Category Archives: Week 6

Min Ju Lee

Mentors: Victoria Hall, Nicole Calakos M.D.-Ph.D

Department of Neurbiology

Habitual behaviors are automatic reactions to a certain stimulus, which enables the brain to reduce cognitive load of performing repeated sequences. While habits can be beneficial, the process of transitioning between goal-directed and habitual behavior is necessary to act appropriately in variety of contexts. Neuropsychiatric disorders with impaired decision-making, such as Obsessive-Compulsive Disorder and addiction, are thought to involve an inability to shift between action control strategies. Still, little is known regarding the circuitry responsible for this transition. We hypothesize that there are neuronal ensembles specific to both goal-directed and habitual behavior. Targeted Recombination in Active Populations 2.0 (TRAP2) method was employed to selectively label neurons activated during goal-directed or habitual action strategy of lever press training. Images were taken with Zeiss 880 Confocal; analyzed using ImageJ to compare location and count of TRAPed cells across groups. Initial montages of the whole brain show different ensembles for habitual and goal-directed behavior in the sensorimotor cortices. Further research would involve capturing both ensembles in the same brain using immunohistochemistry. Our findings hope to elucidate the circuitry involved in action control to help us understand what happens when this adaptive process becomes maladaptive.

Tissue-engineered skeletal muscle is a promising platform for in vitro modeling of human muscle diseases and pharmacological testing. However, most engineered skeletal muscle tissues contain only muscle and fibroblasts, lacking the complexity of native muscle, which also includes motorneurons, macrophages, vasculature, etc. The Bursac Lab has been developing a pre-vascularizing engineered skeletal muscle with contractile function comparable to that of avascular, muscle-only engineered tissues. In an effort to improve this platform, we have started to investigate the effects of Apelin-13, previously shown to improve skeletal muscle function in vivo and angiogenesis of endothelial cells in vitro. The main hypothesis of this work has been that Apelin-13 will simultaneously improve both angiogenesis and contractile function of pre-vascularized engineered skeletal muscle. To test this hypothesis, I have been characterizing angiogenesis of human endothelial progenitor cells (EPCs) using a 16-hour Tube Formation Assay with varying concentrations of Apelin-13. By imaging the resulting endothelial networks and quantifying total tube length and area, I expect to show that supplementation of Apelin-13 can promote angiogenesis of EPCs. If successful, I will continue these studies by treating muscle-EPC co-cultures with Apelin-13 and quantifying the effects on muscle structure, function, and formation and stability of vascular networks.

Drosophila courtship behaviors are primarily governed by two master regulatory genes: fruitless (fru) and doublesex (dsx). Both of these genes undergo sex-specific splicing to produce male-specific transcription factors that drive male-specific courtship behaviors. In the olfactory system, there are three types of olfactory receptor neurons (ORNs) that express fru and regulate male-specific courtship behaviors: Or47b, Or67d and Ir84a. It is our hypothesis that olfactory sensation can alter courtship behavior by reprogramming the chromatin state around fru and dsx. In order to examine this hypothesis, Chromatin Immunoprecipitation (ChIP) using antibodies for RNA Polymerase II was performed on both Ir84a mutant and wild-type flies. Analysis of the qPCR results using fru primers from both mutant and wild-type flies suggest that **the chromatin conformation around fru in the Ir84a mutants was more closed than that of the wild type. This difference in chromatin state translates to a variance in expression rates of fru and thus influences the behavioral traits of the flies.

**this is what the expected result is but there are two other potential results

Reprogramming endogenous mRNA by Crispr Associated Fragment Trans-splicing (CRAFT)

Nicolas Rey

Mentors: David Fiflis, Aravid Asokan, Ph.D.

Departments of Biomedical Engineering, MGM, Surgery

Gene therapy is a strategy to correct monogenic disorders through the delivery of nucleic acids that encode a healthy copy of the mutant gene. Classically, a working copy of a defunct gene is introduced via viral vector; most commonly an adeno-associated viral capsid with a packaging capacity of 4.8kb. Hundreds of genes have coding regions larger than 4.8kb (Dystrophin ~11kb) and therefore cannot be delivered by this classical approach. Novel CRISPR Associated Fragment Trans-splicing (CRAFT) technology provides a new gene therapy strategy by efficient editing of RNA via trans-splicing with the help of Cas-13. By manipulating the splicing pathway and inducing trans-splicing, CRAFT is able to replace a full-length protein sequence without having to deliver the entire sequence. Currently, we are targeting Duchenne Muscular Dystrophy (DMD) and Myotonic Dystrophy (DM1) but aim to broaden CRAFT’s application. We are rationally engineering CRAFT to increase trans-splicing efficiency while also scaling up in-vitro testing by moving to patient-derived cardiomyocytes. Our many engineered versions of CRAFT are delivered into cells whose edited RNA can be extracted, reverse-transcribed into cDNA, PCR amplified, and sequenced. Our initial design screen shows strong candidates to move CRAFT forward by transducing it into patient-derived cardiomyocytes and eventually in-vivo.

James J Zheng¹
Mentors: John S Decker¹, Michael D Lynch, MD, PhD^1,2
Departments of Biomedical Engineering¹, Chemistry²

Lectins are naturally occurring proteins found in bacteria, plants, and algae that recognize specific carbohydrates found on many viral envelope proteins. This unique binding behavior allows these proteins to have broad-spectrum antiviral capabilities, neutralizing viruses such as HIV, influenza, Ebola, and SARS-CoV with varying degrees of activity. While multivalent binding between lectins and glycans has been shown to play a key role in viral neutralization beyond what can be explained by binding avidity alone, the mechanisms linking multivalency and neutralization are not well understood. We believe that lectin crosslinking between different envelope protein domains may inhibit cell-entry-associated conformational changes in viral envelope proteins, resulting in viral neutralization. Using rigid-body protein docking simulations between a subset of lectins and viral envelope proteins found on HIV-1 and H1N1, we compared predicted crosslinking to experimental data on neutralization potency and neutralization-modifying glycan deletions. Hopefully, this analysis will enable us to identify a key mechanism of lectin antiviral activity, providing a robust model for engineering improved broad-spectrum antivirals to counter emerging pandemic viruses such as SARS-CoV-2.

Bryan Rego

Mentors: Victoria Goldenshtein, Michael Tadross, Ph.D.

Department of Neurobiology

DART is a novel drug delivery system based on covalent interaction between the HaloTag ligand and the HaloTag receptor that acts as a homing device to enable the delivery of pharmaceutics to specific cell types. Currently, DART can only deliver one drug to one cell type at a time, we are trying to develop an orthogonal DART system for multiplex drug delivery. To achieve this we will use GRIP, a protein display system, and principles of directed mutagenesis to create a library with a large number of protein variants and subject them to selective binding pressure. Next-Generation Sequencing is used to identify the top DNA sequences. To measure the level of binding, cells were plated with a HaloTag vector and a fluorescent dye, and the resulting signal was quantified. Next, we will measure the binding affinity of the top sequences and the cross-reactivity with the original system in neurons. If successful, the dart system would offer potential a deeper understanding of the brain circuits.

Sofia Guerrero

Mentors: Vanessa Simões, Gustavo Silva, Ph.D.

Department of Biology

Oxidative stress, where cells accumulate reactive oxygen species (ROS), is among the most prominent types of harmful environment, damaging cellular biomolecules, fostering cell death, and contributing to neurodegeneration. Cells experience many endogenous and exogenous sources of ROS; however, mitochondria are one of the largest contributors. Functional mitochondria are important for neural and muscle tissue, which require large amounts of energy for proper function. Moreover, defective mitochondria are associated with several human diseases, including Alzheimer’s, Parkinson’s, and muscular dystrophy. Despite this, the mechanisms impacting mitochondrial function in response to oxidative stress are not fully understood. This study aims to elucidate the key role of a ubiquitin enzyme Rad6 in the regulation of yeast mitochondrial function. Through biochemical and fluorescent imaging methods, we demonstrated that deletion of RAD6 increases the number and function of mitochondria. These mitochondria are required for cellular growth as rad6Δ cells become more susceptible to mitochondria targeting drugs. Through further investigation, we will test whether Rad6 regulates the quantity and activity of mitochondria as an adaptive response to the harmful effects of oxidative stress.

Ali Pagliery

Mentors: Richard Wong, Katrina DeWitt, Justin Wright, Ph.D.

Department of Biology 

Sarracenia purpurea is a species of carnivorous plant with pitcher-like structures that collect rainwater. Inside the pitchers lives an inquiline micro-community of organisms that help break down prey that lands in the pitchers, providing the plant with nutrients. S. purpurea ranges from southeast U.S. to Canada, but it is unknown how the micro-community and its importance for the plant’s nutrient uptake varies across latitudes. We hypothesized that S. purpurea from lower latitudes will rely more on digestive enzymes for nutrient uptake, while S. purpurea from higher latitudes will rely more on the micro-community. We will take fluid and leaf samples from plants across a latitudinal gradient. We will study enzyme concentrations, the organismal composition of the micro-communities, and perform a nitrogen analysis to study nutrient uptake. We expect that the plants at lower latitudes will have a higher enzyme concentration, a more diverse inquiline community, and a higher degree of nutrient uptake than plants further north. The plants at higher latitudes will likely have a lower enzyme concentration and a less extensive inquiline community, but they will largely rely on this community for acquiring nutrients. Our findings can be applied to similar aquatic systems that exist on a macro scale.

Neica Joseph

Mentors: Katrina Wilson, Tatiana Segura, Ph.D.

Department of Biomedical Engineering, Duke University

Ischemic strokes account for 87% of strokes worldwide and occur when a blood clot obstructs blood flow to the brain, causing subsequent death of tissue and resulting in long-term disability. Current treatments must be quickly administered after the stroke onset to be effective, resulting in only 5% of patients finding treatments helpful. Therefore, alternative treatments and therapies are highly sought after/would be helpful. One alternative is the use of hydrogels for cellular regrowth. Microporous Annealed Particles (MAP) gels—porous hydrogels that are injected into the stroke infarct—can be used long after the stroke onset to reduce brain inflammation and promote angiogenesis and neurogenesis. In this study, photothrombotic strokes were administered to mice, and MAP hydrogels were injected in the stroke infarct to test the impact of the hydrogels to repair damaged brain tissue. Brain samples were acquired and analyzed, and previous results suggest reduced brain inflammation, a thinner glial scar separating the stroke tissue from healthy tissue, and the recruitment of neural progenitor cells. Further research would involve testing different hydrogel compositions, such as the addition of growth factors, variations in nanoparticle concentrations, and gel porosity.

Isabella Costanzo

Mentors: Julia Palmucci, Jennifer Tenor, Ph.D., John Perfect, M.D.

Department of Medicine, Division of Infectious Diseases

Cryptococcus neoformans, a ubiquitous infectious yeast, proliferates in cerebrospinal fluid (CSF) causing fungal meningitis in immunocompromised individuals. CSF is deficient in nitrogen, a necessary growth nutrient. To overcome this hostile growth environment and maximize fitness, many fungi employ nitrogen catabolite repression (NCR) in which genes required to break down unfavorable nitrogen sources are repressed when more favorable nitrogen sources are present. Other fungal genera like Saccharomyces and Aspergillus have well-studied NCR pathways, but NCR has not been thoroughly investigated in C. neoformans. Current research suggests that Tar1 and Ure3 in C. neoformans may be orthologous to NCR gene repressors in Aspergillus and Saccharomyces (NmrA and Ure2, respectively), and may regulate the GATA transcription factor Gat1—the master regulator of NCR. Tar1 and Ure3 proteins were fluorescently tagged with mCherry and transformed into both wild-type (H99) and Gat1::GFP backgrounds to determine the localization of these proteins during nitrogen replete and deplete conditions and co-localization with Gat1. Co-localization of Ure3 or Tar1 with Gat1 would indicate the C. neoformans NCR pathway is similar to Saccharomyces or Aspergillus, respectively. Negative results may indicate a unique regulatory system in C. neoformans. Further experimentation would examine additional NCR interactors and investigate its effect on C. neoformans on survival in CSF.

George Romero

Mentors: Jiaxuan Qi, Richard Mooney, Ph.D.

Department of Neurobiology – Duke University School of Medicine

Previously studied neurons and cell types within the brain have been characterized using methods that are considerably slow, inefficient, and expensive. To present, there lacks a cell type targeting technology that is both widely accessible and efficient. Technology X is a novel RNA-based cell type targeting technology that can target and manipulate specific cell types within the brain. In vivo functionality of Technology X has not been completely confirmed and is under testing across multiple cell types and between different model organisms. We tested the hypothesis that Technology X will successfully target and express within dopaminergic neurons of the zebra finch. Technology X, tagged with the green fluorescent protein (GFP) indicator, was virally injected into the VTA and SNc (regions densely populated with dopaminergic neurons) of the zebra finch. In our results, we plan to see widespread and exclusive expression of GFP within the dopaminergic cells of the VTA and SNc when viewed under confocal microscopy. This would corroborate that Technology X is capable of successfully targeting and manipulating specific cell types within the brain.

Zach Pracher

Mentor: Dave McClay, Ph.D.

Department of Biology

The developmental gene regulatory network (GRN) of the green sea urchin (Lytechinus variegatus) has been extensively investigated to illuminate the genetic interactions underlying embryonic development and has led to many insights in embryonic development and regeneration. Although the putative metalloprotease Astacin-4 is widely expressed in urchin embryonic blastocoelar cells, its developmental function and position in the established developmental GRN remains unknown. In our study, inhibiting Nodal with an antagonistic drug throughout embryonic development revealed that Nodal signaling between 4 and 6 hours post fertilization is critical to downstream Astacin-4 expression, blastocoelar cell formation, and overall embryo development. Separately, we repressed Astacin-4 expression using a morpholino to determine the potential role of Astacin-4 in embryonic and blastocoelar development and function. Embryo morphology and gene expression patterns were subsequently assessed using in situ hybridization and microscopy techniques. We hypothesize that repressing Astacin-4 inhibits normal blastocoelar cell formation, and we generally expect to elucidate the role of Astacin-4 in the epithelial-mesenchymal transition of blastocoelar cell precursors. Understanding the regulation and function of Astacin-4 in L. variegatus can enhance our knowledge of the genetic relationships underlying immune system development as well as evolutionary conservation of immune system development across the animal kingdom.

Nadeska Montalvan

Mentors: Martina Zafferani¹, Amanda E. Hargrove^1,2

¹Department of Chemistry
²Department of Biochemistry

Out of the total RNA transcribed in cells, only 1.5% is translated into proteins. About 70% of the rest of the human genome is transcribed into non-coding RNA (ncRNA). While the roles of most ncRNAs remain unknown, several ncRNAs have been found to be overexpressed in various cancers which makes them attractive therapeutic targets. Small molecules have been successfully developed to bind to RNAs. However, it remains unclear whether small molecules selectively bind to different RNA sequences with similar structural motifs. Thus, we aimed to screen a large in-house small molecule library against three RNA sequences with triple helix structures, namely MALAT1, NEAT1 and PAN. We hypothesized that most molecules that would bind to one RNA would bind to the other two, indicating low selectivity. We used high-throughput screening to determine which small molecules bound to each RNA. Surprisingly, the screening revealed several small molecules that selectively bind to each of the three triple helices. Additionally, findings yielded a 100-fold higher hit rate than the average rates of high-throughput screenings against general RNA targets. Current efforts are focused on evaluating the newly identified selective small molecules against their respective RNA target to determine any possible unique properties of selective binders.

Author: Shibani Mallik

Mentor: Nina Tang Sherwood, Ph.D.

Department of Biology

Spastin is a microtubule-severing protein important for microtubule degradation and growth. Spastin mutations in humans are known to cause Autosomal Dominant Hereditary Spastic Paraplegia (AD-HSP), a neurodegenerative disease of the motor system. Ubiquitous deletion of spastin in Drosophila also causes motor defects in adult flies, as well as defects in synaptic bouton morphology and function. While spastin is thought to be expressed in neurons, the true site of action of Spastin remains unknown. This study used the CRISPR-Cas9 genome-editing tool to generate spastin deletions in only neurons or glia using tissue-specific drivers, along with ubiquitous drivers, to discover Spastin’s site of action. This was accomplished through a series of genetic crosses, larval dissection, and immunofluorescence microscopy to visualize boutons at the neuromuscular junction. This study also evaluated the efficacy of using CRISPR-Cas9 as an editing tool in Drosophila. If Spastin is required only in specific tissues, then the larvae with certain tissue-specific drivers will display the mutant phenotype. A ubiquitous driver should also display the mutant phenotype, depending on the functionality of the CRISPR-Cas9 system. This study has important implications for future therapy development for AD-HSP and future projects on the spastin gene, among others implicated in microtubule development.

Megan Stone

Mentors: Jennifer Li, Lindsey Glickfeld, Ph.D.

Department of Neurobiology

It has been thought that varying subtypes of interneurons have different roles in controlling the neuronal circuits that drive visual perception.  This has been primarily studied through the activation or inhibition of specific interneuron populations through the use of optogenetics (which has limited clinical applications) or non-selective pharmacology (which is prone to off-target effects).  This study aims to further understand the roles of parvalbumin (PV) and somatostatin (SST) expressing interneurons in the mouse primary visual cortex (V1).  This will be achieved by selectively inhibiting their activity through a recently developed technique: Drugs Acutely Restricted by Tethering (DART).  Unlike optogenetics or non-selective pharmacology, this technique will allow us to selectively inhibit specific interneuron populations in a clinically feasible manner.  Thus, we expect to see suppressed responses in PV and SST cells, following an electrical stimulus, in comparison to the control.  Overall, these findings will contribute to the overall understanding of the function of  PV and SST interneurons in neuronal circuits and add to the knowledge of mechanisms driving perception and visually guided behaviors.  Additionally, this study seeks to validate the use of DART as a technique to manipulate specific neuronal populations within V1.