Category Archives: Week 2

What can we learn from chimps?

Chimpanzees share almost 99% of our DNA, yet the way our brain functions, our morphology, our phylogeny, our phenotype, and multiple other factors differ greatly. So how can this be the case? The drivers for these differences lay within the non-coding parts of our DNA – specifically the gene regulatory system. These DNA sequences, instead of coding for a specific protein, tell our genes when to turn on and at what level they should be expressed at what time. Because of these nucleotide differences, genes in the human forebrain have allowed humans to grow smarter, and genes in chimpanzees have allowed them to build a stronger and more resilient immune system.

This summer, I will be working with the Wray lab under Micah, a first-year graduate student at Duke. Even though I will only be working on a small part of the greater project, the end goal of this research project is to investigate evolutionary differences between chimpanzees and humans at the gene regulatory level. More specifically, chimp-human hybrid induced pluripotent tetraploid cells will be cultured to see how DNA from the chimp will interact with DNA from the human, and to see how the gene regulation in the hybrid cells differs from just the human or chimp cells. From this, we can tell what factors affect gene regulation in both species that lead to the different phenotypes observed. This technique can then be further used to look at disease specific differences that could give us insight into how chimpanzees are better adapted for some diseases than humans are, and why.

Even though I hope to see this project to completion eventually, my specific role in the lab this summer will be to work with human induced pluripotent stem cells (hiPSC’s) to differentiate them into neural progenitor cells. To complete this, iPSC’s must first be thawed and cultured in petri dishes. Their stem cell properties must be confirmed using antibody markers that detect specific transcription factors. Next, the stem cells must be differentiated into neural progenitor cells (NPC’s) using a range of different factors such as noggin to direct their gene expression. The identity of these cells must then be confirmed using antibody markers once again for specific transcription factors expressed by human NPC’s. Once these human NPC’s have been produced, multiple tests can be run to gain insight into their cis and trans gene regulatory systems to get a better understanding of how they work in vivo.

I look forward to getting more comfortable with the lab, learning new bench techniques, and making an impact on the greater scope of this project.

A Question Years in the Making

In 2004, Dr. Nina Sherwood and her lab discovered the spastin gene in Drosophila in a gain-of-function screen. Spastin was found to diminish microtubules in the Drosophila larval neuromuscular junction (AKA, a synapse) when overexpressed, consistent with the fact that spastin is a microtubule-severing protein. So, with this logic, a loss-of-function mutation in spastin should prevent microtubule degradation, right? Wrong! The same results were discovered in the deletion of spastin, and this is because microtubule degradation by Spastin allows for the formation of free nucleation centers to allow for the growth of new microtubules. The Spastin loss-of-function mutation also showed smaller, bunched boutons at the presynaptic terminals (see picture below), unlike wild-type linear boutons, and behavioral changes like those observed in humans. Later findings by Dr. Emily Ozdowksi implicates the role of another gene, pak3, in the mutant phenotypes conferred by Spastin loss. Pak3 was recently found to act in the subperineurial glia. Loss of Pak3 alongside the loss of Spastin restored the wild-type phenotype, suggesting some interplay of these two genes. The question remains now, does spastin function in the glia, the neuron, or both?

This summer, I am working in Dr. Sherwood’s lab, along with my lab partner, Jayden, on identifying the site of action of spastin. What I am perhaps most excited about in this project is the use of the CRISPR-Cas9 system to determine spastin’s site of action. My interest in biology was sparked my freshman year of high school after doing a short paper on CRISPR’s role in genome editing, and to finally be involved in such research myself is something my 15-year-old self would have never seen coming. 

To accomplish such a task, we will be forming a lengthy series of crosses between different fly lines to both express the Cas9 protein in specific tissues using the UAS-Gal4 system (another method of inducing gene expression using “technology” adopted from yeast) via the use of tissue-specific promoters and fly stocks that express sgRNA (guide RNA, the instructions used by the Cas9 protein) to make partial or complete spastin deletions. Jayden and I formulated some crosses for two glial drivers, and we were just amazed at how quickly we were able to pick up all this new terminology and information in just a week. Once we achieve our desired phenotypes, we will perform larval dissections and immunostaining to observe the neuromuscular junction. While so far, larval dissections are proving to be extremely difficult to execute, I am very excited to see what this project will reveal after years of research on this gene!

Figure 4 from Sherwood et al. (2004)

Figure 4 from Sherwood et al. (2004). This picture shows the difference in synaptic morphology of the wildtype (figure 4a) and the complete spastin deletion (figure 4b).

 

lncRNA – An unknown world

We all know the basics about RNA and DNA. However, did you know that out of all the RNA that gets transcribed, only 1.5% gets translated into proteins? These types of RNA are referred to as non-coding RNA, or ncRNA. The lab I’m working at this summer, Hargrove Lab, is researching long non-coding RNA, or lncRNA, which is defined as ncRNA that is over 200 nucleotides in length.

LncRNA is a new, unknown, exciting world in the scientific research community. There is not much known about their functions, mechanisms, etc. I find this to be fascinating, as the research I will be doing this summer will help find out more about this hidden, unknown world.

My research project entails screening a large small molecule library (with thousands of small molecules in it) with certain target lncRNA tertiary and quaternary structures. The targets we are investigating are lncRNA structures that are known to have a disease-causing effect in the body. The goal of our research is to find small molecules that effectively bind to the targets in order to be able to manipulate/work with these lncRNA structures.

This can get really tricky though. In addition to the fact that we are looking into so many small molecules, we want to find out not only if they bind, but a plethora of other details as well. Can we find a small molecule that only binds to one specific target and no others (Is it selective)? Does the small molecule bind the same way at different pH levels? Does the small molecule bind better in one concentration than another?

It is a large task at hand, but a fascinating one too!

I’m not bugging around!

I was never a fan of insects and spiders, but sometimes you have to face your fears for the sake of science. This summer, I’m working with the Bernhardt Lab to help gather data for our Bass Connections project: ‘A City and Its River: Durham’s Ellerbe Creek Watershed.’ This project combines the fields of chemistry, biology, and social science to seek to understand if/how wealth and social status affect the state of the environment for Durham’s Ellerbe Creek watershed residents. So you may be asking, where do the bugs come in?

I’m working alongside a graduate student to look at 3 river sites in Durham. The developed Northgate and Glen Stone sites are in Ellerbe Creek and our third site, also known as our reference site, is New Hope Creek in the Duke forest. We’re using different techniques to catch samples of fly larvae within the water, right above the water, and then far above the water. I will be taking the sticky traps that we use to catch flies far above the water and using them to measure weekly biodiversity and abundance across all three sites. Then, I will use physical data such as stream temperature, precipitation, storm surges, etc. to see if they have any correlation with biodiversity and abundance. Basically, my general aim is to see why biodiversity and abundance differs across all three sites. I hypothesize that urban stream sites will see lower biodiversity due to storm surges and higher temperatures, but this is yet to be seen. My work will involve counting and identifying insects on the sticky traps each week. I’m not particularly fond of bugs, but I am fond of my research question, so I just have to pull through!

Ubiquitin, Neurodegeneration, and the Powerhouse of the Cell

This summer I will be investigating Rad6’s role in the fermentation and cellular respiration metabolism of yeast. Now, you may be wondering how this is relevant, why this is interesting, or why you should care. Let me give you some background.

As a reminder, I am working at the Silva Lab which focuses on protein and ribosomal ubiquitination in response to stress! Ubiquitin is a very important and highly conserved small protein in the cells of all eukaryotic organisms. Ubiquitin’s function is well…ubiquitous. It has many functions in many places throughout the cell, but the one you have probably heard of is protein degradation, where ubiquitin is used to flag proteins that need to be degraded by the proteasome, perhaps because they have been damaged or are no longer of use to the cell. Findings suggest that a large array of neurodegenerative diseases show aggregates of ubiquitin-flagged proteins (Tai and Schuman 2008). This indicates how important ubiquitin pathways are for the function of neurons! Ubiquitin is an incredibly important protein and it serves a prominent role in translation (one the lab is focused on elucidating) when cells are exposed to oxidative stress. 

One of the lab’s main proteins of interest is called Rad6 in yeast. Rad6 is a ubiquitin-conjugating enzyme or an E2 (for those that are relatively familiar with ubiquitination). It’s involved in all sorts of pathways, such as chromatin silencing at telomeres, protein degradation, histone ubiquitination, and DNA repair. Its homolog in humans is called UBE2A and its malfunction is linked to several diseases, including X-linked intellectual disability type Nascimento and Late-Onset Parkinson’s.

Back to my research project. Why am I investigating Rad6 in the context of yeast? Well, yeast are one of the few organisms that prefer fermentation over cellular respiration. They can switch their cellular metabolism to cellular respiration depending on their environment. Before I came along, the lab found some interesting and unusual observations (more on that in my upcoming chalk talk) in the rad6∆ strain of yeast when it is in fermentation. In addition, mutations in the human UBE2A protein that lead to X-linked intellectual disability type Nascimento were found to affect the function of mitochondria in neurons (Verstreken et al. 2013). As you know, mitochondria are pretty important for cellular respiration! My job will be to take these initial observations and see if I can try to figure out what is going on here. Whatever findings result from this project, I am truly excited to contribute to the lab’s overarching goal of investigating Rad6 and ubiquitination during oxidative stress!

The Ubiquitin-Proteasome System (UPS) from Tai and Schuman 2008

The Heebie – Species

This summer I will be working in Rausher Labs. Much of their research deals with different evolutionary biology concepts, and their main focus is plants. My project specifically is looking at species boundaries, which I am just now starting to wrap my head around. Since middle school, many of us were told that if ” Two organisms cannot make viable offspring, they are from two different species”. However, the lines are more blurred than that. There are examples (especially in the plant world) that prove this wrong. I work with one of the biggest examples everyday in the lab, morning glories.

When you look up the scientific names of many plants, like your common maple, or sunflower, you’ll find the full scientific name including the species of the plant However, it is a little harder to find a clear scientific name for morning glories. This is because different kinds of morning glories are constantly interbreeding. So now they have just became a  tangled mess of what used to be distinct species. That is not the only issue. These hybrids can make viable offspring as well, so the previous rule I learned in high school has been made blurry.

The goal of the experiment is to see what specific species boundaries there are for morning glories. Also to see if there are any specific phenotypes that are always passed to hybrids and if there are any that are rarely passed down successfully. We are doing this by growing different kinds of morning glories in the greenhouse, many of them hybrids made from other plants we grew in the lab. Everyday we collect seeds, detangle, an score the flowers of each plant. We are looking at the limb and throat color of the plants, specifically we are looking to see if the limb and throat colors turn out white, or if the bloom pink limbed and purple throated flowers.

 

 

Drinking in the Dark

The experiment I will be primarily working on uses a relatively common animal model called drinking in the dark or DID. We’re trying to see if there will be a genotypic difference between the amount drunk by the mice which could provide insight into the relationship between microglia and excessive alcohol consumption. 

This is a chronic experiment so this means we’re giving ethanol to mice during the dark cycle of the day for 6 weeks. The mice we’re working with are a transgenic line of myD88 mice. Myd88 is a 2 pronged pathway that downstream produces inflammatory cytokines. In our mice, we also use the cre-lox system. This allows us to selectively knock out one arm of the myD88 pathway only in microglia in some of the mice (cre positive) and the other mice are fine (cre negative). In some of our mice (the knockout ones), the amount of inflammatory cytokines is severely depressed but not completely eliminated since one arm of the pathway is still functioning. Preliminary data shows that female mice, in general, drink more. This isn’t necessarily surprising because from what I’ve learned sex differences are not uncommon when conducting microglial experiments. It will be interesting to see if this trend continues for the whole six weeks or if a significant genotypic difference will emerge as well when analyzing the rest of the data. 

Mechanism: Easy Question, Not So Easy Answer

The last time I had to come up with a mechanism, it was on a (very tough) organic chemistry exam that I didn’t do very well on. Yet, whether it’s quantum mechanics or enzyme kinetics, asking how something works is one of the most fundamental questions in science.

Over the past couple of months, researchers have worked at remarkable speeds to figure out how the novel coronavirus (SARS-CoV-2) that has quarantined the world for 13+ months works. Particular attention has been paid to the all-too-familiar spike protein, which contains a particular region that can bind to a specific receptor (ACE2) on human cells. What makes this virus particularly infectious, however, is its ability to evade the immune system and pre-activate its spike proteins for cell membrane fusion. Thus, any effective treatment for SARS-CoV-2 would need to interfere with this process in which the virus can efficiently infiltrate human cells.

Griffithsin (abbreviated GRFT) is a red algae-derived protein that exhibits broad antiviral behavior against a wide variety of viruses, and scientists have most recently been interested in its ability to inhibit HIV cell entry. GRFT works by binding to various glycosylation sites (sugar scaffolding) present on all kinds of viral proteins, and has been shown to be effective against cousins of the current coronavirus, such as SARS-CoV and MERS. However, the detailed mechanism by which this protein inhibits coronavirus infection is not particularly well understood. This is especially true for SARS-CoV-2, a virus which remains every bit as mysterious as it is new.

Structural depiction of griffithsin (dimer form) (Xue et. al. 2012)

This summer, I’ll be figuring out how griffithsin blocks SARS-CoV-2 from entering cells, working to understand the complex interactions between GRFT and the coronavirus spike protein that allow for this unique behavior. Given my past history with figuring out mechanisms, it seems like a daunting task, but I have no doubt that I’ll learn a lot about experimental design along the way.

Monkeying Around With the Animal Model

This summer I’ll be looking at the degree to which social behavior is heritable in the Amboseli populations of baboons, including the age at first groom and age at first agonism of maturing baboons. Because age at first groom is correlated with baboon survival and thus fitness (see below,) we’re really curious to see the extent to which this behavior is determined by genetics.

The problem is that there are plenty of cofactors that can basically confuse statistical models we use to interpret our data. It’s difficult to know if one mother’s offspring tend to groom earlier because of their genetics or because they have higher access to resources because of their mother’s social position or because they have more maternal aunts, for example. Because of the statistical noise that all of these covariables introduce into standard linear models, we use something called the animal model to increase the statistical power of our tests by accounting for the possiblity of this covariation.

Something I’m especially looking forward to investigating is if the heritability of social behavior varies between various groups of studied baboons in the Amboseli basin. Because it lies at the boundary between Papio cynocephalus and P. anubis, most baboons there are hybrids to some degree. While we know that more anubis-like individuals experience competitive advantage against their more cynocephalus-like neighbors, it’s not clear why this is the case. Regardless, understanding the causes of this could be important to predicting demographic trends of baboon populations in East Africa.

Baboon distribution in Africa. Note that P. anubis and P. cynocephalus meet in Kenya. Photo courtesy of ResearchGate.

Relevant Papers:

  1. Alberts S.C. 2019. Social influences on survival and reproduction: Insights from a long‐term study of wild baboons. Journal of Animal Ecology 88:47–66,
  2. Silk J.B., Alberts S.C., Altmann J. 2003. Social bonds of female baboons enhance infant survival Science 302:1231-1234
  3. Archie E.A., Tung, J. Clark M., Altmann J., Alberts S.C. 2014. Social affiliation matters: both same-sex and opposite-sex relationships predict survival in wild female baboons. Proceedings of the Royal Society B: Biological Sciences 281:20141261
  4. Kruuk Loeske E. B. 2004. Estimating genetic parameters in natural populations using the ‘animal model’ Phil. Trans. R. Soc. Lond. B359873–890
  5. Charpentier, M.J.E., Fontaine, M.C., Cherel, E., Renoult, J.P., Jenkins, T., Benoit, L., Barthès, N., Alberts, S.C. and Tung, J. (2012), Genetic structure in a dynamic baboon hybrid zone corroborates behavioural observations in a hybrid population. Molecular Ecology, 21: 715-731. https://doi.org/10.1111/j.1365-294X.2011.05302.x

The Circuitry of Immunity

I’m sure the last thing anyone wants to read about right now is more immunology. A lot of people (myself included) have thought something like this at least once in the past few months: “If I hear the word ‘antibody’ one more time, I will walk out of this room right now.” Well, the bad news is I’m still going to be talking amount immunology, but the good news is my research has to do with a different kind of immune system! While antibodies are essential to fine-tuning humans’ adaptive immune response to very specific pathogens, there is another, broader kind of immune system called the innate immune response. The innate system recognizes general biological traits associated with pathogens, like glycan (a component of many bacterial cell walls), unlike the very specific adaptive system. Given the recent explosion of coverage concerning antibodies, it would be easy to think the innate response is just irrelevant, but that wouldn’t be doing justice to the evolution and ubiquity of innate immunity. Immunologists and developmental biologists discovered that innate immunity is way older than adaptive, and is present in a vast array of organisms compared to adaptive immunity, which is really only present in vertebrates (that’s us!). Because adaptive immunity likely evolved from the innate response, it also means that the two are far more interconnected than anyone previously thought.

Because the innate system is so old, it’s had a lot of time to diversify the molecules and cells involved, making it hard to draw evolutionary connections between the immune systems of humans, which have both innate and adaptive immune systems, and those of sea urchins, which have only an innate system. So instead of looking only at the kinds of immune cells and molecules produced, essentially the “end results” of immunity, developmental biologists and immunologists have turned to gene regulatory networks (GRNs) that determine when and how these immune cell types develop. Through these GRNs, we can better understand both how immune systems evolved and what role each gene plays in immune cell development and function.

Recently, the McClay Lab discovered a gene that is very highly expressed in a certain cell type of the embryonic sea urchin innate immune system. There’s also a kind of “master signal” at the beginning of sea urchin development which has been really thoroughly investigated over the past couple of decades. My work in the McClay Lab this summer focuses on finding out if there is a significant connection between this original “master signal” and this specific “end product” gene in the immune cells. If the genes can affect each other, it means there is probably a GRN connecting them, but we have very little idea of how many gene components are in the circuit, what they are, or what they do – all out there to be discovered. If there isn’t an observable connection between the master and end genes, then the end gene could be the tail of a completely unexpected GRN, which poses an equally exciting opportunity for research and discovery! I’ll be using a battery of microscopy, molecular biology, and moving-colorless-liquids-back-and-forth techniques to get at this GRN, and probably producing some really cool pictures of colorful embryos along the way (stay tuned)! Although this project may seem daunting, characterizing this genetic circuitry could help us better understand the incredible harmony between diversity and unity in immune systems across all domains of life, and provide some really awesome insights into how to analyze rapidly evolving biological systems, like the immune system. The past week working on this project has been a complete joyride, and I can’t wait to keep it going through the summer!

Work Together Now!

Have you ever tried to get a group to work together? If so, how many were you trying to reconcile? With that image in your head, imagine trying to do that with hundreds or even thousands of people. Now, make those people cells which can’t speak and who each want to move in a random direction independent of the group, and you’ll begin to see the wonder that is collective cell migration.

This summer, I am working in the Hoffman Lab studying collective cell migration (CCM). CCM is the process by which hundreds (if not thousands!) of cells move together as sheets, groups, or chains in the same direction with the same velocity. However, each cell is independent and can generate propulsive forces without needing outside help. So, how then can so many cells all stay connected and coordinate their movements? Our lab wants to discover the molecular mechanisms that enable this incredible phenomenon.

Specifically, we are interested in how mechanotransduction, or the conversion of mechanical forces into biochemicals signals, mediates this process.  Many key proteins have been identified that deform when the proper force is applied, changing their structure and function. These force-dependent conformations can regulate biochemical pathways that influence complex cellular processes, like the cell coordinating its movement with the larger group. These “mechanosensitive” proteins allow the cell to turn local forces into chemical signals that can impact and influence the entire cell!

But one of the big challenges facing this field is identifying which proteins are involved and how this process is regulated. Much of the actual molecular mechanics of it all is still poorly understood. Until we really have a tighter grasp on these mechanisms, we are hindered in our ability to manipulate CCM, both to understand it better and harness it for future applications in cancer biology, wound healing, and regenerative medicine.

This is where I come in! This summer I am using molecular cloning to engineer fusion proteins which will allow us to study the dynamics of some of these mechanosensitive proteins in live cells. You can think of fusion proteins as the Frankenstein’s monsters of the protein world. By cutting and pasting the DNA using traditional molecular cloning techniques, we can take the mechanosensitive proteins we are interested in and attach a fluorescent protein to the end of it. Because this protein is fluorescent (lights up when hit by the right wavelength of light), we can use this “biosensor” to see the protein in live cells! With this, we can better understand its localization and dynamics in the cell and its involvement in CCM. Additionally, by creating biosensors where the mechanosensitive protein has a single amino acid substitution whose effect on the protein is already known, we can further study the function of our protein in CCM by examining the mutant’s effects relative to the normal biosensor.

So, that’s my plan for the summer. Stay tuned to see if I can actually brighten up the world a bit!

Bonus: These fluorescent proteins lets us take really beautiful pictures of cells and cell sheets! Photo courtesy of Hoffman Lab.