Zinc pyrithione (ZPT) is a zinc conjugate that has been used as a preservative in industrial manufacturing supplies for preventing microorganismal growth on materials such as latex and plastics, as well as in shampoos and cosmetics. Little is known about the antibacterial effects of ZPT on common bacterial pathogens, especially when in the form of a solid surface barrier. Here we analyzed the inhibitory effects of ZPT in the form of a solid surface coating, provided by Cao Inc., on the bacterial strains E. coli and E. faecalis. Noufa Khan and I spread plated these bacteria on agar plates that contained a coat of 0%, 2.5%, or 5% ZPT underneath the agar. This indirect ZPT contact did not inhibit E. faecalis by any of the three ZPT materials, while E. coli was inhibited by 5% ZPT only. Noufa and I next tested the effects of direct contact of ZPT on bacterial growth by adding a designated amount of E. coli or E. faecalis directly to 3x3 cm squares of films containing 0%, 2.5% or 5% ZPT, three separate times, each in triplicate. After incubating for 24 hours, we removed the bacteria from these squares by vortexing and sonication into tubes containing liquid media, and plated them after serial dilution onto trypticase-soy agar plates. We counted the colonies to determine the extent of inhibition, if any. With direct ZPT contact, 2.5% and 5% ZPT inhibited E. coli and E. faecalis up to 95-99.9% relative to growth on the 0% ZPT control, with a higher concentration of ZPT showing a higher inhibition rate. These results demonstrate ZPT inhibits growth of common bacterial strains upon direct contact. An application to this research would be to use ZPT as an antibacterial plastic coating on door handles and other commonly touched surfaces.

The eyecup consists of retinal pigment epithelium (RPE), choroid, and sclera. Eyecup tissue is used as a proxy for RPE. RPE metabolism is assumed to be dominant in eyecup preparations, but this has yet to be proven rigorously. In this study we probe the contribution of retinal pigment epithelial cells to eyecup metabolism. We approach this question by determining metabolic flux in eyecup tissue from control and mice injected with 50 mg/kg of the selective RPE cell toxin sodium iodate (NaIO₃). Seven days later we sacrificed mice and dissected eyecup tissue into physiological buffer, then quantified extracellular flux of glucose and lactate using spectrophotometric assays or intracellular flux using gas chromatography-mass spectrometry. We compared glucose flux in eyecups from saline-injected control mice to eyecups from NaIO₃-injected mice. NaIO₃ treated eyecups released 42% less lactate from media than controls (p<0.05), despite negligible glucose consumption. Surprisingly, most glycolytic and tricarboxylic acid (TCA) cycle metabolite levels were unchanged by NaIO₃ injection. NaIO₃ treatment did however significantly decrease levels of lactate and the TCA cycle metabolites malate and fumarate. Flux from glucose to lactate and malate was also decreased by NaIO₃ treatment. Our results suggest that lactate export in the eyecup is partly due to retinal pigment epithelium metabolism. However, metabolite levels and flux were partly maintained, implying that RPE metabolism may not be dominant in the eyecup. Remaining glucose metabolism may be from endothelial cells, or microglial cells recruited to the eyecup after NaIO₃ treatment. These findings may give insight into diseases affected by RPE metabolism, including some forms of retinitis pigmentosa and age-related macular degeneration. However, further analysis is still required to fully understand the role of RPE metabolism in the eyecup.

Mammalian retinas function in a more hypoxic environment than most tissues. The retina thus does not exclusively use oxygen as a terminal electron acceptor in the electron transport chain (ETC). As a result, expression of proteins involved in energy metabolism may be affected. We determined the levels of metabolic proteins of various tissues, and found that compared to eyecup (consisting of retinal pigment epithelium and choroid vasculature), kidney, and cerebellum tissue, the retina expresses higher levels of Hexokinase I. This suggests that glycolysis may occur faster in the retina than in other tissues. We also observed higher levels of the ETC proteins cytochrome c and subunit 4 of cytochrome c oxidase levels compared to both the eyecup and cerebellum. This implies an increased capacity for electron transport in the retina, despite a lower O2 tension. We also investigated post-translational protein modifications that could be affected by a hypoxic tissue microenvironment. Lysine succinylation is one such modification, and is controlled by regulators of energy metabolism such as SIRT5. I observed that succinyl-lysine intensities were higher in the retina than in the cerebellum, kidney, liver, and eyecup. Further investigation will be necessary to determine the role that lysine succinylation plays in the retina. Through these experiments we show that retina tissue is well-suited for rapid energy metabolism in spite of its hypoxic environment.

The mechanistic target of rapamycin (mTOR) pathway is a central nutrient signaling pathway involved in regulating cell proliferation and metabolism. Targeting this pathway has promising implications for treating a variety of diseases, especially cancer and age-related diseases. Rapamycin is an allosteric inhibitor of mTOR that has been shown to extend longevity in the fruit fly Drosophila melanogaster. Despite this, rapamycin can interfere with healthy mTOR pathways necessary for survival, which is why further research on rapamycin’s mechanism is necessary to improve clinical usage. Previous research has shown that larval size is significantly decreased in fly larvae following inhibition of the mTOR pathway. However, this effect has not been studied across fly strains with differing levels of rapamycin sensitivity. In the Promislow Lab, we have found that fly strains vary in sensitivity to the effect of rapamycin on developmental timing. We are now comparing variation in sensitivity of development time with variation in rapamycin’s effect on larval size. Using D. melanogaster, we are able to selectively expose larvae to rapamycin from embryogenesis to model the effects of rapamycin. We are collecting larvae at various time points throughout their development, comparing individuals exposed to rapamycin or control conditions. We use ImageJ software to quantify larval size. Because of mTOR’s involvement in growth pathways, we hypothesized that larvae with greater rapamycin sensitivity would have significantly smaller sizes at the same time points compared with larvae with lesser rapamycin sensitivity following rapamycin exposure. These experimental results will provide insight into how rapamycin sensitivity is linked to the phenotypic effects of rapamycin on larval size. They will also help us investigate what contributes to the sensitivity differences seen between genotypes. The spectrum of rapamycin resistance in humans is unknown and so our work could help us identify targets for treatments and therapies of age-related diseases.

Traditional foams are fabricated via stochastic chemical processes that yield homogeneous material properties. Foams can exhibit a wide range of material properties by varying process controls allowing them to be used in many industrial and commercial applications. Previously, additive manufacturing could only produce foam approximations in the form of traditional lattice infill. My work employs viscous thread printing (VTP) of thermoplastic polyurethane (TPU) on a fused filament fabrication (FFF) printer, exploiting the semi-viscous nature of extruded filament to coil producing a new type of printed foam. Specimens were tested under compression to determine uniformity along principal axes and behavior under strain when compared to infill patterns, such as grid and cubic. My work establishes that VTP, using elastic materials, can be used to manufacture programmable stiffness foams as a function of density, suited to a variety of needs and should be considered as an alternative to traditional foams and other printed lattice geometries.

Becoming an adult is a significant life course transition which brings much uncertainty to an individual's life. Previous studies attribute this uncertainty to navigating a growing sense of agency, with more outlets to explore emerging adulthood and personal identities. As young adults increasingly engage with these opportunities, more time is spent away from their families. As a result, they are less susceptible to the parental, familial, and cultural pressures in the home, where parents enforce their expectations for adulthood onto their children. The lifestyle of young adults of Asian immigrant parents who identify as a gender or sexual minority (AAGSM) often conflicts with their parents' ideal of a successful transition into adulthood. Yet, they find themselves poorly represented within gender and sexual minority (GSM) spaces, often downplaying their racial or ethnic identities to feel accepted. Scholars are only beginning to examine the outcomes of this identity conflict with AAGSM young adults, and so far, none have made a clear connection between the transition to adulthood literature and the (in)visibility of Asian-American coming-out narratives. The goal of my study is to understand how parental closeness and expectations for emerging adulthood affect how AAGSM young adults perceive their preparedness for adulthood. This goal will be met by conducting semi-structured interviews, asking participants to reflect on their parents' and their own expectations for adulthood with the consideration of their AAGSM identity and relational closeness. As I am using grounded theory to conduct my analysis, the anticipated results of my study have yet to be determined. Focusing on AAGSM young adulthood is a promising area for future research, as it holds important implications on how to accomodate the intersectional experiences of individuals as culture continues to evolve. 

Why do identical twins have different lifespans? Beyond genes, what else might influence the aging process? Variation in any phenotype is due to the combined effects of genetic variation and environmental variation. In fact, there are two types of environmental variation—one is extrinsic environmental variation, such as food, temperature, etc., and the other is intrinsic environmental variation, which can lead to subtle differences in behavior, such as how much an individual eats, how long it sleeps, etc. We hypothesize that these differences can be predicted by an individual’s underlying metabolism. There are thousands of molecules that make up the structural and functional building blocks of all organisms, a domain known as the metabolome. Previously, many studies have shown that genotypes vary in lifespan, but even within a single genotype there is enormous variation in lifespan. Here we address how intrinsic environmental variation influences aging by controlling the genetic and extrinsic environmental variation under lab conditions. We designed an experiment using Drosophila melanogaster, and since Drosophila has a natural tendency to climb upwards against gravity, and climbing ability of flies decreases with age, we hypothesized that we might use climbing ability as a biomarker of future mortality risk. Using mid-life climbing ability, we separated genetically-identical flies and then analyzed each group’s lifespan. We found that within a genotype, strong climbers had a longer lifespan than non-climbers. Finding strong support for this hypothesis led us to propose that the metabolome between climbers and non-climbers might be different. Our goal is to understand the role of intrinsic variation in aging. If we can find metabolites that associate with climbing ability, and as we have shown, climbing ability is associated with aging, we might be a step closer to explaining how intrinsic environmental variation influences aging.

Throughout our lives, we generally base our idea of age upon someone’s ‘chronological age’, or how many years they’ve been alive. This, however, is not always the best indicator of aging, as people reach social and biological milestones at different ages. As an alternative, someone’s ‘biological age’ can be more representative of their progression through life. As such, research has focused on identifying biomarkers of biological age to help us better understand aging. Recent work in our lab has sought to determine the impact of several metabolites - biomolecules used for metabolism - on the biological age of the fruit fly, Drosophila melanogaster. Among the metabolites studied, histamine - a neurotransmitter involved in wakefulness and visual processing - had one of the strongest correlations with lifespan, suggesting that it plays a role in aging. In this study, we attempted to manipulate the biological age of female D. melanogaster by altering either their metabolic levels of histamine, or their perception of histamine. To do this, flies were given food supplemented with histamine or with the antihistamine hydroxyzine, a competitive inhibitor of histamine receptors. These experimental conditions were compared to control food that lacked additives. Treatment was administered continuously starting at 4 weeks and the lifespans of flies in each condition were measured. Based on our previous results, we expected to see a negative effect of added histamine on lifespan and an increase in lifespan in response to antihistamine. Our study could highlight histamine’s role in aging and lay the foundation for demonstrating that biological age can be influenced by a single metabolite.

The mechanistic target of rapamycin (mTOR) is a protein kinase that is closely linked to growth and nutrient control in a multitude of organisms. Inhibiting TOR with the drug rapamycin has been shown to increase lifespan in many species. In the fruit fly, Drosophila melanogaster, rapamycin slows development, an outcome that is perhaps closely related to its effect on lifespan. Recent work in the Promislow lab on larval development has shown that the effect of rapamycin varies greatly across different genotypes, from no impact in the time of development to a nearly doubling of development time. However, it has not yet been determined which of the three larval stages is most sensitive to rapamycin. My project attempts to answer this question. I tested the delay in larval development of larvae treated with rapamycin across six different fly genotypes, four that are known to be sensitive to rapamycin treatment, and two that are resistant. After transferring eggs to food containing rapamycin or control food, I collected larvae over three days and staged them based on specific characteristics of each stage. The data were compared between treatments and genotypes to see if there was a delay in specific larval stage development that resulted in the overall delay seen in previous experiments. These data were analyzed using R, and the results indicate that there is a significant delay in development of the first instar larvae of the sensitive strains, and no delay in the resistant strains. Based on these results, I will next use single cell sequencing of first instar larvae raised on rapamycin-treated or normal food, with the goal of better understanding the specific mechanisms by which rapamycin leads to a decrease in larval development time, and the genetic basis of variation in the response to this treatment.

 In humans, gait changes with age; changes that have been associated with the onset of disease. We also see age-related changes in the fruit fly, Drosophila melanogaster, which shows a decrease in the ability to climb vertically. However, the effects of age on walking patterns of flies on a flat surface, which more closely mimics human walking, have not been fully characterized. In my research, I use D. melanogaster as a model to investigate such effects. During the past year, I followed cohorts of D. melanogaster over their lifespans and recorded videos of them walking in an enclosed arena. A wide-field camera captured the entire arena while a higher resolution camera captured the leg movements of individual flies. I analyzed the trajectories of each fly from the wide-field videos to evaluate walking velocity and duration. Based on my preliminary analysis, I have discovered that flies walk less frequently and at slower average speeds with increasing age. As a next step, I am analyzing the high resolution videos to investigate the possibility that changes in gait might explain the slower walking velocities at older age. To do this, I trained a neural network using multi-pose animal estimation software to track the movement of individual legs in relation to the fly’s thorax. This will allow me to look at gait (step length, swing duration, stance duration) as well as coordination. I expect to see age-related changes in gait and a loss of limb coordination over fly lifespan, which might explain why flies walk slower with increasing age. With the findings from my study, I hope to establish a foundation for how gait changes with age in D. melanogaster.

The human brain is highly sophisticated and its functions are influenced by a multitude of factors, many of which play a role in the complex aging process. Certain individuals appear to possess more resilience to environmental and biological stressors as they age compared to others. However, why they are more resilient is not understood. Resilience refers to an individual's capacity to respond to stress (physically, psychologically, emotionally) by resisting damage and bouncing back. In my research in the Promislow lab, I use the fruit fly, Drosophila melanogaster, to explore the intricate process of aging. In this experiment, I applied a biological stressor on the flies halfway through their lives and examined mortality and motor function as measures of health to study resilience throughout the fly’s lifespan. I stressed the flies with a sublethal dose of paraquat, a neurotoxin that causes oxidative stress and mitochondrial dysfunction upon acute exposure. If the stressed flies return to the mortality and motor function levels of the control flies, this tells us the flies are resilient. I hypothesize that all of the flies that receive the paraquat dosage will experience an increase in mortality and a decrease in motor function when compared to the control flies. While I think the majority of the flies will fail to recover from these stressed levels, I hypothesize a small number of flies will return to the mortality and motor function of the control flies, demonstrating resilience. In my project, I aim to understand how flies can recover from biological stressors, and how their ability to recover changes throughout their lives. My long-term goal is to understand how exposure to biological stressors affects the aging process, and in particular, how and why resilience varies with age.

Recently, the Promislow lab found that levels of metabolites in the carnitine pathway can be used to estimate age in the fruit fly, Drosophila melanogaster. This ‘metabolite clock’ not only predicts an individual’s age, but also shows that when an individual’s predicted age is older than its chronological age, it has a higher mortality rate than other flies its age, and vice-versa. The carnitine pathway is required for energy production via fatty acid oxidation, for which carnitine also removes cellular waste products, and which may influence aging. I hypothesized that higher levels of carnitine would be associated with a longer lifespan, sustained by ongoing energy production and reduced cellular toxin accumulation. To test the effect of the carnitine pathway on fly aging, I measured the lifespan of flies while either supplying additional carnitine, or treating with the carnitine biosynthesis inhibitor etomoxir. I expect flies treated with supplemental carnitine to live longer than control flies, and that etomoxir-treated flies will live shorter than control flies. Approximately 125 female Drosophila melanogaster were assigned to food vials in each condition, plus a control condition that lacked added carnitine or etomoxir. I recorded deaths every two days, transferring remaining flies to fresh vials. Once all flies are dead, I use survival analysis to determine if either treatment affects lifespan, thus testing for a role of the carnitine pathway in fly mortality. Should the results support my hypothesis, I may explore the role that fatty acid oxidation has in aging, or to what degree the metabolome clock is affected by manipulation of the carnitine pathway.

Arrhythmogenic cardiomyopathy is a life-threatening inheritable disease that can result in sudden cardiac death or heart failure. One cause of this heart disease is a pathogenic mutation in the desmosomal protein named desmoplakin (DSP), which is a critical component in humans for maintaining the structural integrity of adjacent cells. Since over 70% of pathogenic DSP variants have been found to be premature truncating variants (ptvs), we hypothesize that DSP haploinsufficiency leads to arrhythmogenic cardiomyopathy. Engineered heart tissues (EHTs) from patient specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were previously generated from two unrelated patients with different DSP-ptv mutations. Max EHT twitch force measurements suggested that the DSPptvs were weaker compared to wild type. To control for genetic differences in patient derived iPSC-CMs, the DSPptv mutations of these two patients were created in an unrelated normal patient WTC iPSC line using CRISPR/Cas9 in order to study these mutations in a genetically isogenic background. iPSC-CMs from these two isogenic lines are currently being used to generate EHTs to determine the impact these DSPptvs have on cardiac tissue function. DSP protein levels of these isogenic iPSC-CMs are also currently being analyzed to determine the effect that these mutations have on DSP protein abundance. We expect the max EHT twitch force measurements to be weaker and lower levels of DSP protein found in the DSP isogenic EHTs and iPSC-CMs compared to wild type. If these experiments suggest haploinsufficiency, we will overexpress DSP in the mutant lines to determine if restoring normal DSP protein levels can rescue contractile function. Overall, this study will help us better understand the mechanisms of DSPptv-mediated arrhythmogenic cardiomyopathy, in hopes to potentially identify a novel therapeutic treatment for these patients.

Recent advances in neural recording technology are allowing scientists to record data at unprecedented scale. For example, the International Brain Lab (IBL) is a consortium of 22 labs working together to produce mouse brain recordings using high-density Neuropixels probes. In total, the IBL stores data from over 1000 sessions which provide a picture of neural activity across the entire mouse brain. At this scale, traditional static images and visualizations fail to communicate the results of neural analyses for brain wide activity and time-dependent behavior. As a tool for researchers in the IBL and undergraduate courses, we are developing 3D visualization software, the Virtual Brain Lab (VBL), capable of rendering a complete view of neural activity in the mouse brain in a simulated interactive laboratory. In my role as a developer, I am building tools to (1) rescale trial-averages around events and replay neural responses with variable event timings and (2) replay a single trial recorded in the IBL. Our simulation is built using the software package ‘Unity,’ allowing easy construction of custom 3D environments and publishing to diverse devices from laptop browsers to tablets, to virtual reality headsets. Displaying event-averaged neuron activity and single-trial replays will help IBL researchers spot anomalous data and holistically view their recorded sessions. Additionally, researchers can use our software to generate high-quality figures and videos for papers, outreach, and presentations. Finally, a host of key neuroscience concepts, such as sensory and motor coding and correlated variability are only communicated to students via dry lectures or textbook figures. Expensive lab classes in which students perform neuroscience experiments to rediscover these concepts are inaccessible to schools with fewer resources. Using our framework for visualization, we can build and distribute simulated tutorials and labs for students at little to no cost – reducing barriers for neuroscience education.

Pathogenic missense mutations in the myosin heavy chain 7 (MYH7) gene are the most common genetic cause of hypertrophic cardiomyopathy. While several pathogenic variants have been modeled and studied extensively, the majority of MYH7 variants are classified as variants of unknown significance (VUS) in variant catalogs such as ClinVar, because they lack sufficient clinical and functional data for variant effect interpretation. To model and determine the functional significance of additional MYH7 VUS, I will employ gene-editing techniques to generate single variant repair templates for several VUS of interest in human induced pluripotent stem cells (hiPSC). Following heterozygous knock-in of a variant transgene into the endogenous MYH7 locus, I will differentiate hiPSCs into cardiomyocytes using established, efficient cardiac directed-differentiation protocols for comparison to wild-type hiPSC-derived cardiomyocytes across multiple metrics. Variant effect on contractile function will be measured in engineered heart tissues (EHTs). In addition, cardiomyocyte cell size will be measured across wild-type and variants using flow cytometry. This functional data will inform VUS interpretation as benign or pathogenic and provide healthcare professionals and patients with clinically actionable information.