Category Archives: Week 2

The great American philosopher Alicia Keys once declared that “Big lights will inspire you” on a track with Jay-Z.

It’s this sentiment that I’ll be spending my summer trying to extrapolate to my mice. See, my project involves optogenetic stimulation of the brain. Optogenetics is a brain stimulation technique that relies on light. Basically, a surgeon puts some implants in a specific region of the mouse brain, we hook up the mice to some optic fibers, and then we use light on that brain region to stimulate it.

Optogenetics is a powerful technique because it allows us to pinpoint certain regions and see how behavior is altered by either activation or inhibition of those regions. In my project, we’re trying to see how activation and inhibition in the prefrontal cortex affects operant learning (learning to perform a task to obtain a reward). To test this, I’ve spent some time training mice to press a lever under different reward schedules. First, they learned that one press of a small lever leads to the release of one pellet of food. After 3 days of that schedule, I ramped it up to 5 lever presses for one pellet of food, and then finally to 10 lever presses for one pellet of food. The first day, they’ll take two hours and maybe press the lever 20 times or so, but within 2 days of training, they’ll press it 50 times in less than 10 minutes. It’s just like us humans— if we’re hungry and want food, we’ll figure out what it takes to get it.

After the first 9 days of training and eagerness to see the results of the experiment, I initialized the optogenetic stimulation for the first time on Saturday. The operant conditioning chambers in the lab are each equipped with motion sensors to track when the mice put their little heads in the feeding area and each box is connected to two computers that control the illumination and track how many times each mouse presses the lever to obtain a food pellet. Right now, I’m focused on collecting data and processing it so that I can start to make graphs for the analysis this upcoming week.

If I’m able to observe a significant change in the lever pressing behavior in response to illumination, we could develop a more detailed model of the operant learning pathway that’s being studied in other projects in the lab as well. The results from Saturday and Sunday will be my first optogenetic results of the summer, and I’m looking forward to seeing if the data I gathered can implicate the implanted brain region into the operant learning pathway.

As everyone by now has heard, the U.S is experiencing an epidemic. I’m not talking about infrastructure or climate change or income inequality, rather I am referring to the current opioid crisis. You see, a couple of decades ago big pharma companies created drugs that resemble the effects of opium, a drug known for its pain killing effects as well as its capability to start wars. These drugs where marketed as same pain killers that where much more effective then aspirin or ibuprofen. Soon, doctors were prescribing these pills left and right to people with chronic pain, post-surgery pain, or even people with bad tooth aches. Eventually however people got hooked. You see, drugs that are very similar in molecular structure to heroin started getting people addicted. Who would’ve guessed that? People started to take more of these drugs to get their “fix” and this only increased their tolerance. Many of these people would turn to stronger opioids like heroine or fentanyl. These drugs have a high likelihood of overdose with chronic use. It is no surprise then that overdose has surpassed car accidents as the leading cause of death in the U.S.

What can be done about this tragedy? While many have suggested tighter regulations on big pharma, who are we kidding by expecting these regulations to become law? Instead, the best alternative is to try to stop people from wanting the drugs in the first place. In my lab I have been assigned to test different drugs on rats to see if it stops the cravings for opioids. By using the classic Skinner Box, rats are trained to self-administer Remifentanil, a type of opioid, in a manner that mimics addiction. Whenever the rat presses the correct lever, a dose of Remifentanil is intravenously injected into the rat via a catheter. Once these rats are dependent like a human addict, we will give them either the test drug (we are testing different drugs in different individual studies) or saline (as the control group). If it is found that rats injected with the test drug stop self-administering Remifentanil, it would mean that this drug could potentially be prescribed to opioid addicts as a way to curb their addiction. If successful, the drugs we are testing could save countless lives from potential overdose.

The use of drugs to treat behavioral traits is not knew. Instead of therapy or lifestyle changes, many people with anxiety or depression will opt to take SSRI’s to treat their mental illness. This has proved to be more practical, and often more effective than therapy and has greatly helped millions of people. While all mind-altering drugs have potential side-effects, the illness or addiction is often worse than the occasional dizziness or mood swings. While it may seem ironic, the truth of the matter is that our only hope to fight drug use is using more drugs. Welcome to the 21^st century everyone!

To know how something differs when it’s broken, it must first be known how it excels when it’s fully functional.

Autism is a spectrum disorder characterized by deficits in communication and social behavior, most commonly diagnose in boys. While autism is heritable, and there have been numerous genes identified with direct correlation to autism, genes alone cannot explain development of the disorder. Hundreds of environmental factors have been implicated in autism, like chemicals, drugs, fuels, heavy metals, and dietary factors. Though, when studied in isolation, like many studies have done, either genes or environmental factors alone are only weakly predictive of autism onset.

My mentor has created a mouse model of combined prenatal environmental exposure to link the effects of environmental factors and maternal stress to alteration of brain histology causative of autism. Specifically, this summer I will be studying a region of the brain called the anterior cingulate cortex (ACC), which is important for emotion processing, learning, and memory. The focus is on cells called microglia, the brain’s resident immune cells necessary for neurologic immune response, proper central nervous system function, and are crucial for the formation of synapses. So far, the normal developmental pathway of microglial cells in the ACC has never been characterized, and it is unknown how prenatal stressors alter synapse number at different developmental time points. That’s where my data will hopefully come in. After characterizing the normal developmental pathway of microglia in developing mice brains over five developmental time points, the data will allow us to consider how toxins and prenatal stressors alter this normal developmental pathway. Likewise, it is unknown whether alteration in synapse number is due to lessened synapse formation or heightened microglial engulfment (elimination) of these synapses.

To answer these questions I’ve been working on staining brain tissue to be able to look at microglial cells through a confocal microscope and then reconstructing these 3D images through various computer programs. I like to think of it as a fancy photography project, one which will hopefully result in some really awesome conclusions and a colorful poster. I’ve been loving my time so far, and can’t wait for what’s to come.

Hello readers! Welcome to Week 2 of my science blog about my research experience. This week I get to talk about the science that my lab is exploring as well as my specific project. To start, I’ll begin with some background:

Lung cancer causes the most deaths per year out of all cancer types. Nearly 150,000 men and women are projected to die due to lung cancer in 2019 in America alone (American Cancer Society). Furthermore, lung to brain metastasis (the spreading of cancer from the lung to the brain) is a common occurrence and particularly poor prognosis for patient survival. My lab studies the mechanisms of brain metastases by exploring the signaling machinery that goes awry in cancer. We are focusing on a specific kinase (a protein that causes chemical shifts in other proteins, acting like a master signaler) and its role in metastasis. Jake and I are using a line of cancer cells that naturally metastasize to the brain for our research. Jake made this line earlier by injecting cancer cells into the hearts of mice, isolating tumors that spread to the brain, and repeating this process until the cancers reliably migrated to the brain. I think that’s a really elegant way to design a cancer line that is specific to our research.

As far as my research goes, I am dividing my work into two projects. For one of the projects, I am replicating a number of experiments that Jake completed earlier in a different cell line. His results were really promising, and it would help the story immensely if we could show the same effects in a lung cancer cell type with a different “driver” mutation (the major contributor to the tumorigenic phenotype). This project is fairly straightforward since the path has already been laid out, and the process is going smoothly so far.

My second project is much less structured, at least so far. Earlier, Jake compared RNA sequencing data between our metastasizing cancer line and the “parental” line (the same cancer line without the repeated intracardiac injections) to find a list of about 100 genes that appeared to be upregulated in the metastazing line compared to the parental line. Jake and previous students explored some of these genes, but most of them haven’t been analyzed yet. My project is to pick some of these unexplored, upregulated genes and perform tests to determine their levels of upregulation, impact on metastasis, and any interesting new pathways they may be involved in. I chose about 30 of the genes and designed PCR primers for all of them in order to perform an RT-qPCR experiment to quantify their levels in the metastasis line and parental line. The preliminary results were really interesting. In fact, one of the genes appears to be upregulated over 70 times in the metastasis line! Multiple other genes were also upregulated, usually on the order of 2-4 times. These results will need to be repeated to confirm their legitimacy, but my next step is to focus on these genes and figure out why they are upregulated. My plan is to look at the transcription factors linked to these genes and try to trace a path back to the kinase we are focusing on. That would make a cool story!

I am really excited about my work so far. While I have run into speed bumps in different experiments on different days, in general the projects are going smoothly. I really hope I can make a significant contribution to Jake’s work and the work of the other members of the lab. Even if the data I collect doesn’t show anything interesting, I am still learning so much about the daily life of a researcher and what it means to be exploring cancer. Next week I will interview the PI of my lab, Dr. Pendergast, and share that here. See ya next week!

-Brennan

We have all had various experiences that have had a significant effect on who we are and how we act. Whether it was the childhood memory we will never forget, or the friendships we formed throughout school, our experiences definitely shape us. In a similar fashion, the neural connections within our brain change in response to experience. Just like when someone makes an impression in clay, our brains’ circuitry changes in response to new experiences.

This idea is referred to as “synaptic plasticity” and is a major focus of the Calakos lab, where I am working this summer. The lab focuses on how experience influences behavior, but also how in neurological conditions, the mechanisms of synaptic plasticity can go awry. More specifically, I am focusing on the condition of dystonia, which is a movement disorder in which muscles may contract uncontrollably and is the third most common movement disorder.

Past research within the lab has shown that there is a specific protein pathway known as eIF2alpha which is associated with synaptic plasticity and may be correlated with dystonia. Think of this protein pathway like a set of instructions that helps regulate our cells. This pathway typically responds when cells are experiencing high stress. It has been hypothesized that in dystonia, this pathway becomes dysregulated early on in development.

However,  it remains uncertain whether and when targeting eIF2α signaling can improve dystonia. It is also important to determine exactly where in the brain selective vulnerability to altered eIF2α signaling occurs. Therefore, for my research project, I will be using western blot to determine if there is a dysregulation in eIF2alpha in a mouse model of dystonia compared to their littermate controls. I will analyze the brain tissue from mice for expression of eIF2alpha at various time-points throughout development including the day of birth, 5 days after birth, 14 days after birth, and 21 days after birth to determine if there is a period of susceptibility in which pathway dysregulation occurs. I will be analyzing four main brain regions: midbrain, striatum, cerebellum and cortex, regions previously implicated in dystonia, to determine if there is a specific brain region in which the pathway’s dysregulation is most predominant. I feel that my experiences working in this lab on this project will definitely shape my future decisions and maybe even change a few of my neural connections along the way.

This summer, I am working in Dr. Gong’s Biomedical Engineering research lab on a project focused on examining place cells in the hippocampus of mice. Place cells play an important role in spatial and episodic memories and the current project looks at how visual spatial manipulations affect how place cells are fired.

A 1-photon microscope was used to record the firing of the neurons. First, an Adeno-associated virus that carries the protein GCaMP6f was injected in the hippocampus of the mice. This particular virus was selected because GCaMP6f has a unique ability to bind to calcium ions and fluoresce. As a result, when the concentration of calcium is high, more of the GCaMP6f is bound to Ca2+, resulting in increased fluorescence that can be picked up by the 1-photon microscope. Since Ca2+ increases during an action potential, we can correlate the increases in fluorescence to periods of neural activity.

The physical experimental set-up consisted of a styrofoam cylinder that served as a linear treadmill, on top of which the mouse was placed. A virtual reality display was shown on monitors in front of the treadmill and the mouse controlled its movements in the virtual reality through its actions on the wheel. The purpose of the virtual reality was to allow for manipulation of what the mouse sees in ways that couldn’t happen outside of a computer screen. After a training period, the mice ran through randomized trials that consisted of either running on an unaltered track or on a track with a visual spatial manipulation.

After running the experimental trials, an algorithm called constrained non-negative matrix factorization was used to identify areas of fluorescence that may represent neurons. However, the computer is not always accurate and may outline a group of pixels that are not neurons. For example, the outline might circle two neurons, the space between two neurons, only part of the neuron, or part of a neighboring neuron. Currently, my primary job is to identify whether the computer-generated outlines are actually neurons or not. To do this, I pay attention to nearby pixels that brighten and dim together. If the outline encapsulates a group of pixels that fulfill those criteria, then it is most likely a neuron and I select it.

After all this data is processed, we will be able to identify place cells and determine their function by correlating their respective calcium transients to locations in space. With this information, we will be able to better understand how place cells react to visual manipulations. This information will be useful in looking at how vision corrects for errors, in better understanding how the brain uses vision, and in further understanding how the brain works in general, allowing future researchers to compare this data to disease models.

A special thanks to my mentor Emily Redington for her help with this blog post!

When I first sat down with Lindsey Glickfeld, she explained every unknown neurobiology term with a diagram, which I will try to emulate throughout this blog post. I think trying to explain the Glickfeld Lab’s focus on the synaptic organization of the mouse visual cortex with words might be a bit tiring on the eyes. I want to give a little background on the mouse visual cortex since the Glickfeld Lab uses the mouse model; I hope the diagram and its caption below is more appealing than a big blob of words!

(A) Follow the right side of the flow chart . . . Visual processing begins in retina, then to the dorsal lateral geniculate nucleus (dLGN) in the thalamus. From dLGN, the visual information moves to the primary visual cortex (V1) and then to the higher visual areas (HVAs). (B) A zoomed in diagram of V1 and different HVAs. Please ignore the red . . . most of this blog’s focus is on V1 to LM, AM, and PM.

Before I dive a little deeper into my project, I want to define some neurobiology jargon that is essential to understand this research at the Glickfeld Lab. Surround suppression can be defined as the “neuron’s initial increase in firing is followed by a decrease in firing as the stimulus become progressively larger,” and visual receptive fields can be defined as “a portion of sensory space that can elicit neuronal responses when stimulated.” But, maybe this diagram of surround suppression on one specific neuron might make a little more sense; the receptive field is drawn as the red and green figures and the stimulus as the white and black gratings.

Surround suppression is shown through the initial direct relationship between firing rate and stimulus size following an inverse relationship between the same variables.

Now that the background is out of the way, I’ll start to explain what I plan to research this summer. I am working under Jenny Li, my mentor, who is focused on the pathway from the primary visual cortex (V1) to higher visual areas (HVAs). Past research has looked at V1 and three HVAs that receive the strongest direct input from V1: lateromedial (LM), anterolateral (AM), and posteromedial (PM). However, there was one HVA that caught researchers’ eyes: PM. Unlike the other two HVAs, PM had significantly less surround suppression and bigger receptive fields (see below for yet another diagram!). Jenny is interested in these differences between higher visual areas.

To discover and eliminate variables that may play into this phenomenon, I will be measuring the width of axon spread from V1 to HVAs. I will accomplish this by performing burr hole surgeries for viral injections, perfusions, brain slicing, and finally imaging. If there are differing widths of axon spread, it could be a possible anatomical explanation for why PM show less surround suppression and larger receptive fields than other prominent HVAs.

In the bigger picture, the mouse’s visual system is different from the primate’s  visual system: lower acuity, lack of trichromacy and fovea, and more. However, the similarities do outnumber the differences. Furthermore, it is much easier to monitor and manipulate specific cells types and circuits in mice, helping us advance towards to goal of understanding how vision works. By studying the mouse’s cortical circuits rather than its vision, researchers can discover fundamental principles of cortical processing that may be universal across species. 

Some coronal brain slices!

Mounted coronal slices!

How many moons does Jupiter have? What is a group of frogs called? How many total steps does the Eiffel Tower have? If you know the answers, congrats! If you don’t, take a guess! If you’re curious about the answers, keep reading…because my project is about that—curiosity!

When it comes to learning, we as humans constantly seek out and consume information, whether we’re aware of it or not. When we look at a traffic light, we’re learning from that stimulus so we can make our next move—stop or go. The Adcock Lab studies learning and the many cognitive elements behind it, such as attention, reward, and you guessed it—curiosity. The information-gap hypothesis posits that curiosity is a sense of “deprivation that arises from the perception of a gap in knowledge and understanding” (Lowenstein, 1994). Basically, when we don’t know something, our interest is piqued, driving us to learn and consequently resolve that lack of knowledge.

The Adcock Lab has previously conducted research on curiosity, such as identifying neural networks that correspond to anticipation and attention when information is withheld or delayed. My mentor Abby has also recently done a study in which she characterized certain determinants of curiosity—what spurs it, and what maintains or prolongs it. By implementing real-time self-reports of curiosity into her experiment, in which participants produced guesses on the content of art videos, she was able to track how curiosity changes over the duration of information arrival.

My research project is an offshoot of Abby’s, looking at other mediators of curiosity. It has been largely established that there is a link between curiosity and memory, and another link between engagement and memory. Curiosity has been seen to enhance learning and memory both for information of interest (Kang et al. 2009, Wade & Kidd, 2019) and incidental information (Gruber et al. 2014), while choice and active engagement was seen to enhance memory with both intentional (Voss et al. 2011) and incidental encoding (Murty et al. 2015). However, the three-way relationship between curiosity, agency, and memory has not been as clear; my project, therefore, is to establish that link. In the experiment, autonomy and active engagement will be manipulated to see differences in levels of self-reported curiosity during the previously mentioned art video task. Participants will then be called back to perform a memory test in order to examine differences in recall.

My project will also explore how the unique dispositions of people respond differently to these manipulations in engagement. This “trait curiosity” is well-defined in Kashdan et al. 2017, where certain people may view curiosity as positive, constantly asking questions and being fascinated with the workings of the world, while others view it as more negative—asking questions only when the uncertainty of a lack of information is too uncomfortable to ignore. By linking active engagement, curiosity, and memory, and consequently analyzing that link with respect to dispositional differences in curiosity, this research will hopefully have strong implications in education. Curiosity could be better incited through greater student autonomy or engagement in the classroom setting, enhancing learning beyond rote memorization. The self-awareness of one’s own curiosity could also result in a greater motivation to close the information gap, leading to a stronger internal desire to learn instead of for extrinsic incentives like grades. The potential impact of this research is exciting, and as the days pass, I become more and more curious on the conclusions of my project!

Answers: Jupiter has 79 moons, a group of frogs is called an army, and the Eiffel Tower has 1710 steps!

All animals use Wnt growth factors for controlling cell fate decisions as the organism develops. The wingless gene (wg) encodes the Drosophila Wnt growth factor, Wingless (Wg) protein. In Drosophila, a loss of function mutation in the wingless gene causes a disruption in epidermal patterning during embryogenesis. At the end of embryogenesis, a wild type fly larva would have a cuticle pattern, produced by the epidermal cells, that consists of repeating segments that alternate between a belt of denticles and a region of naked cuticle (see image below).

Cuticle patterning on a wild type fly.

A complete loss of function mutation in the wingless gene eliminates the regions of naked cuticle that separate denticle belts, resulting in a continuous area of denticles (see image below). In both flies and humans, Wnt growth factors associate tightly with cell membranes, but they are able to control cell fate decisions at a distance from their origin points. In the fly, some mutations in the wingless gene disrupt the cell to cell movement of the protein without altering its signaling. For example, the wg^NE2 mutation restricts the range of protein movement, and so is not able to control cell fates at the normal distance seen in the wild type.

Region of denticles with no naked cuticle to create proper segmentation.

The Bejsovec lab has identified two suppressors that improve the cuticle patterning of the wg^NE2 mutant embryos. For my project I will be working with these suppressors to characterize how they impact the movement of the Wg^NE2 protein and to test whether they affect movement of the wild-type Wg protein. As a part of my project I will be doing antibody staining for the Wg^NE2 protein and viewing my preps using a confocal (fluorescent) microscope.

A hope that stems from my project is that these same suppressors will also have impacts on the movement of wild type Wg protein. The discovery of movements in the wild type Wingless protein could possibly lead to groundbreaking discoveries given that the Wingless growth factor is very similar to the Wnt1 growth factor in humans, which is required to pattern the central nervous system. In mice, knocking out the Wnt1 gene results in lack of development of the cerebellum, and death of the mutant embryos (to read more about this, click here) Understanding how Wnt proteins move will help us understand how our nervous system is patterned during development. 

A special thanks to my principal investigator, Dr. Bejsovec, for helping me in this blog post.

Credit for the images and information below.

Dierick, H. A. & Bejsovec, A. Functional analysis of Wingless reveals a link     between intercellular ligand transport and dorsal-cell-specific signaling. 10

Delta-catenin is a protein that was believed to be neuron specific but spoiler alert it is in astrocytes as well! In the catinen-cadherin complex, delta-catenin is important in cell adhesion by cadherins between pre and post synaptic neurons. Literature on delta-catenin has shown that it can coordinate changes in perisynaptic processes (Arikkath, J., et al). Recently delta-catenin was discovered in astrocytes posing the question of whether it is important for neuron-astrocyte interactions by the same or similar mechanism?

Astrocytes are a type of glial cell that regulates neural functions. Astrocyte morphogenesis is correlated with synaptogenesis or the uptake and removal of synapses (Strogsdill, J.A., et al). Astrocyte release factors have been shown to affect synaptic processes such as glutamate uptake and ion homeostasis. I am working on answering the question of whether direct interaction between astrocytes and neurons affects astrocyte morphogenesis or, in other words, astrocyte complexity?

CTNND2 is the gene that encodes delta-catenin in both neurons and astrocytes. Over-expression in astrocytes results in stickier astrocytes with more adhesions and less complexity. This summer I am working on experiments to knockdown CTNND2 and then test the effects on cell adhesion. One set of tests involves entering a mRNA knockdown of CTNND2 into astrocytes alone to see the resulting effects on astrocyte morphology compared with wild type astrocytes. Preliminary results have showed a decrease in astrocyte complexity which supports my hypothesis. Knocking down CTNND2 in neuron culture will also provide insights. My project is to run a series of combination co culture experiments: wild type astrocytes with CTNND2 knockdown neurons, CTNND2 knockdown astrocytes with wild type neurons, and CTNND2 knockdown astrocytes with CTNND2 knockdown neurons. These permutations will provide some data on the importance of cadherin and delta-catenin in cell interactions.

The next set of experiments I am working on are done in order to image the effects of knocking down CTNND2 in the brain of mice. With a technique called Post Natal Astrocyte Labeling by Electroporation (PALE) astrocytes can be visualized with a confocal microscope to analyze their complexity. One experiment is to genetically introduce a flox into the genome to knockdown CTNND2 mice. This allows for the researcher to choose which cells they don’t want to express the floxed gene. Another aspect of the project is to knockdown the gene in the anterior cingulate cortex (ACC) of the brain which is associated with autonomic processes as well as attention, decision making, and other thinking processes. The ACC is linked to autism in several ways, so visualizing the function of delta-catenin in astrocyte morphology in this region could provide some insight into how the mutation of certain genes leads to cognitive abnormalities. This project fits within the larger goal of the lab which is to understand the interactions of cells in the brain. How cells pass information back and forth is the first small step in understanding how people retain information, make decisions, and have such complex cognitive abilities.

As high school biology teachers often remind us, we’re more closely related to other animals than we might generally like to think. We all eventually, when traced back far enough, stem from a single ancestor. And despite our distinct and innumerable differences, just a few, significant similarities are enough for such blasphemy to begin to station in truth. One of these, found in all bilateral animals, and the subject of my investigation for the following weeks, is the presence of the transcription factor, brachyury.

In an embryo, specifically of deuterostomes like humans, the first feature to develop is the anus. The embryo then invaginates at the anus—turning itself inward—and gradually stretches towards the mouth at the other end of the embryo to form an early version of the gastrointestinal tract in a process known as gastrulation. We know that brachyury plays a critical role in this stage of embryo development, though much about the protein remains an enigma. We know where brachyury is expressed, though we do not know what processes it controls; we know that it’s presence is essential for proper development, though we do not know why that is and what causes the failures observed in its absence. My project at the McClay Lab will be to examine further these characteristic of brachyury and its role in gastrulation, to seek answers to the many questions surrounding this crucial protein, yet shrouded in mystery.

These past two weeks have been dedicated towards locating brachyury in embryos undergoing gastrulation. Knowing where it is, how it is expressed, and at which stages one may observe each pattern of expression will be the first step I take towards learning more about it. We hope that this investigation will eventually lead to a deeper understanding of gastrulation and embryo development as a whole, and unlock some of the many secrets to the complexity of our being.

Cancer pain delivers a message. A vile, biting message to patients that life itself is seeping through their fingers. A disheartening, damning message to medical specialists that even their powers are limited. It thrusts itself as a physical manifestation of the unseen, of your interiors being eaten away. It is common to mistaken pain as merely pain neurons being stuck in the ‘on’ position, but it is much more complex than that: pain that develops from an acute injury is actually the result of hyperactivation of the peripheral pain neurons responsible for inflammation. Previously thought of as merely bystanders, these pain neurons have been revealed to actively participate in the progression of the carcinogens. With cancer pain being one of the most intense and difficult brands of pain to treat, further research is warranted more than ever.

My project this summer in the Ji lab branches off of my mentor’s ongoing project on the role of STING, a protein that activates interferons, in suppressing pain and reducing neuroinflammation. STING holds a central role in the innate immunity–it produces a protective response by inducing mediators such as cytokines or chemokines. These molecules can then either produce or suppress neuroinflammation. One of the most important mediators that STING activates is type I interferons, which are proteins that boost the immune system in fending off viruses or cancer. As a result, activating STING has potential to not only decrease tumor growth but also dampen neuroinflammation, the underlying etiology of pain.

My mentor and I have been running experiments on mice to not only affirm the role of STING in producing interferons that decrease sustained pain but to also eliminate the possibility that other mechanisms are contributing to this effect. Mice that experience chronic pain have a heightened ability to feel pain from that stimuli that wouldn’t induce pain in normal conditions. To determine the exact threshold where these mice are first able to sense pain, I have been conducting a test called the Von Frey assay. A factor that can lead to false signs of pain in mice is anxiety, so I have also been conducting an open field test to track the movement of mice and then analyze signs of anxiety, ultimately to show that these mice are not any more anxious than normal. So far, the hypothesis that activating STING decreases pain has been supported, but further experiments are necessary to identify the exact mechanism by which STING is able to do so.

Modern clinical medicine relies heavily on the blood-based diagnostic tests that measure the amount of protein biomarkers present in circulation to make clinical decisions. In hospital settings, this is most commonly done by enzyme-linked immunosorbent assay (ELISA). While accurate and sensitive, ELISA requires considerable resources, infrastructure, and expertise to perform. The D4 assay is a miniaturized, self-contained assay that can measure protein biomarkers in blood with ELISA-like performance without the need for equipment other than a smartphone and can be performed with minimal user training. Assay reagents are inkjet printed onto a glass chip coated with a special “zero-background” polymer coating, which acts to minimize biomolecular noise (making the assay very sensitive) and stabilize the reagents even without refrigeration. The assay is user-friendly because adding a liquid sample (blood, serum, cell lysate) to D4 chips automatically drives the assay to completion. Furthermore, the assay is very portable since a cellphone-based detector utilizes the phone’s camera lens to readout the fluorescence on the D4 assay. For a more in-depth explanation of how the D4 system works, read Joh, D.Y., et al. Because of the EpiView-D4 system’s portability, it has huge implications for healthcare in low-resource settings like Liberia.

Over the summer, I’m helping with two D4 assay projects. The first project is developing a point of care test for breast cancer. I’m optimizing a D4 assay that detects HER2, a protein found on breast cancer cells; this protein is important because if breast cancers are found to be HER2-positive, then they are likely to respond to so-called “anti-HER2” cancer drugs (e.g. trastuzumab), which is potentially life-saving. In the long-term, my goal is to make a multiplexed assay that targets the four major clinically-relevant biomarkers for breast cancer: these are ER, PR, HER2, and Ki67. This capability is standard care in the United States, but unavailable in many developing countries (where the majority of breast cancer deaths now occur). By having a single, low-cost, and user-friendly assay that profiles all four markers simultaneously, clinicians in the developing world will be able to match different breast tumors to the medications which they are most likely to respond to. The second project is developing a point-of-care test that rapidly identifies Methicillin-resistant Staphylococcus aureus (MRSA). MRSA can resist antibiotics due to expression of an altered penicillin-binding protein, PBP2a. Current methods for identifying MRSA versus methicillin-sensitive strains are based on culturing the bacteria, and this typically requires at least a day or longer.  If a point of care D4 assay can be developed to detect PBP2a, MRSA diagnosis and treatment could become more efficient.

When I was placed in the Volkan lab and took a tour back in May I was fascinated, but I had my doubts. What could we possibly learn about human neurobiology by studying Drosophila? Can I get a handle on these complex biological techniques in 8 weeks? How am I going to dissect a fly brain??

Upon arriving, my mentor Qichen really helped bridge the gap between the work we plan to do in the lab and my understanding of neuroscience. He explained to me that the human brain as we know is an incredibly complex system. The brain consists of around 80 billion neurons and 100 trillion specific synaptic connections, making the study of its development and organization highly complicated (Barish et. al., 2018). He explained to me that this makes Drosophila the ideal model organism and its olfactory circuit the ideal system to study to get a better understanding of how neurons sort and make very specific connections to allow for the proper function of the system. Through previous research, our lab has identified a family of proteins dubbed DPRs, defective proboscis response proteins, and their binding partners called DIPs, DPR interacting proteins, to be essential for regulating the positioning and structure of glomeruli in the olfactory system (Barish et. al., 2018).

This summer, my project will focus on the role of one specific protein in this family, DIP-alpha. My major goals are to discover its expression pattern, in specific olfactory receptor neurons, and to genetically perturb its function. The first goal will be accomplished using genetic labeling and antibody staining of both the Drosophila antennae and brain, and the second goal will be attained by down-regulating DIP-alpha in specific classes of olfactory receptor neurons to investigate what happens in the protein’s absence. So far, I have been honing in on my dissection skills and familiarizing myself with the staining and imaging process. In the coming weeks, I hope to get some fascinating data that leads us closer to understanding the development of this circuitry. And if I don’t, at least I can dissect a fly brain!

On my very first day my amazing PI, Dr. Sanders, handed me a sheet of paper with three goals for this summer. I was very surprised that I was able to start working on my first goal, learning mammalian cell culture and proper aseptic technique that same day. Today, I am incredibly happy to say that I have completed that first goal. I have worked with a line of human embryonic kidney cells (HEK293). I can now successfully and independently (much to my mentor’s delight) split, count, thaw, make media for, and collect protein from these cells.

I will be using my mammalian cell culture techniques throughout the rest of the summer in an effort to meet my two other goals and to answer important scientific questions. One of my goals is to determine if HEK293 cells lacking a  LRRK2 gene are more sensitive to environmental toxicants and DNA damaging agents. To do this, I am planning to treat wild type cells with LRRK2 and cells that have had the LRRK2 gene removed with the same doses of certain toxicants and DNA damage inducing agents and analyze the differences. Some of the many common toxicants I am planning to use are rotenone, hydrogen peroxide, and mitomycin C. If I am able to complete this project and get promising results (fingers crossed), I can move on to my third and final goal.

My last goal is to determine whether LRRK2 deficiency leads to cell cycle checkpoints in the presence or absence of the same environmental and DNA damaging agents that were used to determine cell viability before. I will be checking the levels of unique proteins associated with different parts of the cell cycle to see if the checkpoints are working properly. I will be using DC protein assays and Western Blots in order to carry out these goals, as well as my mammalian cell culture technique to keep my cells happy throughout.

Why is this my project? My lab, the Sanders lab, has discovered the importance of mitochondrial DNA damage in neurons affected in Parkinson’s disease. The LRRK2 gene is known to be a genetically significant factor for the onset of Parkinson’s disease. Although scientists know that it plays a role in many Parkinson’s patients, they do not fully understand what it does. My lab believes that LRRK2 may play a crucial role in the mitochondrial DNA damage they found earlier and may play a critical role in the cell cycle when such DNA damage (produced by the exposures in vitro) is present. This is why I am looking at both cell viability and cell cycle checkpoints in HEK 293 cells with the LRRK2 gene and the same cells without the LRRK2 gene. We hope to understand this gene better, so we can understand how it is affecting Parkinson’s patients and to try and find a way to reverse the damage it does.

While many people (not Dr. G) fear snakes or spiders, I have been afraid of antibiotic resistant bacteria since I read a book about it in middle school.  But unlike my other fears, I don’t want to avoid antibiotic resistant bacteria.  I want to do something about it.  That’s one of the reasons why the Brennan lab stood out as a good match for me this summer.

My project in the Brennan lab is primarily focused on a bacterial toxin called HipA (high persistence A), a protein that mediates multidrug tolerance through a mechanism known as persistence.  Essentially, during times of stress, such as the presence of antibiotics in an environment, HipA causes bacterial cells to enter a dormant state in which all cellular activity stops.  During this time, antibiotics are not effective against them because the functions targeted by antibiotics are shut down.  After a period of time, the levels of HipA in the cell decrease, and the cell returns to normal functioning.  These cells are known as persisters because they survive antibiotic treatment.  You can learn more about bacterial persistence here.  The Brennan lab is collaborating with a drug discovery company to find molecules that bind with HipA, potentially reducing its ability to induce dormancy.  In the future, these findings could lead to improvement of antibiotics.

My project involves isolating HipA from E. coli that are engineered to overexpress it, or make much more of it than they usually would.  HipA is a kinase, which means that it transfers phosphate from ATP to other molecules, and its activity can be measured by its ability to autophosphorylate, in which it uses its kinase abilities to phosphorylate itself.  I can treat HipA with the drug precursors to determine if any of them can inhibit HipA’s autophosphorylation, since if HipA cannot phosphorylate itself, this is an indication that it has lost its function as a kinase.  My longterm goal is to determine which, if any, of the drug precursors inhibit HipA’s function.  You can read more about HipA and phosphorylation here.

Field trip to the lemur center!

In 2014, Malaysia Airlines Flight 370 disappeared. The world held its breath as week after week, crews in the air and in the water searched for clues. Every once in a while, a glimmer of hope: search teams in the sky spot what they think is debris from the missing plane–only to find out that it’s trash. Carelessly discarded plastic garbage that now wanders on the water like men lost at sea.

Amid the frustrations of the search for Flight 370, a spotlight shone on another tragedy–the fact that we have turned our oceans into massive dumpsters. We dump an estimated 8.8 million tons of plastic into the ocean every single year (National Geographic). The problem is that plastic isn’t biodegradable–with a lifetime of more than 400 years, the plastic garbage in our oceans isn’t going anywhere unless we do something about it.

The fact that our beautiful oceans have become garbage dumps is an outrage in itself, but our negligence has even more far-reaching effects. Every year, marine animals are strangled by plastic debris. Birds starve to death because their guts, so full of plastic, physically can’t hold food. Chemicals from microplastics may even travel up the food chain into the fish tacos on your dinner plate and eventually end up inside you.

This summer, I’m working on a Bass Connections project on the bioremediation of plastic pollution. Our project was inspired by a 2016 paper written by scientists in Japan who isolated a bacterium that secretes enzymes which break down PET, the plastic found in single-use water bottles (See the 2016 paper here). The goal of my project is to create a library of mutant enzymes and select the enzymes which can most effectively break down PET. Hopefully, this project can contribute to future work on a solar-powered bio-reactor that can be used to clean up areas in need of plastic removal.

I’m really excited about my project! And really concerned about the state of Planet Earth…

If you’re interested in learning more about our planet’s plastic problem, I recommend this article and this article, both from National Geographic. And if you’re not into reading, check ’em out anyway! The pictures are pretty darn powerful.