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• Kyle Bledsoe
• Clementine Chen
• Jessica Cramer
• Daphne Hansell
• Sam Kim
• Gillyoung Koh
• Angie Quiroz
• Gwendolyn Rewoldt
• Shriya Sai Shivakumar
• Jo Smith
• Chloe Tang

Kyle Bledsoe

Advisor: Adam Williamson

CRISPR-Cas9 edited cell line library to understand the fundamental biology of Batten Disease

Batten Disease is a set of pediatric, neurodegenerative lysosomal storage diseases caused by mutations in any of fourteen different CLN genes. Even though all but one of the genes have been identified, the fundamental biology underlying the role of these proteins and the disease is still not well understood. Additionally, very few models of the disease has been done in human cells. The Williamson lab focuses on the basic biology of the immune system through studying phagocytosis, an ancient method used to clear debris inside cells. Lysosomes digest the material gathered through phagocytosis, so knowing the role of these proteins in phagocytosis is critical to understanding their disease phenotypes. To understand more about the proteins and their role in phagocytosis and Batten disease, I will begin making a library of CRISPR-Cas9 edited cell lines. This library will create a way to study Batten disease and the data generated will greatly increase the knowledge about the role of CLN proteins and Batten disease itself.

Clementine Chen

Advisor: Bárbara Bitarello

Evaluating the evidence for adaptive evolution of the TAS2R38 bitter taste receptor gene in primates

Background: As one of the senses that enable people to discern and differentiate diverse flavors, taste is facilitated by taste receptors. There are two types of taste receptors, with type I in charge of detecting salty tastes and type II receptors detecting sweet, umami, and bitter tastes. TAS2R, the gene family encoding type II taste receptors, specifically account for bitter taste receptor function. These receptors are characterized as G-protein coupled receptors (GPCRs). Within this protein family, 25 protein-coding genes and 11 pseudogenes are present in humans. Collectively, they are capable of interacting with a vast array of molecules, each receptor exhibiting unique molecular affinities.

Due to the fact that bitterness is important for animals to potentially prevent swallowing poisonous substances, we believe that the TAS2R genes that code for bitter taste receptors may have evolutionary significance to humans and other primates. Especially, the gene TAS2R38, which binds to 23 known ligands, is one of the most studied among the TAS2R family. This gene is specifically associated with the perception of the taste of phenylthiocarbamide (PTC), a chemical whose structure closely resembles goitrin, a naturally occurring chemical in the cabbage family, including vegetables such as broccoli. Due to genetic polymorphisms within populations, individual perceptions of PTC's taste can greatly vary. Some scientists speculate that human ancestors' ability to perceive the taste of natural chemicals, similar to PTC, would have assisted in avoiding toxic plants, thereby suggesting the possible evolutionary significance of the TAS2R38 gene. Previous studies showed that, in humans, there are two distinct TAS2R38 haplotypes at moderate frequencies, suggesting the presence of long-term balancing selection. However, a recent study proposes that demographic events in human evolution can account for the recent evolution of this gene, thus restricting balancing selection to the distant evolutionary past of TAS2R14.

Question & Hypotheses: Due to the contradictory results from prior studies, we aim to gain clarity on whether the TAS2R38 gene underwent adaptive evolution (positive selection and/or balancing selection) in humans and other primates and, if so, under which timescales. If so, are there specific sites that show increased rates of evolution? We hypothesize that the evolutionary significance of each site is related to their ligand binding ability, and we anticipate observing an elevated dN/dS value (the ratio of nonsynonymous substitution rate to synonymous substitution rate, a measure of adaptive evolution) at sites that determine ligand specificity. We also want to test if there are any primate lineages where we find evidence for accelerated evolution for the TAS2R38 gene. In addition, another test will be conducted to determine whether TAS2R38’s evolution has been influenced by the specialized diets of humans and other primates as we hypothesize that feeding habits could play a role in shaping the genetic variations of the TAS2R38 gene.

Preliminary Findings and Continuing Work: Throughout the past year in the Bitarello Lab, we’ve been working to conduct a phylogenetic analysis of 53 primate species by creating phylogenetic trees using the aligned genes and finding the sites of TAS2R38 genes of primates and humans that are suggested to have been subjected to adaptive evolution. By using molecular evolution models in HyPhy and PAML, we confirmed by different tests that specific sites of TAS2R38 have strong evidence of positive selection. Moving forward, the branch test and branch-site test are our next steps to work on. Using the branch model, we will assess the variation in the rate of evolution (dN/dS value) across different branches in the phylogenetic tree, enabling the identification of lineages under positive selection. On the other hand, the branch-site model allows us to confirm the evidence of positive selection at individual sites along specific branches, providing a more fine-grained picture of the adaptive molecular evolution of the TAS2R38 gene.

The findings from phylogenetic analysis could offer new perspectives in various scientific domains. Understanding the evolutionary importance of the TAS2R38 bitter receptor gene enhances our comprehension of cultural evolution across human history and the impact of dietary preferences on human evolution. Additionally, identifying the sites subject to adaptive evolution aids in comprehending the mechanism of ligand binding in the bitter taste receptor or other selective pressures, hitherto unknown, that may be driving such accelerated evolution, and this knowledge can assist in the development of medications with a higher tolerance for patients.

Jessica Cramer

Advisor: Gregory Davis

Using RNAi to Knockdown Insulin-Like Peptide Signaling Pathway in the Pea Aphid (Acyrthosiphon pisum)

The Davis Lab is investigating the pea aphid, Acyrthosiphon pisum, which exhibits a reproductive polyphenism in which individuals of the same genotype develop into asexual and sexual forms depending on environmental conditions. During the summer, when days are long, aphids reproduce asexually via live birth (viviparously) but, as it gets closer to winter and the days get shorter, aphids can sense this change and give birth to offspring that develop to reproduce sexually. These offspring are both male and female, the latter of which will lay eggs (making them oviparous) that are able to withstand the winter frost. In the spring, these eggs will hatch into another round of asexually reproducing aphids. This switch in reproductive modes is thought to be mediated by an unknown biochemical factor known as “virginoparin,” which is produced by the mother and delivered to her embryonic progeny prior to birth.

Previous work in the Davis lab using the anti-insulin pathway drug wortmannin has suggested that insulin signaling may be required to specify asexual fate, with the tantalizing possibility that insulin-like peptides (ILPs) produced in the aphid’s brain may constitute virginoparin. Wortmannin, however, is capable of interfering with multiple signal transduction pathways other than the insulin pathway. A study done by Cuti et al. (2021) found that ILP1 and ILP4 are synthesized by a group of neurosecretory cells (NSCs) known as Group 1. These ILPs are potentially delivered to the embryo by the mother via the mother’s nervous system, as they are released very close to the site where the embryos are developing. We propose to test whether ILPs are required for specifying asexual fate by injecting double stranded RNA (dsRNA) against ILPs to trigger RNA interference (RNAi), a technique that has been used successfully in aphids to interfere with gene function, and block the ability of the mother aphid to deliver ILPs to her developing embryos. In order to do this, we would try both injecting dsRNA directly into aphids and delivering dsRNA via plant-mediated feeding. Both techniques have successfully been shown to transmit dsRNA and activate RNAi in aphids (e.g., Ding et al. (2017)). We  expect that with the mother aphid no longer able to transmit the ILPs to the developing embryos, they will default to the sexual fate. By blocking both the ability of the mother to send ILPs to the embryos and the ability of the embryos to receive the ILP signal, we hope to gain a clearer understanding of the possible role that ILP signaling plays in the aphid reproductive polyphenism.

Daphne Hansell

Advisor: Bárbara Bitarello

Here, we introduce balselr, a comprehensive R package designed to bridge this gap. Balselr will operationalize the NCD approach, enabling researchers to identify regions of the genome under LTBS efficiently. This will allow researchers to leverage both single-locus data and broader genomic data with an accessible, easy-to-use interface, even for individuals with basic proficiency in R. The package will not only performs calculations of NCD1 and NCD2 statistics but also facilitate the preparation of input files based on commonly used data formats in the field, such as VCF files for SNP data. The package will also provide help pages and tutorials on GitHub.  By making using the NCD statistic more accessible, balselr aims to catalyze advances in our understanding of the prevalence, targets, and broader implications of LTBS in population and evolutionary genetics.

Sam Kim

Advisor: Gregory Davis

Investigating Insulin-like Peptides as the Maternal Signal in Determining Acythosiphon pisum Reproductive Fate

Understanding the mechanism of developmental plasticity can provide insight into phenotypic evolution and the ability to adapt to one's environment. In the G. Davis Lab, the pea aphid, Acythosiphon pisum, serves as a model organism for studying developmental plasticity in that this species exhibits several cases of discrete plasticity, a phenomenon known as polyphenism. For example, in the so-called reproductive polyphenism, aphids produce asexual versus sexual progeny during cyclical parthenogenesis in response to seasonal changes in night length. Specifically, short summer nights cue the production of asexual females, which reproduce viviparously. In contrast, long fall nights cue the production of sexual females, which reproduce oviparously.

            This summer, I plan to study the reproductive polyphenism of aphids and the mechanism that mediates the effect of night length on reproductive fate. Previous studies suggest that short nights cue a maternal signal that specifies the asexual fate when received by embryonic progeny. This asexual-promoting maternal signal is known as virginoparin and is the focus of my study. Two competing hypotheses suggest that virginoparin is either juvenile hormone (JH) or insulin-like peptides (ILPs), respectively. Available evidence suggests that the JH is sufficient to promote but not maternally required to specify asexual fate, while ILPs are required to specify asexual fate. If insulin is virginoparin and JH acts to up-regulate insulin, it would explain why JH is sufficient but not required to specify asexual fate. My goal this summer is to apply both JH, and Wortmannin, a drug that inhibits the insulin pathway, to aphids to potentially establish that the sufficiency of JH to specify asexual fate is in fact dependent on the insulin pathway, providing support for this model.

Gillyoung Koh

Advisor: Bárbara Bitarello

Investigation of Long-Term Adaptive Evolution In TAS2R14, A Promiscuous Bitter Taste Receptor In Primates

One of the research areas in the Bitarello lab is on the T2R or TAS2R gene family, which are genes coding for Type II taste receptors that allow organisms to taste bitterness via binding of bitter agonists to their corresponding receptor(s). We are specifically focusing on TAS2R14 (Taste receptor type 2 member 14), which is very promiscuous and binds to more than 150 known, diverse bitter agonists. This is roughly twice the second-highest number of known ligands that a bitter taste receptor from the T2R family can bind to. One study by Bitarello et al. (2018) suggested long-term balancing selection (preservation of certain alleles in a population over long periods of time) may have played a role in the evolution of TAS2R14 (the gene that codes for this receptor) in the primate lineage. 

We are testing to see whether adaptive evolution in TAS2R14 occurred and in which codons. If we see significantly high rates of nonsynonymous substitutions / synonymous substitutions (dN/dS) in codons involved in recognizing and/or binding to bitter agonists, this would suggest that adaptive evolution targeted codons involved in binding specificities of the receptor. We hypothesize that we will find codons involved in receptor-agonist interactions to undergo long-term adaptive evolution. This stems from the widespread view that being able to taste bitter compounds has an adaptive role in preventing the ingestion of toxic compounds, which are often bitter. Additionally, we want to see if phylogenetic groupings based on taxonomy or dietary preferences have undergone certain selective pressures. 

In the past year, we ran several phylogenetic tests to assess whether this gene, overall, has undergone adaptive evolution in primates and, if so, in which sites specifically. We used HyPhy, a software package that implements several codon substitution models. From HyPhy, we used GARD, BUSTED, FEL, MEME, and SLAC. GARD looks for breakpoints in the data, which are signs of recombination and can lead to false positives in the data concerning adaptive evolution. After finding no breakpoints, we used BUSTED to look for broad evidence of adaptive evolution. The BUSTED results we obtained suggested that there is evidence of adaptive evolution, without pointing to specific sites or lineages in the tree. Then, we ran FEL, MEME, and SLAC to find codon sites under adaptive evolution using a p ≤ 0.1 criterion. We discovered suggestive evidence of long-term adaptive evolution, but we want to obtain additional supporting evidence to either support or not support our hypothesis. 

Moving forward, we will use PAML, a package of programs to use for analyzing our DNA sequences, to find codon sites under adaptive evolution and compare with the found codon sites from HyPhy. We aim to find a reliable set of specific codons in TAS2R14 that have been targeted by long-term adaptive evolution by overlapping results from different models and assess whether these codons correspond to areas of the receptor that interact with the bitter agonists. We will also use branch models to assess which primate lineages specifically have been subject to this long-term adaptive evolution for TAS2R14, and whether this corresponds to different diets observed in primates. 

Angie Quiroz

Advisor: Thomas Mozdzer

Stomatal Conductance in Relation to Fluorometry in Phragmites Australis

The Mozdzer lab has been looking at Phragmites Australis, an invasive marsh species that can be used as a model organism due to their dominant and efficient growth globally. Their ecological perseverance and genetic diversity is crucial to understanding how their genetic makeup plays a role in determining favorable characteristics which can be used to modify and support coastal wetlands. There are various components and areas of focus regarding this common reed, therefore, it is critical that I get exposure to gathering different types of data from relative growth rate to light curves which I can use to connect and interpret my own project. Over the course of the summer, I will be looking at stomatal conductance and fluorescence which measures, chemical reactions and physiological aspects of the plant by using the Li-600 Promoter/Fluorometer. I hypothesize that plants with greater nocturnal stomatal conductance are susceptible to a greater rate of withering. This will give us more insight to how plants with a different genetic makeup may respond to future levels of elevated carbon dioxide. Given that Phragmites Australis is an invasive marsh species, there have been countless efforts to remove it from coastal wetlands, however, these experiments could shine light on previous studies that also hint at the benefits of this common reed. The money that goes towards eliminating Phragmites Australis could instead be used for more experiments to determine ways in which the presence of invasive marsh species could become more beneficial to surviving the effects of climate change on coastal wetlands. 

Gwendolyn Rewoldt

Advisor: Thomas Mozdzer

Investigating heritable trait variation in functional model plant Phragmites australis

My project is part of the bigger C-EVO project which is investigating how rapid evolution in response to global changing factors and ecological processes are potentially interacting and influencing each other using salt marsh Phragmites australis as the study organism. Salt marshes are a very productive ecosystem with a limited number of species and many ecosystem engineers, like Phragmites. While results from prior studies indicate that Phragmites traits related to C cycling exhibit heritable variation, my current project is gathering data on traits and their variation between the 198 unique genotypes with a focus on leaf canopy traits to further assess heritable versus non-heritable traits. Here at ��Ӱֱ��, we are conducting a huge common garden experiment where we have taken 198 individuals with unique genotypes to assess if measures of individual plant traits are correlated with C cycling and if they are heritable. Using methods like collecting SLA samples, measuring stem heights, and potentially measuring leaf thickness, my measurements will be used to parameterize a model to further evaluate the potential influence of evolution on C cycling. I hypothesize that we will see different measurements of leaf canopy traits that are correlated with genotypic variation. In the following years, the project will continue by crossing each dam with a different sire, creating a F1 generation with known parentage from which functional trait data can be collected to estimate heritability.           

Shriya Sai Shivakumar

Advisor: Bárbara Bitarello

Assessing the impact of smoking behavior on genetic blood trait variation via alcohol intake through multi-ancestry analysis

Genome wide association studies (GWAS) have associated thousands of loci with quantitative human blood trait variation. Blood trait loci and related genes may regulate blood cell-intrinsic biological processes, or alternatively impact blood cell development and function via systemic factors and disease processes. Observational studies show conflicting results. Studies have shown that both alcohol and smoking behaviors impact blood cell traits. Smoking was linked with higher red blood cell and hemoglobin count, and alcohol showed altered blood cell traits and function. Clinical observations linking behaviors like tobacco or alcohol use with altered blood traits can be subject to bias, and these trait relationships have not been systematically explored at the genetic level. They could be subjected to unmeasured or unrecognized confounding factors. Previous blood traits GWAS adjusted for both tobacco and alcohol use behaviors based on presumed multi-trait impacts (instead of specifically blood traits) but the effects of these behaviors on specific blood traits or developmental lineages are unclear. The Vuckovic et al Cell 2020 GWAS was not adjusted for smoking or alcohol use, and has increased power, giving the opportunity to analyze the determined effects of these behaviors on blood traits.

Using a Mendelian randomization (MR) framework using GWAS summary statistics, we can confirm causal effects of smoking and drinking on different blood lineages. Using Multivariable MR (MVMR) and causal mediation analyses, we can identify mediating or confounding traits to explain the causal effect. MVMR can also help establish a causal direction between smoking and alcohol as they are highly genetically correlated. We previously did this for GWAS from entirely European cohorts (Shivakumar et al. 2023) and showed that there is a casual direction from Smoking to Drinking, and that drinking has a significant negative effect on erythroid traits. After correcting for the exposure of alcohol, we negated all the significant effects of tobacco use on blood cell traits.

However, the majority of GWAS and overall collected samples/data are based on a white European ancestry, leading to a dangerous lack of representation in the studies and associations.This has been shown to impact the utility of tools like polygenic risk scores and CRISPR editing in groups not represented in the original studies, so diversifying studies is critically important.  Moving forward, we will replicate our analyses in a multi-ancestry approach. Using the filtered meta-analysis summary statistics and polygenic risk score from the Saunders multi-ancestry meta analysis of 3.4 individuals, we will analyze ​​nicotine and substance use and check for disparities among different GWAS. We hope to confirm that what we observed in Europeans is true for other groups as well. Collectively, our findings might demonstrate a novel role for genetically influenced behaviors in determining human blood traits, revealing opportunities to dissect related pathways and mechanisms that influence hematopoiesis.

Jo Smith

Advisor: Thomas Mozdzer

Heritable trait variation in relative growth rates and herbivory among Phragmites australis genotypes from two populations

Salt marsh ecosystems provide many vital ecosystem services, but their existence is threatened by accelerating global change factors including rising concentrations of CO2, nutrient pollution, and human development. The common reed, Phragmites australis, is considered to be a new model organism for both invasive species and plant physiology. Recent research has demonstrated that exposure to both near future levels of CO2 and nutrient enrichment have reduced intraspecific levels of genetic diversity and altered plant traits suggesting that populations are rapidly evolving. In order to evaluate the heritability of plant traits, 120 unique genotypes of P. australis were collected from two populations in MD, USA, the Smithsonian Global Research Wetland (Edgewater, MD) as well as Parker’s Creek (Prince Frederick, MD). A total of 240 genotypes were grown in a common garden at ��Ӱֱ��.The common garden allows me to evaluate heritable traits by removing confounding environmental factors. Given previous research that shows plants exposed to elevated nitrogen have an increased relative growth rate as well as a higher number of aphids per plant. These findings hypothesize that genotypes that were treated by nitrogen will have a higher growth rate and aphid density than plants that were not exposed to elevated nitrogen. I measured plant relative growth rate by measuring the height of 3 tagged shoots from each pot. In addition, given evidence of differential herbivory, also measured the presence and severity of different types of herbivory between the plants. Herbivory was categorized between sucking insects such as aphids, chewing insects and animals, and stem boring insects. This research aims to provide insights into which traits are heritable, to provide further insight to the evolutionary ecology of salt marshes as a whole.

Chloe Tang

Advisor: Tamara Davis

Methylation patterns of multiple DMRs in 12.5 dpc wild type and methyltransferase mutant mouse embryos

Mammals have evolved to have many forms of gene regulation and genomic imprinting is one of the forms that only allows the expression of one parental allele from an organism’s genome. Imprinted genes are regulated epigenetically through DNA methylation, facilitated by DNA methyltransferase (Dnmt1), where a methyl group (CH3) is added to one allele to generate a differentially methylated region (DMR). The methylated allele is silenced, leading to differential gene expression of the parental alleles. Methylation at primary DMRs, which is inherited at fertilization, is stable and symmetrical across development whereas methylation at secondary DMRs, which is acquired during early embryogenesis, is less stable and asymmetric. The proper establishment of methylation patterns is crucial for embryonic development as abnormal  methylation patterns can lead to inappropriate gene expression, developmental disorders and diseases such as cancer.

In addition to different methylation patterns between primary and secondary DMRs, previous research suggested that methylation is well maintained at primary DMRs in mouse embryos with a hypomorphic mutation of Dnmt1 (p allele) but is dramatically reduced at secondary DMRs. These pieces of evidence implied that Dnmt1 might function differently at different loci. We noticed that secondary DMRs have greatly reduced methylation in P allele mice and wanted to understand the reason behind this. We hypothesize that the increased hemimethylation levels are due to reduced Dnmt1 fidelity. To understand the underlying mechanism of this methylation loss, I am analyzing the methylation patterns at multiple primary and secondary DMRs in 12.5 days post conception (dpc) wild-type and Dnmt1 mutant mouse embryos to test this hypothesis.