particular, to unravel the ways that living cells beget new cells by the process known as cell division?  In the James laboratory, we seek to understand “why normal cells get it right every time they divide, and why cancer cells get it wrong every time”.  A worst-case scenario, played out again and again in cancer, occurs when a normal cell “forgets” how to stop dividing, and begins to proliferate incessantly and relentlessly.  We address this fundamental problem in the fungus Aspergillus nidulans because basic features of cell division evolved early on, and proved so effective in promoting survival that they’ve been conserved with little or no change in increasingly complex creatures, including us.  In fact, fungi and humans are similar enough that we could literally swap many genes without harm to either organism, like trading sparkplugs between a Volkswagen and a Ferrari.

This summer the excitement is palpable as we close in on two genes, discovered by us, that harbor previously unreported roles in cell division.  Mutant cells lacking these genes let slip the brakes on cell division, allowing cells to divide more readily than normal.  By extension, this means that the normal versions of these two genes must act as restraints to keep cell division from getting out of control.

One of the two new cell division genes, called snxA, is a conserved gene in many other species, where it is best known for its role in escorting messenger RNAs (mRNAs) out of the confines of the nucleus, where mRNAs are made, and into the cytoplasm where these RNA messages are translated into proteins.  In this process, snxAparticipates with several other proteins that together govern the export of mRNAs out of the nucleus.  To better understand snxA activity, it is important to know how snxA works with these other proteins to influence mRNA export and cell division.  To this end, Julia Palmucci (’18) and Elliot Rodriguez (’18) are studying some of these other proteins, as they describe below.

The other new cell division gene, called GYF, is also conserved across the evolutionary spectrum from fungi to humans, but comparatively little is known about its function.  This summer, Morgan Brown (’19) is completing an initial study of this gene, as she explains below.

### Elliot Rodriguez (’18)

As mentioned above, our lab studies cell cycle control, specifically proteins involved in the shuttling of messenger RNA (mRNA) from the nucleus into the cytoplasm. Along with the star protein of our lab, snxA, we also study the THO/TREX protein complex. THO/TREX is an 8-protein complex responsible for binding nascent mRNA and escorting it through nuclear pores with the assistance of snxA. The THO/TREX complex is the focus of my research. Our lab has worked to characterize some of the subunits that comprise the THO/TREX complex. By characterizing each subunit of the THO/TREX complex, it will provide us with a better understanding of the role each subunit has in the complex as well as how the complex interacts with other proteins such as snxA. My research in particular involves characterizing THO complex subunit 5, or thoc5.

Thoc5 is evolutionarily conserved in higher eukaryotes, however the exact roles of Thoc5 in transcription and mRNA export are still unclear. In adult mice, thoc5 is essential in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis. In Drosophila, thoc5 mutants are viable but have spermatogenesis defects. Thoc5 is present in fungi and mammals but is absent in the popular model organism budding yeast, Saccharomyces cerevisiae. Studies of thoc5 in fungal model systems are limited, which makes Aspergillus an especially relevant model system for studying thoc5. The main goals of my research are to complete the initial characterization of thoc5 mutants as well as to investigate potential thoc5 interactions with the snxA shuttling mRNA-binding protein that has been the long-term focus of our lab’s efforts.

I am also assisting Julia with her work with another THO/TREX subunit, thoc6. Our lab has shown that the absence of thoc6 causes a mislocalization of snxA. Normally snxA remains in the nucleus, but the absence of thoc6 delocalizes snxA to the cytoplasm through an unknown mechanism. I am adding a green fluorescent protein tag to thoc6. This will allow us to visualize thoc6 in Aspergillus using fluorescence microscopy. By tagging thoc6 and other proteins such as snxA, we hope to better understand the interactions between these proteins as well as the unknown mechanism responsible for the mutant phenotype.

### Julia Palmucci (’18)

This has been my first week as a summer research student in the James Lab.  The earlier part of my summer was spent doing research of a much different sort in the Peruvian Amazon with Dr. Trillo’s Tropical Terrestrial Biology course.  The class brought us to the one of the most biodiverse areas in the world where we identified over 100 species of birds, observed numerous mammals—including nine species of monkeys, and learned how to characterize insects and amphibians. We were also given the opportunity to use the pristine natural resources of Cocha Cashu Biological Station in Manú National Park for independently designed research studies.  Utilizing the area’s untouched mature forest (500-700 years old) and the more successional forest (about 150 years old) formed as a result of the meanderings of the nearby Rio Manú. I compared soil composition and insect diversity across differently aged forests.

The research I am undertaking for the remainder of this summer involves the interaction between snxA and the THO/TREX complex.  Specifically, I am studying a subunit of the THO/TREX complex—THOC6. This subunit is one of seven THO components in animals, and we believe Aspergillus nidulans may be the least complex organism that contains THOC6. Other common model eukaryotes used in molecular genetics research such as the budding yeast Saccharomyces cervisiae lacks thoc6.  Thoc6 is a fascinating protein, composed of multiple WD40-repeats that confer a β-propeller structure important in mediating its interactions with other proteins. A β-propeller protein (seen below) is composed of 5 to 8 “propeller blades” each with 4 to 8 β-pleated sheets.  A common misconception is

Basic structure of the aptly named β-propeller protein (Fulhop and Jones, 1999)

that each WD40 repeat translates directly to one blade, but this is not the case. Instead, each repeat results in a majority of one blade and some fraction of the next, allowing for a gene with numerous WD40-repeats to have fewer blades. Additionally, the approximately 40 amino acid WD40 repeats are notoriously diverse in sequence between their starting ‘GH’ and ending ‘WD,’ and even these namesake characteristics can vary as well.

Previously, it was unclear if the gene we were studying, AN1056, is actually the ortholog of thoc6 in higher organisms. Much of the research on THOC6 focuses on the pathology of thoc6 mutations in humans. For example, several thoc6 mutations cause intellectual disability. In addition, thoc6 defects confer stress sensitivity in the fruit fly Drosophila melanogaster. This past year, however, our lab found that a deletion of AN1056 results in a cold-sensitive phenotype and delocalization of snxA into the cytoplasm, indicating that this protein may be associated with snxA and may be involved in cell cycle regulation. This summer we have begun accumulating answers to this puzzle in hopes to determine more conclusively if AN1056 is indeed THOC6, and how this protein interacts with snxA.  Using a new program that more accurately predicts WD40, we have discovered tantalizing commonalities between AN1056 and the sequences of thoc6 in flies and humans, including the same number of similarly arranged WD40-repeats.

My goal for the remainder of the summer is to determine the function of THOC6 and its interaction with snxA using fluorescence microscopy.  I will use strains carrying several fluorescent tags, including green-fluorescent snxA and red-fluorescent thoc6 to monitor defects in snxA localization during the cell cycle in wild type and Δthoc6 mutants using the Nikon Ti-U inverted epifluorescence microscope.  These tagged strains will be helpful for determining the role of THOC6 and clarify the reason for the defective phenotype exhibited by the Δthoc6 mutants.

### Morgan Brown (’19)

Morgan (left) and Dr. James (right)

While my labmates have been focusing on proteins that function alongside snxA, my research looks to characterize snxA itself. Much of our previous work has involved mutated strains of A. nidulans that suppress defects in regulators of the CDK1 mitotic induction pathway, which controls whether or not a cell can enter mitosis. Other phenotypes of these strains include extreme cold-sensitivity and decreased levels of mRNA, and we’ve been referring to these as snxA1 and snxA2, as though there were mutations within the snxA gene of these strains that caused the defects. However, we were stunned and flabbergasted to discover that, rather than a simple point mutation, the disruption of the snxA locus actually resulted from a reciprocal translocation! This means that the arm of Chromosome II where snxA is located, swapped with an arm of Chromosome I. Consequently, the snxA locus was broken within the first intron, separating the promoter region and first exon from most of the coding region. At the same time, the Chromosome I breakpoint occurred within the fourth exon (out of five) in a novel unstudied gene, AN6228. We know only that this gene contains a GYF domain, which is known to mediate binding to proline-rich motifs in certain other proteins. Now that we find  that the “snxA mutant” is actually two mutations in unrelated genes, I am recreating the GYF gene truncation and comparing it to a deletion of snxA to determine how each affects cell cycle control. This will allow us to tease out the contribution of each to the overall mutant phenotype.

A diagram of the reciprocal translocation occurring between snxA and GYF.

To determine the contribution of each disrupted gene, snxA and GYF, to cell cycle regulation, I first deleted GYF from a wild-type background. It grew perfectly fine at cold temperatures, while strains in which snxA was deleted couldn’t grow at all, so our cold-sensitive phenotype was clearly a result of the snxA truncation. A deletion of snxA was shown to rescue cell cycle defects, leading us to believe that snxA (and not GYF) was responsible for cell cycle rescue. To our lasting surprise, a deletion of GYF also suppressed these defects. This suggests that the reciprocal translocation serendipitously occurred within two unrelated genes that both happen to play a role in regulating the G2-M transition of the cell cycle. It is important to note that a gene deletion completely eliminates its function, but since the translocation breaks GYF in the fourth exon, this truncated allele could retain all of its function, partial function, or no function. So, in order to resolve this question, I have engineered the truncated version of GYF into a wild-type strain, and am now testing the phenotype.

Enter a caption

Currently I am performing genetic crosses to generate strains carrying both the GYF deletion or truncation with mutations in G2-M cell cycle regulators. Furthermore, today I am tagging GYF with Green Fluorescent Protein (GFP) and other fluorescent tags in order to characterize the location and function of this novel gene. It’s been busy over here in the mold lab, but it’s been exciting as well!

An image of the lovely protoplasts that we will be injecting our mutations into. Aren’t they adorable??

# Music, Media, and Aggression

Media exposure is ever-present in our society – its effects have been studied in relation to a number of human behaviors. In most research, media exposure is often operationalized as how often individuals view/partake in TV Shows, Movies, and Video Games. While this is not a bad measure of media exposure, I can’t help but think that there is a gaping hole in it – namely exposure to auditory media. That’s right, music. Music is a generally understudied aspect of media exposure in the realm of psychology. This summer, music is being studied (by Dr. Chris Barlett and Doug Kowalewski) in regards to its relationship with aggressive behavior. In addition, several other studies are being undertaken to further enrich the media exposure literature – in terms of both adding music and the entire paradigm itself.

Music has been studied in psychology rather minimally – especially in comparison to other types of media. In the past, social psychologists have primarily studied music lyrics and their effect on pro-social or aggressive behaviors/attitudes. Knowing this, the first project being undertaken this summer is a meta-analysis of all past studies that have looked at manipulating lyrical content and its effect on aggressive behavior. To do this, I am searching the library’s online databases – as well as Google Scholar – to find all semi-relevant studies. Then I’m going through each individual one, coding the songs that were used, and locating the results. Coding the songs usually takes the longest – I’m essentially inserting standardized musical information for each song (i.e. – the song “It’s My Life” by Bon Jovi is in English, has 234 words in its lyrics, 1 aggressive word, 3 aggressive phrases, is at 120 beats per minute (tempo), is 3:44 long, has 4 (out of a scale of 5) bass, has traditional instrumentation (not electronic), has a simple harmony, is sung by a male, is performed by a band of 5 members, and is Rock). I’m coding each song similarly (and with even more variables) so that at the end of the study we can see what other aspects of the music, other than lyrical content, have an effect on aggressive behavior. We’re just at the beginning of this project (it’s our big, overarching one), so we’re not sure how it’s going to work out!

In addition to the meta-analysis, I’m also combing through correlational data that we collected on Amazon Mechanical Turk (an international survey site that provides data cheaply and quickly) that has implications for both media exposure and, more specifically, music exposure. We’ve already submitted for publication a study, using this data, looking at the relationship between violent media exposure and cyberbullying behavior. In addition, I’m also coding the songs that participants listed as their three favorites in a manner similar to the way I’m doing the meta-analysis songs. The goal is to see if there is any relationship between particular elements of the song and the participants’ self-reported aggressive attitudes and behaviors. We’re still going through this project too, but the data looks pretty good and we’re hopeful to find results that we can look at along with the meta-analysis data.

Speaking of cyberbullying, we’re also very close to submitting for publication a paper regarding the Barlett-Gentile Cyberbullying Model (BGCM). The longitudinal research, taken over ten waves, provides insights into the learning mechanisms that underlay the BGCM. Specifically, our results show that as individuals cyberbully, they learn that they are relatively anonymous when they do so, and that their physical strength doesn’t matter. This learning leads to positive attitudes regarding cyberbullying behavior, which in turn leads to increased cyberbullying behavior. Our results also show that this increased cyberbullying behavior predicts even higher feelings of anonymity and the belief that physical strength doesn’t matter online. This final finding is a very important addition to the BGCM, as it shows that the model continues on over time.

Overall, psychology research involves a lot of looking at past work, finding how you can contribute to the literature, and designing studies that will allow you to do so. It also helps to be interested in the topic you’re researching (I for one really enjoy the music and media aspects of our work). So far, the experience of researching music, media, and aggression has been thoroughly enjoyable and rewarding.

I better get back to it – there’s plenty of more music to listen to!

# The Wrath of Con(ceicao)

Research in the field of mathematics does not have fancy labs or fancy procedures like other fields. Unlike how the name of this post suggests, math research is neither stressful nor aggravating. While mathematics research does sometimes involve staring at equations erratically scrawled on a chalkboard, our tools are not limited to mundane methods like chalkboards and paper. Research in mathematics is a puzzle. It is finding trends and proving them. It is making relations between an unknown solution and known theorems to further knowledge of both the known and unknown.

This summer, Dr. Conceicao is working with two math majors: Sam VanFossen and Rachael Kelly, whom you can see busy at work in the pictures at the bottom of the page. Our research is based off of the Markov equation $x^2+y^2+z^2 = xyz$. The solution $(x, y, z)$ to the equation is known as a triple. All integer solutions to Markov’s equation have been known for over 100 years using a simple recursive formula that allows a single solution to “branch” to other solutions. We, however, took a different approach to this equation. What if $x, y$, and $z$ were polynomials, not integers? More specifically, what if they were monic polynomials? A monic polynomial is a polynomial whose leading coefficient is 1. For example, $5t^2 + 3t + 2$ and $t^2 + 3t + 2$ are both polynomials but only the latter is monic.

Now, in order for polynomials to satisfy Markov’s equation, we must work in what is called a finite field. The “standard” field has infinite numbers. 1, 2, 3, 4… and so on. We can count upwards indefinitely. In finite fields, however, there are a set, or finite, number of elements. In our research, we work with primes of 1 mod 4. The “mod” of a number is the remainder of that number. Thus, 5 mod 4 is equivalent to 1 mod 4. We require our primes to be 1 mod 4 because -1 is a quadratic residue of numbers 1 mod 4. That is to say that i exists in the finite fields of primes that are 1 mod 4. i is the number such that $i^2 = -1$. In the standard field, i is known as an imaginary number because it does not exist; however, it is possible for i to exist in finite fields. In mod 5, -1 is equivalent to 4, since both -1 and 4 are divisible by 5 when 1 is added to them, and, as we know, $4 = 2^2$. Thus, in mod 5, 2 = i. Further, $3^2 = 9 = 4$ in mod 5. Therefore, both 3 and 2 are i in mod 5. exists in all finite fields created by numbers that are equivalent to 1 mod 4 . For example, 5, 13, 29, and 37 are primes that are equivalent to 1 mod 4.

Using this information and returning to Markov’s equation, we can prove that all monic solutions stem from a triple in the form $(f, f + \beta, f^2 + \beta f - 2)$ where $\beta = 2i$ and f is some monic polynomial. Thus, we have an infinite number of primes with an infinite number of trees with an infinite number of entries on each tree. And we aren’t even considering nonmonic solutions. Now that we have effectively found all solutions, what can we say about them? What other relationships do they have? A generating function is able to generate polynomials down a set “branch” of the tree. Using this function, we are able to have an explicit formula for the polynomials.

A classic conjecture that we would like to investigate is about the uniqueness of Markov polynomials. Does every polynomial appear on each tree as the maximum in a triple exactly once? Another question that we would like to investigate is if all Markov polynomials are reducible (or factorable). We are trying to understand the subfamily of Markov polynomials, $F_n$, defined by the generating function. Further, we can use the generating function to determine the values of n for which $F_n$ has roots modulo a prime. For example, i will always be a root of $F_3$ and $F_n$ if $n=ko+3$, for some integer o and all integers k. The value of o is known as its order. Rewriting the generating function as a Pell equation is useful in finding the order of certain roots. As such, we can create parallels between the Pell numbers, divisibility and Markov polynomials.  For example, in the finite field with p elements the order of β, or 2i, is the index of the first Pell number divisible by p. Similarly, the order of i for a specific prime p is found using Fibonacci numbers.

To further our research and save hundreds of hours, we use a program called Sage (when/if it works) to perform lengthy (to say the least) computations for us. We then enter the data on Excel spreadsheets, where we look for patterns in the data. When we find patterns, we then look for ways to prove that these patterns occur in every possible case or ways to generalize these results. Researching this topic has been a rewarding and illuminating experience, and we look forward to discovering more about these fascinating polynomials!

# NanoParticle Lab

AND THE NOMINEES ARE:

RICH GAWEL

Despite their incredibly small size, nanoparticles have already been identified to serve in a diverse range of applications, with further uses continually being investigated.  One of the most heavily researched applications of nanoparticles is their potential use in targeted pharmaceuticals. For example, the small size of gold nanoparticles allows them to penetrate through porous vasculature at specific sites within the body, such that they can selectively accumulate within that site. If we coat nanoparticles with certain drug molecules, we can potentially target that drug to the area of interest rather than allowing it to concentrate in other locations throughout the body where it can produce harmful side effects.

New cancer research is exploring the attachment of chemotherapeutic drugs to the surface of gold nanoparticles in order to apply EPR to selectively target the drugs to the tumor site.

Despite being very small and sounding very complicated, [some types of] gold nanoparticles are relatively simple to synthesize in large volumes. This is important when producing large quantities of nanoparticles for industrial applications.

A little bit of citrate into a little bit of gold gives us a big batch of bright red gold nanoparticles! (Yes…gold nanoparticles are red, not gold)

This brings us to my current work. We are currently exploring the potential of using gold nanoparticles as carriers for antidepressants, specifically the Selective Serotonin Reuptake Inhibitor (SSRI) fluoxetine (Prozac ®).

Fluoxetine

We have been examining two methods by which we can prepare a fluoxetine-loaded polymer that can attach to the surface of our gold nanoparticles. Most nanoparticles cannot exist on their own; they need some type of molecular coating – a surfactant – to provide stability. In the nanoparticles with which I am working, negatively charged citrate provides charge screening against other negatively charged particles. While it is possible to attach molecules individually to the nanoparticle, it would involve replacing the citrate stabilizer with the drug, thus reducing the charge stability of these particles, causing them to aggregate (which is BAD!) Therefore, we are exploring methods to incorporate fluoxetine into a polymer that we attach to the gold nanoparticle. The polymer acts as a coating to prevent the particles from colliding with each other (which is GOOD!)

Just because we work in an analytical chemistry lab, it doesn’t mean we can’t do some synthetic organic chemistry too!

Mix some polymer with some nanoparticles à get some polymer coated nanoparticles.

As nanotechnology is a relatively new concept in the context of pharmaceutical delivery, a substantial amount of work is being performed in order to quantify the amount of drug molecules attached to each nanoparticle. Unfortunately, we can’t simply grab a nanoparticle and count the number of drug molecules, so we have to employ some other methods. Now that we have synthesized our fluoxetine-linked gold nanoparticles, the bulk of our work involves quantifying the amount of fluoxetine per nanoparticle. One of these methods involves the bromination of fluoxetine and methyl orange dye to perform spectrophotometric measurements.

When there is more fluoxetine, more Br2 is consumed; therefore there is less Br2 available to inactivate methyl orange, such that the reaction will have a greater absorption of light.

One of the concerns about nanomaterials and many pharmaceuticals is their persistence in the aquatic environment once released via wastewater. For this reason, we plan to assess our fluoxetine-nanoparticle conjugates on a variety of aquatic model organisms, such as frog tadpoles.

APHRA MURRAY:

I am new to the lab this summer, so I’m looking to build off a previous lab member’s project. Celina’s work looked to quantify the number of polymers, specifically polystyrene sulfonate (PSS) that were wrapping around each gold nanoparticle. The work that she did involved developing and optimizing a dialysis stage to “clean” the nanoparticles in order to remove any excess polymers in solution. Over the course of these eight weeks, my project, while still in it’s initial stages, will look to explore the effects of changing the identity of the salt during the coating stage.

I have looked at how changing between the salts lithium, potassium and sodium chloride respectively changes either the “thickness” of the polymer coating or even the number of polymers associated with the nanoparticles. The first trial has just been completed and the data is currently being processed! The results suggest that there is a difference in the number of polymers per nanoparticle, but more trials will have to be conducted in order to determine whether this is statistically significant.

Some of the difficulties that I have run in to are both the pace of the dialysis and the difficulties associated with making large batches of monodisperse particles. The dialysis process, while efficient, lasts up to a full day meaning that it’s roughly a week-long window in order for one trial of results to be collected. This means that any alterations to the procedure take a while to be observed. The monodisperse particles, however, are more important. Given that analytical techniques are being used to calculate the number of polymers per gold nanoparticle, the surface area and thus the size of the nanoparticles are important to control. During the first couple of weeks in this lab therefore, I worked on refining the production of 250 mL batches of spherical nanoparticles.

Two weeks ago, the lab also attended the regional ACS Conference in Hershey, PA. Even though the day started at 6.30 (yes A.M) I thoroughly enjoyed my first Chemistry conference. The days were divided into several different larger topics, but some of my favourite talks that I attended were a guide to a chemical history walking tour in Paris and another talk about the future of 3D printed personalized medication!

FONTAINE MCFEATERS:

This summer I am continuing my work on polyelectrolyte quantification on the surface of gold nanoparticles. I am additionally studying the thermodynamics of the binding interactions between polyelectrolytes and gold nanoparticles using an instrument called the Isothermal Titration Calorimeter. The isothermal titration calorimeter, or ITC for short, is a way of carrying out small-scale titrations to determine key thermodynamic properties such as binding constants, enthalpy, and the stoichiometry of a reaction. In other words, it is basically a very expensive thermometer. The ITC works by injecting a very small amount of a specific polyelectrolyte over time into the sample cell, which contains the gold nanoparticles. The instrument measures the heat that is absorbed or released for each injection. At the moment, I am working on testing different concentrations of polymer to see which concentrations cause the nanoparticles to aggregate, or deform, before the solutions are used in the ITC. Once the optimal concentrations are found, I will be able to use those solutions to measure the binding interactions.

My other project looks at the quantification of polyelectrolytes on the surface of gold nanoparticles. We begin by synthesizing the nanoparticles via a seed-mediated growth method. The nanoparticles are then coated with a polymer, polyanetholesulfonic acid, also known as PAS. Last summer we did the same procedure with a different polymer, polystyrene sulfonate (PSS). We decided to use PAS due to its similarities in molecular structure to PAS. Ultimately we are testing to see if a polyelectrolyte with a smaller molecular weight, such as PAS, will vary in the quantification of polyelectrolyte on the surface of the nanoparticles compared to polyelectrolytes with a larger molecular weight, such as PSS. The coated nanoparticles must undergo a vigorous purification process to make sure the excess polymer is removed and we can quantify the polymer that is tightly bound to the surface of the nanoparticles. I am currently working on optimizing the purification process for PAS coated nanoparticles, as the particles are more prone to aggregation due to the smaller mass of the polymer.

After undergoing the purification, the particles are analyzed using Inductively-Coupled Plasmon Optical Emissions Spectroscopy, or ICP-OES. The particles are passed through the instrument where they are exposed to a plasma flame that is about the temperature of the surface of the sun, or around 15,000 degrees Fahrenheit! The hot plasma breaks all of the bonds in the solution, exciting the electrons. The ICP-OES measures the intensity of the light that is given off as the electrons return to their respective ground state. We are ultimately able to determine the number of polymer units per gold nanoparticle by the concentrations that the instrument works out from the intensities. This involves many Excel spreadsheets and a lot of patience!

When I’m not in the lab, I have spent most of my time supporting the best hockey team on the planet, the Pittsburgh Penguins, as they won the Stanley Cup for the second year in a row! LGP!

AND THE WINNER IS:

# Lookin’ Fly

We don’t always look this cool, but when we do, we’re doing a gel extraction.

Hey everyone, this is Connor McLoughlin (’17) and Rachel Wigmore (’18) and we’re working in Dr. Hiraizumi’s lab this summer. The main focus of our research is the genetic basis for the expression of a class of enzymes called dipeptidases which breaks down small peptides into amino acids. Why is this research relevant? In diseases such as Alzheimer’s, Crohn’s, and Celiacs, symptoms are correlated with low dipeptidase activity. By better understanding the genetics of dipeptidase expression, we may learn more about their connection to human diseases.

To accomplish this, we study the dipeptidase B gene (Dip-B) in Drosophila melanogaster, or the fruit fly, as a model system. Specifically we use the strains NC25 III and CL55; NC25III  has DIP-B enzymatic activity that is only one-tenth that of CL55.

We’re focusing on two possibilities for the difference in enzymatic activity between the strains. First, NC25 III produces fewer messenger RNA (mRNA) that is translated into protein. Second, the two strains produce the same number of enzyme molecules but DIP-B of NC25 III is catalytically less active than that of CL55. How do we address these possibilities?

For the first possibility, an efficient and practical method to quantify Dip-B mRNA transcripts was needed. To do this, a process called Reverse Transcription- Polymerase Chain Reaction was used to make many copies of cDNA (copy DNA) from mRNA. The quantity of cDNA produced is a function of the quantity of mRNA in the samples. To compare the samples, a quantitative densitometric assay was developed. The cDNA was subjected to agarose gel electrophoresis, which separates DNA fragments by size in a matrix of electrical gradient, with a stain specific for DNA. The separated cDNA is imaged using a ChemiDoc machine, which detects the stain intensity by a densitometric scan. Densitometric intensity is a function of DNA quantity. The protocol for the densitometric quantitative assay using an external standard is under refinement but results are promising, as shown below.

The second possibility for the difference in activity levels is that NC25III might have a less active DIP-B enzyme in comparison to CL55. Will Ueckermann, a former member of the research lab, obtained the coding sequence for Dip-B in CL55 and NC25III.  Compared to the gene sequence in the NCBI database, both strains contained SNPs or single base mutations. Will’s findings suggest that NC25III strain has a non-conservative missense mutation which changes the hydrophilic amino acid serine into a hydrophobic amino acid isoleucine. This change can very well affect the structure of the DIP-B enzyme molecule. We are sequencing more independent samples to confirm the sequence data, so stay tuned until the end of summer for further updates!

In eukaryotes (such as plants, fungi, Drosophila melanogaster, and humans), non-coding sequences (introns) are spliced out during transcription of DNA into mRNA. However, sometimes some introns are retained to generate different mRNA molecules (mRNA isoforms). This process, referred to as alternative splicing, is not unusual and Drosophila melanogaster is not an exception.

Dip-B gene has been reported to produce several mRNA isoforms. Four of the isoforms (A, B, C, and D) are documented in the NCBI database; our lab found evidence for two more isoforms called E, and A/C that are diagrammed below. What makes Dip-B different from conventional alternative splicing is that the coding region for all mRNA isoforms are identical, but the mRNA isoforms vary in the sequence that precedes the coding region called the 5’ Untranslated Region (5’-UTR).

Isoforms A and C contain identical transcription initiation site, but what makes them interesting is the presence of introns in their 5’-UTR. Presence of introns in the 5’UTR in eukaryotic genes is not  uncommon, with 4000/14000 Drosophila genes and 35% of human genes having this feature. The function of introns in 5′ UTR is unknown, but it sure would be interesting to learn more about them during research here!

We have performed many experiments involving PCR amplification of isoforms A and C and gel imaging of PCR products. There have been good days…

Actin is the external standard of Connor’s Quantitative Densitometric Assay, so we need a lot of pure actin, shown in this gel.

…and not so good days.

An attempt to image Isoforms A and C.

Our main goals for the rest of the summer include sequencing non-coding regions, specifically the 5′ UTR of the Dip-B gene, as well as to refine the protocol for a densitometric quantitative assay. We hope to isolate more Dip-B DNA to be sent out for sequencing to confirm the existence of the SNPs Will found.