UMSS20 Biomedical Sciences
UMSS20 Presentations by Category
UMSS20 Social Sciences and Humanities
UMSS20 Physical and Mathematical Sciences
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1003. The role of BPs’ gp33 immunity repressor in the downregulation of Mycobacterium chelonae genes
Undergraduate Presentation. Author(s): Anna Schumann, Dakota Archambault. Mentor(s): Sally Molloy.
Abstract: Pathogenic mycobacteria are the leading cause of death worldwide. Mycobacterium abscessus causes lung infections and often is completely resistant to antibiotics. Increased research on antibiotic resistance mechanisms is important for developing new treatments. Prophage, viral genomes integrated into bacterial genomes, contribute to virulence in most pathogens M. chelonae determined that prophage BPs alters expression of 7.4% of bacterial genes. M. chelonae carrying prophage BPs also has increased antibiotic resistance. It has not been reported how prophage changes bacterial gene expression and antibiotic resistance. Prophage gene products may alter bacterial gene expression. Prophage BPs expresses high levels of a repressor that binds to the regulatory regions of BPs lytic genes to prevent their expression. We identified similar repressor binding sites in the regulatory regions of four M. chelonae genes that are downregulated in the presence of BPs, one of which may contribute to antibiotic resistance. This project aims to determine if the repressor binds to M. chelonae sequences to downregulate expression by creating a reporter plasmid that can detect expression from the four regulatory sequences in the presence and absence of the BPs repressor. We have PCR amplified the four regulatory sequences, and four negative control sequences. We are currently inserting those sequences using Gibson cloning, into the reporter plasmid encoding a green-fluorescent protein (GFP).
1007.* Mechanisms of the Hyperinflammatory Response to Influenza Infection
Undergraduate Presentation. Author(s): Kodey Silknitter, Brandy Soos, Con Sullivan, Benjamin King. Mentor(s): Ben King.
Abstract: Influenza A virus (IAV) is an enclosed, RNA virus that infects the respiratory system and is responsible for 21,000-41,000 deaths annually in America. Upon initial infection, the virus infects lung epithelial cells, resulting in an initial anti-viral inflammatory response by the innate immune system. As the infection continues, it elicits a long-term inflammatory response intended to reduce the severity of infection that can also result in tissue damage. Understanding how the innate immune system responds to IAV infection may lead to new influenza therapies. Neutrophils play an important role in the innate immune system, but little is known about how they function during IAV infection. The zebrafish is a recently established model to study IAV infection and offers unique advantages over other models because the function of neutrophils can be studied in vivo. We hypothesize that neutrophils have an important role in the response to IAV infection. To test this hypothesis we will examine the role of neutrophils during IAV infection by comparing responses from WHIM mutant (Tg1(- 8mpx:cxcr4b-EGFP)) zebrafish that lack functional neutrophils and sibling control zebrafish that have functional neutrophils. A preliminary survival analysis suggests WHIM mutants infected with IAV have a lower survival rate than controls infected with IAV. We will determine the level of infection and expression of candidate genes over a time course of infection. These studies will help us understand the genetic mechanisms that regulate the inflammatory response to IAV infection and the roles neutrophils have in that response.
1009. Defining the role of Beta-arrestin in ERK activation during JCPyV infection
Undergraduate Presentation. Author(s): Sarah Nichols, Colleen L. Mayberry, Melissa S. Maginnis. Mentor(s): Melissa Maginnis.
Abstract: Human JC polyomavirus (JCPyV) infects up to 80% of the population and establishes a persistent, lifelong infection in the kidneys. In individuals with severe immunosuppression, JCPyV can spread from the kidneys to the central nervous system, resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), for which there are no effective treatments. JCPyV internalization is mediated by 5-hydroxytryptamine 2 receptors (5-HT2Rs) and clathrin-mediated endocytosis. Previous data gathered by the Maginnis laboratory details the importance of the 5-HT2R-related endocytic protein β-arrestin and activation of the mitogen activated protein kinase (MAPK) signaling cascade, specifically extracellular signal-regulated kinase (ERK), in facilitating JCPyV infection. Further, it is known that the MAPK adaptor protein, growth factor receptor-bound protein 2 (GRB2), is crucial for initiation of MAPK signaling. Specifically, GRB2 activates the MAPK protein Ras, followed by a series of phosphorylation events, and subsequent activation of ERK. Thus, as ERK is a critical regulator of JCPyV infectivity, and as ERK can be activated by GRB2, GRB2 may be necessary for ERK activation during JCPyV infection. Understanding how ERK is activated in cells challenged with JCPyV will enhance our understanding of viral pathogenesis. The goal of this project is to define whether activation of the 5-HT2Rs, β-arrestin, and GRB2 activate the ERK signaling cascade to drive viral infection in SVG-A cells. Preliminary data suggests no dependence on GRB2 for successful viral infection. Understanding how JCPyV hijacks host cell signaling to promote survival will fill critical gaps in knowledge that could provide a platform to develop antivirals.
1011.* Mouse Telomerase Reverse Transcriptase as a Marker of Adult Stem Cells
Undergraduate Presentation. Author(s): Josh Passarelli, Gabriel Jensen, Kristy Townsend. Mentor(s): Kristy Townsend.
Abstract: Improper energy balance and metabolism is associated with a variety of maladies and diseases, including diabetes and neuropathy. Adult stem cells (ASCs) are likely important for the adult brain to coordinate energy balance, in part by supporting the turnover of new neurons in the brain’s energy balance center, the hypothalamus. Currently, this field of stem cell research is greatly hindered by the lack of a specific and unique marker of ASCs in the brain. We hypothesize that mouse telomerase reverse transcriptase (mTERT), which is important for persistent stem cell replication in other adult tissues, can be used as a specific and unique marker of quiescent ASCs. Here, we characterize mTERT+ cells and their progeny by performing co-staining experiments with tissues from mTERT-GFP direct reporter and lineage tracing mouse lines. mTERT+ cells in mTert-GFP direct reporter mouse brains occasionally express known adult stem cell markers. These cells are found in well known stem cell niches and interestingly in niches where the potential for resident stem cells is less understood, such as the choroid plexus. Lineage-traced cells are widely dispersed throughout the brain and have been found to coexpress markers of mature cell types. Based on these results, we propose that mTERT+ adult stem cells residing within the adult mouse brain repopulate various regions of the brain with pericytes, epithelial cells, and other unknown mature cell types.
1012. Characterization of Ncf1 Mutants in a Zebrafish Model of Innate Immune Function with Human Influenza A Virus Infection
Undergraduate Presentation. Author(s): Lily Charpentier, Brandy Soos, Benjamin King. Mentor(s): Benjamin King.
Abstract: Seasonal influenza A virus (IAV) infections and their associated respiratory diseases are the cause of an estimated 650,000 deaths each year, according to the World Health Organization. The zebrafish is a powerful vertebrate model to study innate immune function and host-pathogen interactions as the function of neutrophils and other phagocytes can be characterized in vivo. Preliminary studies have shown an increase in neutrophil respiratory burst activity to eliminate the invading pathogen, yet little is known of all of the mechanisms involved in neutrophil function. The NADPH oxidase complex, of which neutrophil cytosolic factor 1 (Ncf1) is a key component, regulates reactive oxygen species (ROS) to control neutrophil response to viral infection. Although necessary to fight infection, this elicits a hyperinflammatory response that can damage the infected host epithelial tissue, leaving high-risk individuals with increased mortality rates. Our hypothesis is that a fully functional Ncf1 protein is required for neutrophil function, but morpholino knockdown of the gene will limit the amount of damaging ROS hyperinflammation in host tissue. Our preliminary studies of system IAV infected embryos indicate that the survival of ncf1 morphants (MO) was increased compared to scrambled morphant control groups. We are currently conducting TCID50 assays to monitor viral titer over a span of 96 hours post-infection, and qRT-PCR studies to assay the expression of genes associated with neutrophil function in ncf1 and control MO. These studies aim to increase our understanding of neutrophil function that may eventually lead to new therapies for treating IAV infection.
1015.* The Functional Role of Glia in Peripheral Metabolic Tissue
Undergraduate Presentation. Author(s): Emma Paradie, Magdalena Blaszkiewicz, Joshua Havelin. Mentor(s): Kristy Townsend.
Abstract: Obesity and its comorbidities have reached pandemic levels, resulting in an increased prevalence of diabetes, which is often accompanied by peripheral neuropathy. While the brain is responsible for coordinating the balance between energy intake and energy expenditure, peripheral nerves are responsible for communicating between the brain and peripheral tissues, including adipose (fat) tissues. There are non-neuronal glial cells in the central and peripheral nervous systems (CNS, PNS, respectively) whose functions are vital for maintaining homeostasis, forming myelin, and providing support for nerves. Myelin is a fatty substance that wraps around nerves and acts similar to the insulation around a wire, allowing for saltatory conduction and increasing the speed at which electrical signals are sent through the nervous system. Demyelination of nerves leads to loss of proper nerve function and therefore contributes to neuropathy. While much remains to be discovered about cross-talk between glia and other cell types in the CNS, there is even less known regarding glia-nerve communication in the PNS. We have shown that a subset of peripheral nerves innervating adipose tissues are myelinated, and we can visualize what appear as specialized glia called Schwann cells, which regulate axonal myelination. Schwann cells are known to work with macrophages in lesioned peripheral nerves to clear myelin debris – vital for axonal regeneration. Schwann cells are also known to be able to switch between various phenotypes, but the functions and mechanisms of all of these Schwann cell subtypes have yet to be solidified, especially in adipose tissue. To date, we have optimized a unique protocol for immunopanning adipose tissue to isolate Schwann cells and other glial subtypes, which we are currently confirming with qPCR to assess cell type gene expression. Fluorescence-activated cell sorting of subcutaneous white adipose from young versus aged mice has revealed differing numbers of Schwann cell populations, coinciding with our previous findings of decreased innervation in adipose with aging.
1017. Characterizing Epsins in Pheromone Gradient Tracking
Undergraduate Presentation. Author(s): Sarah Latario. Mentor(s): Dr Joshua Kelley.
Abstract: Endocytosis is the process by which cells take up extracellular components through the internalization of regions of the cell’s plasma membrane. Epsins are proteins that aid in the control of this process by helping to curve the membrane during endocytosis. These proteins recruit and interact with other membrane proteins while binding the cytoskeleton, resulting in the membrane curving in the correct direction at the right time. This process is also used during cellular polarized growth such as the kind used by Saccharomyces cerevisiae during its response to pheromone. During this response, a G-protein coupled receptor (GPCR) recognizes the extracellular pheromone gradient and activates downstream signaling proteins that lead to polarized growth towards the high pheromone concentration. We suspect that epsins are involved in controlling this response by regulating downstream signaling molecules during endocytosis, resulting in the polarized growth observed in yeast responding to a pheromone gradient.They also may play a role in membrane localization of downstream proteins during this response. By deleting epsins, we hope to determine their exact role in the yeast’s pheromone response pathway and if their activity may rescue defects during other steps in the pathway.
1018. A Vascularized Medium for Passive Monitoring of Bacterial Growth
Undergraduate Presentation. Author(s): Benjamin Chasse. Mentor(s): Caitlin Howell.
Abstract: Currently, infection in a person is often not detected until symptoms are present, or bacteria appear in their fluids. The purpose of this work is to develop a system which can detect the growth of bacteria in vivo in an early and non-invasive manner. To test this idea, a vascularized agar matrix was 3D printed to resemble the characteristics of a circulatory system. And, similar to how a circulatory system delivers nutrients and removes wastes from surrounding tissues, the 3D printed channels exchanged diffusible compounds with the surrounding matrix. The detection of signaling molecules from the surface was tested by 3D printing channels in LB agar, inoculating one system with wild type E. coli, and leaving the other without bacteria as a control. Both systems were incubated at 37ºC for 24 hours. The channels of each were then injected with 1.0x PBS and were sampled and replaced four times over the following 58 hours. The samples were then analyzed using HPLC to assess differences between the diffused molecules in each. These results may be applied to help to create a detection system to help identify the presence of bacteria sooner and without the need for invasive techniques.
1019.* Investigating the Role of Serotonin Receptor Scaffolding Protein Calmodulin in JC Polyomavirus Entry and Infection
Undergraduate Presentation. Author(s): Francois Levasseur, Kashif Mehmood, Colleen L. Mayberry, Tristan M. Fong, Samuel T. Hess, Melissa S. Maginnis. Mentor(s): Melissa Maginnis.
Abstract: JC polyomavirus (JCPyV) maintains a persistent kidney infection in 80% of the population. While renal JCPyV infections are asymptomatic, infections can progress to the central nervous system in immunocompromised individuals. At this stage, JCPyV infects glial cells and leads to progressive multifocal leukoencephalopathy, a fatal demyelinating disease for which there are no treatment options. This research aimed to determine the mechanisms by which JCPyV infects human kidney and glial cell types. JCPyV entry into both kidney and glial cells is mediated by serotonin 5-hydroxytryptamine (5-HT2) receptors, yet the mechanisms by which the receptors drive this process is poorly understood. Although it has been established that JCPyV uses 5-HT2Rs for internalization, direct interactions between these receptors and JCPyV has not been established. Using super-resolution microscopy, we have visually determined the localization and interactions between JCPyV and 5-HT2Rs. Additionally, 5-HT2R scaffolding protein, calmodulin (CaM) may have a role in JCPyV infection. CaM is an intracellular protein with many roles in cell activity, including endocytosis, a critical step in viral entry. To determine whether CaM plays a role in JCPyV infection, cells were treated with a CaM-specific inhibitor W7 and JCPyV infection was quantified by indirect immunofluorescence. This research will better define how viruses utilize host-cell machinery to mediate infection. Outcomes of this research could serve as a foundation for the development of treatment strategies for JCPyV infection.
1020.* Impacts of ERK Activation on JC polyomavirus Infection
Undergraduate Presentation. Author(s): Remi Geohegan, Melissa Maginnis. Mentor(s): Melissa Maginnis.
Abstract: JC polyomavirus (JCPyV) infects the majority of the population, causing an asymptomatic infection in the kidneys. In immunosuppressed individuals, JCPyV infects astrocytes and oligodendrocytes causing progressive multifocal leukoencephalopathy, a fatal disease. However, the mechanisms by which JCPyV regulates infection in astrocytes and oligodendrocytes is poorly understood. Current research is limited to in vitro cell culture models due to the specific tropism of JCPyV for the human host. The study of JCPyV in SVG-A cells, an immortalized human fetal glial cell line can be enhanced using a newly developed cell model, normal human astrocytes (NHAs), which provide a more accurate representation of infection in the host. Studies revealed that the progression of infection in NHAs differed from SVG-A cells, resulting in a delay in viral replication. JCPyV utilizes the mitogen activated protein kinase (MAPK) pathway leading to induction of extracellular-signal regulated kinase (ERK) phosphorylation, which is necessary for the progression of the infectious cycle. To determine whether the delay in infection in NHAs was due to differences in MAPK/ERK signaling, both cell lines were treated with an ERK activator and viral gene products T-Antigen (early transcription product) and viral protein 1 (VP1) (late transcription product) were quantified. Additionally, RNA sequencing analysis of MAPK/ERK signaling genes were evaluated using co-expression networks and ingenuity analysis. Our findings indicate that ERK phosphorylation may lead to increase the production of VP1, resulting in increased infection. These data enhance our understanding of viral regulation of gene networks and signaling pathways in the infectious cycle.
1022. Epsocamisio, the Little Gordonia Phage That Could (Without Integrase)
Undergraduate Presentation. Author(s): Lauren Cusson, Libbee Currie, Andre Dagle, Emma Dunn, Michael Wilczek. Mentor(s): Melody Neely.
Abstract: Bacteriophage are the most diverse and abundant organisms, with a total estimate of1031 in the biosphere. Despite the vast populous, current research has only made a small dent in the immense prospect of knowledge awaiting discovery. To address this, phage genomes were sequenced and by using bioinformatic programs the phage genomes were annotated to decipher the function of the genes. From this, comparisons were made to other known phages, aiding in the understanding of genetic mosaicism. One of the sequenced phages, Gordonia terrae phage Epsocamisio, was isolated from a soil sample using enriched isolation. This phage is a Siphoviridae and has a tail of ~150 nm and an icosahedral head of ~75 nm. It is a temperate phage that forms large circular plaques containing a clear center and a turbid halo. The presence of an immunity repressor in the genome is a potential support of this temperate lifestyle. Epsocamisio infects Gordonia westfalica, Gordonia rubripertincta, and Gordonia terrae. After sequencing, Epsocamisio was determined to be an A15 cluster phage with a genome length of 52,314 bp that comprise ~99 ORFs. Its GC content is 62.0% and its genome encodes for ~96 putative genes and ~3 tRNAs. Future study should be conducted on Epsocamisio to potentially find beneficial applications and to better understand A15 type phages.
1034. Antimicrobial Cetylpyridinium Chloride Inhibits Immune Mast Cell Function
Undergraduate Presentation. Author(s): Christain Potts, Bright Obeng, Bailey West, Suraj Sangroula, Marissa Kinney. Mentor(s): Julie Gosse.
Abstract: Cetylpyridinium chloride (CPC) is used as an antimicrobial agent in many popular mouthwashes, at 1500-3000μM levels, and other consumer products. Little is known about the toxicology of CPC on eukaryotes, and such widespread exposure of CPC calls for investigation for more information. Mast cells, found ubiquitously throughout the human body, play a key role in the immune system, physiological processes, and diseases. The Gosse lab has demonstrated that CPC inhibits mast cell degranulation (the release of bioactive substances such as histamine and serotonin) at CPC concentrations as low as 1μM, roughly 1000 times lower than the concentration found in consumer products. We are investigating the molecular mechanisms of this inhibition. Through use of the genetically-encoded voltage indicator ArcLight A242, we demonstrated that CPC does not interfere with the plasma membrane potential of the cells. We have investigated the effect of CPC on antigen stimulated Ca2+ influx into the cytosol through CRAC channels using the genetically encoded calcium indicator pGP-CMV-GCaMP6f. We found that CPC exposure is decreasing cytosolic Ca2+ levels by 40.4% on average with a 0.005μg/mL antigen dose. Another effect that will be studied is the inhibition of ER Ca2+ release into the cytoplasm through ER Ca2+ GCaMP constructs. This research will provide knowledge on the effects that CPC is exhibiting on eukaryotic cell signaling, allowing the prediction of CPC effects on other eukaryotic cell types that share similar signaling pathways. Continuing this research will provide a better understanding of the overall effect of CPC on human and wildlife health.
1035.* Investigating the Potential Relationship Between Black Soldier Fly Larvae Reared on Potato Scrap Substrate and the Suppression of Gram-Positive Bacteria
Undergraduate Presentation. Author(s): Marissa Kinney. Mentor(s): Edward Bernard.
Abstract: The black soldier fly larvae (Hermetia illucens) are becoming a popular, sustainable choice of feed additive for both agriculture and aquaculture. Black soldier fly larvae (BSFL) are favored because their composition can be tailored to fit the needs of the consumer. BSFL digest organic waste, developing on food scraps and manure. Previous studies have shown that BSFL suppress pathogenic bacteria. There is evidence of suppression of Gram-negative bacteria, but a lack of evidence for suppression of Gram-positive bacteria. In the Bernard lab, we worked with black soldier fly larvae reared on potato substrate to test for potential suppression of methicillin-resistant Staphylococcus aureus. We conducted two rounds of a week-long experiment, taking samples for analysis at days 0, 3, and 7. The samples were plated on a general agar (TSA) and Staphylococcus selective agar to determine if suppression of MRSA could be seen throughout the 7 days. Colony counts were determined and will be analyzed using various statistical methods. We also tested if the soluble portion of the BSFL riddled substrate (either inoculated with MRSA and not) had any suppression on the growth of MRSA. This data will also undergo statistical analysis.
1037.* Investigating the Interaction of Streptococcus agalactiae and Candida albicans in vivo and in vitro
Graduate Presentation. Author(s): Katie Patenaude, Siham Hattab, Dr. Robert T. Wheeler, Dr. Melody Neely. Mentor(s): Melody Neely.
Abstract: Treating opportunistic infections can be problematic because we don’t have a complete understanding of the role other organisms found in the localized environment have on the progression of infection. Both the fungus Candida albicans (C.a.) and the bacteria Streptococcus agalactiae (Group B Strep or GBS) are commensals that reside in the vaginal tract, and both can cause opportunistic infections, which often progress to co-infections. What we don’t understand is how interactions between these organisms affects progression of disease or treatment outcomes. Previous in vitro work has sought to analyze synergistic and antagonistic interactions between other commensal bacteria and C.a., but there is not much known about how GBS and Ca interact in co-infections and its influence on treatment effectiveness. Through both in vitro and in vivo experimental methods, we will investigate the influence each have on each other in their individual growth, their virulence, and their resistance to treatment. Zebrafish are an effective model for studying in vivo interactions between these two commensals as they have highly conserved immune factors with humans, can be genetically manipulated, and are transparent as larvae, making it possible to image and track the progression of infection in real-time. By using both in vitro and in vivo experimental methods we will gain a better understanding on how GBS and Ca interact in a host and their influence on treatment effectiveness.
1038.* Delayed healing and Nav1.8 nerve regeneration following corneal injury in Sox11 conditional knock-out animals
Graduate Presentation. Author(s): Cara Sullivan, Jun Lee, Ian Meng. Mentor(s): Ian Meng.
Abstract: Corneal nerve injuries are a common source of ocular pain and discomfort that can result from disease, injury and ocular surgeries. Delayed healing of the corneal epithelium and nerves can lead to extended pain and discomfort. This study investigated the role of the nerve regeneration-associated gene Sox11 in corneal wound healing and reinnervation following acute and chronic injury. Two injury models were used to create corneal nerve injury, double lacrimal gland excision (LGE) which induces chronic dry eye by eliminating the aqueous component of tears and corneal abrasion (CA) which mechanically removes the corneal epithelium and axon terminals. LGE results in persistent corneal epithelial cell damage and retraction of corneal afferent nerve terminals, while the abraded axon terminals in CA are allowed to recover. Corneal fluorescein was used to examine the severity of epithelial damage, mechanical sensitivity was evaluated using a corneal aesthesiometer and nerve terminal density was imaged and Sholl analysis was performed. This study found that Sox11 knockout animals exhibited delayed epithelial healing following both acute and chronic injury. Post-injury mechanical hyposensitivity was observed across phenotypes and nerve regeneration was significantly reduced in Sox11 knockout animals. Expression of key regeneration factors, while normally shown to increase following corneal injury, was diminished following injury where Sox11 expression was knocked down. In summary, our results provide evidence of Sox11’s intrinsic role in nerve development and the impact of its absence on nerve regeneration and epithelial healing.
1039. AAV9-Ighmbp2 Gene Therapy Significantly Improves Motor Performance in Severe SMARD1-like Mouse Model, nmdem3
Graduate Presentation. Author(s): Sarah Holbrook, Greg Cox. Mentor(s): Greg Cox.
Abstract: Autosomal recessive mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been linked to childhood neuromuscular degenerative diseases (NMDs). C57BL/6J-Ighmbp2em3Cx is a SMARD1-like strain, or Spinal Muscular Atrophy with Respiratory Distress, created via CRISPR-Cas9 targeting of the IGHMBP2 gene and hereafter referred to as em3. SMARD1 is characterized by muscle weakness starting in the distal extremities and diaphragmatic paralysis leading to respiratory failure. Most patients are diagnosed in early infancy and die in early childhood. The em3 mouse has more severe muscle atrophy than the historical SMARD1-like model (nmd2J) in the hind limb, diaphragm, and intercostal muscles. The em3 mouse model also has an average lifespan of ~3 weeks compared to the 2J’s ~3 month lifespan. Gene therapy has shown promise in another NMD, Spinal Muscular Atrophy (SMA). In collaboration with the Meyer lab at Nationwide Children’s Hospital in Columbus, OH, we are testing 2 different AAV9-Ighmbp2 vectors. Each has a different promoter with one having a Chicken β-Actin (CBA) Promoter [higher expression levels than endogenous levels] and the other having a truncated Methyl-CpG binding protein 2 (MECP2 aka P546) promoter [expression levels close to endogenous levels expressed by muscles and neurons]. We did p1 intracerebroventricular injections on the severe em3 mice.
1040.* Injury-Induced Nociceptive Sensitization and Recovery in Drosophila
Graduate Presentation. Author(s): Christine Hale, Ramaz Geguchadze, Samia Pratt, Julie Moulton, Courtney Brann, Geoffrey Ganter. Mentor(s): Geoffrey Ganter.
Abstract: Nociceptive sensitization underlies and perpetuates chronic pain, a condition that affects ~50 million adults nationwide. With many treatment options for chronic pain, such as opioid analgesics, carrying numerous deleterious side effects, research into safer and more effective treatment options is crucial. Recently, an injury-induced nociceptive sensitization model was developed using the larvae of the fruit fly, Drosophila melanogaster, in which UV-injured animals become hypersensitive. Using this model, the necessity and sufficiency of a novel pathway, the Bone Morphogenetic Protein (BMP) pathway, functioning in the nociceptors, was revealed. At present, we propose to build upon this knowledge and reveal a more complete mechanism for how nociceptive sensitization occurs by investigating into the potential roles of other novel genes/signaling pathways. In addition to our focus on mechanisms that promote hypersensitivity, we also propose to expand our efforts by bringing new focus to the resolution phase of the nociceptor as its hypersensitivity subsides. To facilitate these efforts, because the larval stages of fruit fly development are relatively brief, we are also developing a methodology that allows longer term experimentation of nociceptive sensitization after injury in adult fruit flies. Recent efforts toward this goal include mapping the adult fruit fly nociceptor distribution by confocal microscopy and developing thermonociception methods for use with adult flies. Our ultimate aim is to better understand the mechanisms of nociceptive sensitization so that better treatment options for chronic pain may be revealed.
1043.* JC Polyomavirus Infectivity is Reduced by Inhibition of Calcium Signaling Pathways
Graduate Presentation. Author(s): Avery Bond, Mason Crocker, Melissa Maginnis. Mentor(s): Melissa Maginnis.
Abstract: JC polyomavirus (JCPyV) is the causative agent of a deadly demyelinating disease known as progressive multifocal leukoencephalopathy (PML). While many people do not know of this virus, it asymptomatically affects upwards of 80% of the population. However, in immunocompromised individuals, JCPyV leads to PML and typically death within one year of PML onset. There are currently no effective treatments for PML, and understanding specifics of the JCPyV infectious cycle will help to identify possible drug targets. Recently, the Maginnis laboratory performed a large-scale drug screen using the National Institutes of Health (NIH) Clinical Collection and assayed viral infectivity using high-throughput In-cell Western techniques. Results from the screen showed that multiple FDA-approved drugs that target calcium signaling pathways were found to reduce JCPyV infection. Additionally, other studies in the Maginnis lab have demonstrated the importance of the calcium/calmodulin pathway during JCPyV infection. To uncover the mechanism of JCPyV infection inhibition, calcium pathway inhibitors will be further examined by treating cells with inhibitors and siRNAs to modify constituents of the pathway and measure infectivity. Overall, our results demonstrate that calcium plays a role in the JCPyV infectious lifecycle. These findings further our understanding of cellular pathways utilized during JCPyV infection and illuminate potential targets for the design of antiviral therapies.
1044. Biointerface of Cellulose Nanofibril Material for Application as Botanical Wound Dressing
Graduate Presentation. Author(s): Marissa McGilvrey, Caitlin Howell. Mentor(s): Caitlin Howell.
Abstract: Cellulose nanofibril (CNF) material has potential for use in a wide variety of biomedical applications including drug delivery, tissue regeneration and wound dressings. Inherent properties of CNF such as high surface area, breathability, chirality, light weight and ability to form hydrogen bonds across cellulose chains or other polymeric matrices contribute to the engineering capacity of CNF. However, there are some characteristics of CNF that are not fully understood such as anti-microbial or anti-fouling. The goal of this project is to study how surface texture and hydration effect pulp and CNF film interface with biofluids and associated protein adhesion which is known to initiate microbial growth. Surface tension of water and blood droplets was measured by finding the angle in which droplet contacts the pulp or CNF film, using ImageJ Droplet Analysis. The effect of hydration on film protein adhesion was observed visually following application of water or blood droplets. Surface texture and hydration level were found to influence droplet surface tension and level of blood protein adhesion. High heat sterilization of CNF was found to further increase droplet surface tension compared to pulp and unsterilized CNF, which creates potential for greater utility in clinical applications. The physical properties of CNF make it a valuable material to create botanical-based wound dressings in which both surface texture and hydration can be modified for a wide range of wound types, from burns to purulent infections. As a renewable resource in Maine, wood-based wound dressings have ability to expand use of this natural resource.
1045.* Cellulose nanofiber/Hydroxyapatite nanocomposites characterization for hard tissue replacement application
Graduate Presentation. Author(s): Sahar Roozbahani, Jacob Halbrook. Mentor(s): Michael Mason.
1046. Muscle Fiber Transmembrane Protein Localization in a Zebrafish Dystroglycanopathy Model
Graduate Presentation. Author(s): Mary Astumian, Prakash Raut, Clarissa Henry, Sam Hess. Mentor(s): Clarissa Henry.
Abstract: Muscular Dystroglycanopathies are progressive diseases, with varying severity, affecting neurological and muscle health. In functional muscle fibers, the transmembrane proteins alpha-dystroglycan and integrin-alpha7, link the inside actin cytoskeleton to proteins in the outside extracellular matrix (ECM). To function properly, integrins need to cluster in the muscle cell membrane and alpha-dystroglycan needs to be glycosylated. The GMPPB gene is essential for this proper glycosylation. GMPPB encodes GDP-mannose pyrophosphorylase which synthesizes GDP-mannose, a precursor for the glycans that glycosylate alpha-dystroglycan. GMPPB is mutated in subtypes of dystroglycanopathies. Dystroglycan and integrin-alpha7 are receptors for the extracellular matrix protein laminin. Previous work showed improvement of laminin deposition and muscle health in zebrafish dystroglycan mutants and integrin-alpha7 mutants after treatment with oxidized nicotinamide adenine dinucleotide (NAD+). NAD+ did not improve muscle health in GMPPB mutants. One hypothesis explaining these results is that in GMPPB mutants, hypoglycosylated dystroglycan cannot cluster because it lacks sugar sidechains, and this interferes with integrin-alpha7 clustering. The localization of dystroglycan and integrin-alpha7 proteins relative to each other at the muscle cell membrane is hypothesized to be important. Using superresolution microscopy, images of Dystroglycan mutants versus GMPPB mutants treated and untreated with NAD+ will be analyzed to assess the average distance between the molecules of dystroglycan and integrin-alpha7 at the myofiber membranes.
1047. Assessment of SIPA1L2 as a candidate modifier of CMT1A identified in human GWAS
Graduate Presentation. Author(s): George Murray, Timothy J. Hines, Abby L. D. Tadenev, Robert Burgess. Mentor(s): Robert Burgess.
Abstract: A case-only genome-wide analysis of CMT1A patients found four intronic SNPs in signal-induced proliferation-associated 1 like 2 (SIPA1L2) to be statistically associated with foot dorsiflexion strength. Furthermore, reduction of Sipa1l2 by siRNA reduced peripheral myelin protein 22 (Pmp22) expression along with other myelination-related genes in the SOX10 co-expression network in the S16 Schwann cell cell-line (Tao et al., 2019). Given that duplication of PMP22 causes CTM1A in humans, reduction of SIPA1L2 may therefore be a promising therapeutic approach. To better understand the normal function of SIPA1L2, to independently validate the genetic association between SIPA1L2 and PMP22, and to assess reduction in SIPA1L2 as a therapeutic strategy for CMT1A, we created a Sipa1l2 CRISPR knockout mouse. We are performing detailed neuromuscular phenotyping in the Sipa1l2 knockout mice, including gene expression analysis in peripheral nerves, to better understand its normal function and to test its place in the Sox10 co-expression network. In addition, the Sipa1l2 knockout mice have been bred with the C3-PMP22 mice, a transgenic model of CMT1A overexpressing human PMP22, which recapitulate decrements in nerve conduction velocity and eventual demyelination observed in patients (Verhamme et al., 2011). We will analyze the progeny from these crosses at four- and six-months-of-age by behavioral, neurophysiological, and histological methods to determine if reducing Sipa1l2 levels alters the C3-PMP22 phenotype. The outcomes of these studies will improve our understanding of the normal function of SIPA1L2 and will serve as an in vivo test of this rare disease GWAS hit and its potential as a therapeutic target for CMT1A.
1049. Efficient construction of atomic-resolution models of glycosaminoglycans using molecular dynamics data
Graduate Presentation. Author(s): Elizabeth Whitmore, Gabriel Vesenka, Hanna Sihler, Olgun Guvench. Mentor(s): Olgun Guvench.
Abstract: Glycosaminoglycans (GAGs) are physiologically-important biopolymers in animal tissue, specifically in extracellular matrix (ECM) as well as on and within cells. GAGs are linear, structurally diverse, conformationally complex, and may contain up to 200 monosaccharides. These characteristics present a challenge for studying GAG conformational thermodynamics at atomic resolution using existing experimental methods. Molecular dynamics (MD) simulations can overcome this challenge but are only feasible for short GAG polymers. To address this problem, we developed an algorithm that applies all conformational parameters contributing to GAG backbone flexibility (i.e. bond lengths, bond angles, and dihedral angles) from unbiased all-atom explicit-solvent MD simulations of short GAG polymers to rapidly construct models of GAGs of arbitrary length. The algorithm was used to generate GAG 10- and 20-mer ensembles which were compared to MD-generated ensembles. End-to-end distance distributions in constructed and MD-generated ensembles have minimal differences suggesting that our algorithm produces conformational ensembles that mimic the backbone flexibility seen in simulation. Additionally, GAG 100- and 200-mer ensembles were constructed within a day demonstrating the efficiency of the algorithm and reduction of time and computational cost compared to simulation.
1050. Caloric Restriction and Fasting Diets have Negligible, or Potentially Damaging, Effects on Cognition in Diversity Outbred Mice
Graduate Presentation. Author(s): Andrew Ouellette, Niran Hadad, Catherine Kaczorowski. Mentor(s): Catherine Kaczorowski.
Abstract: Aging remains the greatest risk factor for developing Alzheimer’s disease (AD). Given the projected increase in lifespan across the globe, the risk of developing AD is expected to increase, along with significant economic and social burden. As such, the demand for interventions that increase cognitive longevity in parallel with lifespan is high. Several studies have reported that caloric-restriction (CR) or fasting increase lifespan and improve cognition in humans and models of aging [1,2], but others have reported no beneficial effect of CR on cognition [3,4]. These discrepancies may be explained by uncontrolled environmental factors inherent in large human studies, and the lack of genetic diversity in previous animal studies where effects of CR or fasting on cognitive longevity studies in one inbred strain of mice may not generalize to other mouse strains, let alone to humans. In this study, we compare the effects of Ad Lib, 1 day fast, 2 day fast, 20% CR and 40% CR on Y-maze working memory and contextual fear memory (CFM) in a genetically diverse population of mice – the Diversity Outbred (DO). We observed no benefit of CR or fasting on working memory or CFM at 24 months, an age when CR and fasting is known to increase survival compared to Ad Lib. Actually, the 40% CR group exhibited impaired long-term CFM compared to groups fasted for 1 and 2 days. These results emphasize the importance of identifying therapeutics to enhance cognitive longevity, as CR enhancement of lifespan may not generalize to cognitive abilities.
1051.* Characterizing the role of IRS1/2 in osteocyte differentiation and function in bone development
Graduate Presentation. Author(s): Vivin Karthik. Mentor(s): Anyonya Guntur.
Abstract: Osteocytes are the most abundant cell type in skeletal bone and are present in the bone matrix. They form a network for sensing mechanical cues coming from the bone remodeling units which involve both the osteoblasts and osteoclasts. The former secretes collagen matrix and minerals and the latter resorbs the matrix after collagen breakdown. Osteocytes have been studied in the context of mechano-transduction biology and signaling, there are very few studies elucidating the role of osteocytes in metabolic homeostasis and bioenergetics. Insulin receptor substrate proteins 1 and 2 (IRS1/2), are scaffolding proteins involved in insulin and insulin growth factor-1 (IGF1) signaling necessary for the anabolic regulation of bone. Previous studies have shown that IRS1/2 have a role in glucose metabolism and homeostasis in the liver, along with being primarily involved in the development of skeletal muscle and differentiation of osteoblasts. In this study, we aim to delineate the role of IRS1/2 in osteocyte metabolism and bioenergetics by knocking out IRS1/2 in osteocytes in mice using Dentin Matrix Protein (DMP1) Cre. Preliminary results through dual X-ray absorptiometry to measure bone mineral density (BMD) in 8-week-old mice show that Cre specific males and females have reduced BMD compared to wildtype littermates. Future studies would evaluate the bioenergetic profile of the IRS1/2 knockout osteocytes in vitro and the bone architecture of the trabecular and cortical bone through microCT analysis.
1053.* Chronic Cortisol Works Through the Transcription Factor Klf9 to Deregulate Immune Response and Metabolism
Graduate Presentation. Author(s): Ian Gans, Elli Hartig, Joel Graber. Mentor(s): Jim Coffman.
Abstract: Elevated glucocorticoid (GC) hormones are associated with a number of disease states. Studies show that high GC during development can have life-long impacts on health. To understand underlying mechanisms, our lab uses treatment with chronic cortisol (CORT) in developing zebrafish, an emerging model for investigating GC signaling. We found that CORT during development alters a set point for the GC signaling axis, leading to continuous induction of elevated GC as well as immune gene dysregulation and impaired blood glucose homeostasis. To identify pathways perturbed by developmental CORT, we used RNA sequencing to compare wild type (WT) animals with mutants lacking either the glucocorticoid receptor (GRKO) or the transcription factor Klf9, a target of the GR. The results show similarities between CORT-treated WT and VEH-treated GRKO suggestive of GC resistance in WT. In Klf9 mutants, immune genes upregulated by CORT in WT were not similarly over-expressed, suggesting Klf9 mediates immune gene regulation by GC. CORT also increased expression of numerous metabolic genes in Klf9 mutants but not WT. To further investigate Klf9’s metabolic role, respiration rate was measured in live fish. Klf9 mutants consumed less oxygen, but mitochondrial respiratory capacity was unaffected. This, coupled with gene expression data, suggests Klf9 may regulate non-mitochondrial metabolic pathways such as glycolsis and/or peroxisomal beta-oxidation. Additional studies are required, but regulation of glycolysis by Klf9 would fit with the gene’s known tumor-suppresive role, and regulation of peroxisomal metabolism could partially explain the role of Klf9 in mediating immune cells’ responsiveness to CORT.
1054. Evidence for Direct Projections of Corneal Primary Afferent Neurons to the Parabrachial Nucleus in Mice
Graduate Presentation. Author(s): Megan Tomasch, Ian Meng. Mentor(s): Ian Meng.
Abstract: As the most densely innervated structure in the body, the cornea can be a useful model for studies related to the sensory processing of pain. The cornea is primarily innervated by small-diameter primary afferent neurons with cell bodies located in the trigeminal ganglia. Central projections from corneal primary afferent neurons to the brainstem trigeminal nucleus, for the relay of craniofacial pain, have been previously characterized. Here, we set out to assess if there is a direct pathway from corneal primary afferent neurons to the parabrachial nucleus, a structure involved in the processing of pain affect and autonomic responses. Using transgenic mouse models, tdTomato/Nav1.8cre and ArchT-GFP/Nav1.8cre, central projections of sensory neurons to the parabrachial region were visualized. The location of corneal-responsive neurons in the parabrachial nucleus was determined using immunohistochemistry for c-Fos protein after the application of mustard oil (20%) to the cornea. Analysis with confocal and 2-photon microscopy revealed the presence of both primary afferent nociceptors and Fos-positive neurons in close proximity. Next, to explore a possible direct pathway between the cornea and the parabrachial nucleus, we placed a neuronal tracer, DiI, on the cornea combined with intracranial injections of a retrograde tracer, Fluorogold, into the parabrachial nucleus. This revealed double labeling of corneal afferent cell bodies in the trigeminal ganglia, a direct projection from corneal afferents to the parabrachial nucleus. We have begun optimizing behavioral assays to evaluate the functional role of this pathway in corneal pain by optogenetically silencing or activating this pathway.
1055.* Comparision of CRISPR-X and EvolvR for Saturation Mutagenesis of HBE1 locus cis-regulatory elements
Graduate Presentation. Author(s): John Butts, Ryan Tewhey. Mentor(s): Ryan Tewhey.
Abstract: cis-regulatory elements (CREs) are essential for the proper regulation of genes and the disruption of these cis-regulatory-gene relationships is associated with a spectrum of human diseases. As such, accurate modeling of disease variants represents an invaluable tool to study regulatory disease. Unlike genetic disease, most regulatory diseases cannot be directly modeled. Therefore an understanding of the logic underlying CREs is crucial to effective modeling. Saturation mutagenesis allows for generation of unbiased CRE variant libraries which, coupled with reporters, can provide expression data to elucidate the CRE’s regulatory grammar. Many techniques exist to perform the described mutagenesis utilizing CRISPR-Cas9. A disadvantage of traditional CRISPR-Cas9 is its reliance on indels. In response, techniques have been developed that utilize CRISPR-Cas9 localization while otherwise maintaining the wild-type enhancer background. Examples include CRISPR-X and EvolvR. We targeted three sites in the HBE1 locus for mutagenesis: the promoter, HS2, and a control. Using K562 HBE1:mCherry cells, the impact of mutagenesis can be determined by quantification of mCherry. Initial CRISPR-X experiments show successful, unbiased mutagenesis of the HBE1 Promoter. While CRISPR-X is designed for mammalian expression, EvolvR has not been performed in mammalian cells and requires modification. The dCas9 of the PX462 V2.0 vector has been converted to enCas9 by site directed mutagenesis. Furthermore, the EvolvR native EcoPol II was cloned and additionally synthesized in codon optimized form to compare effectiveness in the mammalian cells. Ultimately the two techniques will be compared for their use in future saturation mutagenesis experiments to further study the logic underlying CREs.
1057. Discovering Virulence Factors of Candida albicans that Affect Host Responses
Undergraduate Presentation. Author(s): Emma Bragdon, Bailey Blair. Mentor(s): Robert Wheeler.
Abstract: Candida albicans is a commensal yet opportunistic pathogen. It is typically found in the mouth and gastrointestinal tract of humans. While healthy adults are not infected, immune-compromised individuals such as those going through chemotherapy are susceptible to infection. Understanding why these patients become susceptible may help us develop new strategies to prevent and treat these lethal infections. The innate immune system is the initial defense when intruders enter a system and is required for resistance against a pathogen. It recognizes a threat as soon as possible and recruits phagocytes to the threat site. However, C. albicans has known virulence factors, agents that allow an organism to cause disease in a host and escape the immune system. Out of virulence factors of C. albicans, adherence, secreted aspartyl proteases, and pleomorphism play the biggest role in infection. Adhesin promotes the adherence of C. albicans to host cells, secreted aspartyl proteases damage epithelial cells, induce macrophage chemotaxis and cytokine production, and pleomorphism refers to C. albicans ability to grow as a yeast or hyphae. A larval zebrafish model is a great way to understand and visualize how pathogens interact with the immune system. In this project the larval zebrafish model with fluorescent immune cells was used to visualize the interaction between immune cells and C. albicans in vivo. Mutant C. albicans strains lacking virulence factors were injected into the hindbrain of the zebrafish, and infection was observed and analyzed to draw conclusions on virulence factors that are most important in C. albicans infection.
1058. Utilization of a novel approach to decode the mechanisms of a fatal viral brain infection
Undergraduate Presentation. Author(s): Francesca Armstrong, Michael Wilczek, Remi Geohegan, Melissa Maginnis. Mentor(s): Melissa Maginnis.
Abstract: When we hear the word “virus,” we commonly think of viruses that are currently spreading in the environment such as influenza or the novel coronavirus that generally cause acute infections but can result in severe complications. However, there are numerous viruses that reside within the human host that have the potential to reactivate and cause serious disease. Human JC polyomavirus (JCPyV) infects 80% of the population and establishes a persistent, lifelong infection in the kidneys. In immunocompromised individuals, JCPyV can spread to the brain and infect glial cells, astrocytes and oligodendrocytes. Infection of these cells results in the fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Current research of JCPyV primarily uses techniques that require a mixed glial cell line known as SVG-A cells that were specifically designed to support JCPyV infection. Unfortunately, these cells do not accurately model infection in the human brain. Thus, we have established primary human astrocytes as an alternative approach to study JCPyV. This research will utilize biochemical and molecular biology techniques in order to address whether the host cell pathways necessary for JCPyV infection in SVG-A cells are required for infection in primary human astrocytes. These findings have proposed significant differences in the characteristics of JCPyV infection in SVG-A cells versus primary human astrocytes that may be vital in furthering our understanding of JCPyV infection propagation and PML pathophysiology. Furthermore, this research may possibly provide a platform for studying our neuroinvasive viruses with fatal outcomes.
1060.* Lymphatic System as a Conduit for Immune Cells in Fat
Undergraduate Presentation. Author(s): Thomas Szewczyk, Magdalena Blaszkiewicz, Matthew Karp, James Godwin, Kristy Townsend. Mentor(s): Kristy Townsend.
Abstract: The proper function of adipose (fat) tissue requires neural communication between this important metabolic organ and the brain. Therefore, maintaining nerve health in adipose tissue is of vital importance to overall metabolic health. Nerve health is maintained in part by neurotrophic factors – growth factors responsible for nerve survival and neurite outgrowth. We have identified a subset of immune cells in mice that home to adipose tissue upon cold-induced noradrenergic stimulation and express neurotrophic factor. We have named these cells, which express the Ly6c+CCR2+Cx3cr1+ markers, cold induced neuroimmune cells (CINCs). However, mechanisms behind how CINCs home to adipose are not well understood. We studied the role of adipose lymphatics in recruitment of CINCs. Immunofluorescence imaging has been done on Cx3Cr1 reporter mice, which are genetically engineered to express the green fluorescent protein in CINCs, making them easy to visualize in adipose tissues. This imaging has revealed that CINCs reside in lymphatic vessels, are transported along the endothelial lining of lymphatic vessels, and are morphologically distinct from adipose resident Cx3Cr1 cells, as they show dendritic-like extensions when recruited to tissue. Further experimentation will examine the role of the subiliac lymph node (SiLN) in subcutaneous white adipose tissue, as it is a nexus of immune cell crosstalk, and we have shown neurite density to increase in this area after cold stimulation. Surgical removal and denervation experiments of the SiLN will reveal if it functions as a conduit for CINC recruitment to adipose and how this recruitment is affected by innervation.
1062. Innate Immune Response to Influenza Type A in a Zebrafish Model
Graduate Presentation. Author(s): Brandy-Lee Soos, Benjamin King. Mentor(s): Benjamin King.
Abstract: The World Health Organization estimated 650,000 deaths per year occur worldwide from respiratory diseases associated with seasonal influenza infections. Influenza A virus (IAV) can result in severe respiratory complications due to hyperinflammation and poor viral clearance in children, senior adults, and immune-compromised individuals. Our long-term goal is to understand how the innate immune response to IAV infection transitions from clearing virus infection to a hyperinflammatory response that can lead to tissue damage and death. Studies demonstrate the importance of neutrophil recruitment and phagocytosis in response to bacterial and fungal infections yet their role in antiviral responses are poorly understood. Recently, it was demonstrated that IAV infection can be established in zebrafish (Danio rerio) larvae. The zebrafish is a powerful vertebrate model system that has been used to study infection and innate immunity. We are using this model to study the roles neutrophils have in controlling IAV infection and how over-activation of neutrophils during IAV infection trigger a damaging hyperinflammatory response. First, we are examining pathways that control the respiratory burst function and how reactive oxidative species control neutrophil function during IAV infection. Second, we are investigating how hyperinflammation occurs during IAV infection so that therapeutic measures that preserve the antiviral response, yet contain the associated inflammation, can be developed. This work is supported by the National Institute of Allergy and Infectious Disease of the National Institutes of Health under grant number R15AI131202 and the National Institute of General Medical Sciences under grant number P20 GM103423.
1063. Role of MicroRNAs in Regulating the Innate Immune Response to Pseudomonas aeruginosa Infection
Graduate Presentation. Author(s): Liz Saavedra Perez, Brandy Soos, Benjamin L. King. Mentor(s): Benjamin L. King.
1064.* Sclerostin antibody normalizes decreased trabecular bone and increased bone marrow adipose tissue caused by whole-body irradiation in mice
Graduate Presentation. Author(s): Samantha Costa. Mentor(s): Michaela Reagan.
Abstract: Irradiation (IR) negatively affects the bone marrow (BM) through increased osteoclast activity, decreased osteoblast activity, and increased adipocyte expansion. Patients receiving IR are at increased risk for bone deterioration and other skeletal-related events. We hypothesized antibodies to sclerostin (Scl-Ab) would reverse the adverse effects of IR by increasing bone volume and decreasing BM adipose tissue (BMAT), resulting in improved bone quality. In this study, 12-week-old female C57BL/6J mice were exposed to 6Gy whole-body IR or were non-irradiated (Non-IR), then administered Scl-Ab (25mg/kg) or vehicle weekly for 5 weeks. Tibial µCT analysis confirmed IR decreased trabecular bone volume (Tb.BV/TV) by 43.1% Scl-Ab increased Tb.BV/TV by 2.45 fold in IR and 2.22 fold in Non-IR mice compared to vehicle. Femoral histomorphometry revealed IR did not affect the number of osteoblasts or osteoclasts, but rather the overall efficacy of these cells. Scl-Ab improved bone parameters by increasing osteoblast function (e.g. bone formation rate and mineral apposition rate). BM adiposity within the femoral metaphysis and whole tibia increased following IR; Scl-Ab diminished this effect in the femoral metaphysis (p=0.0182) and distal tibia (p=0.002).
Overall, our data support the hypothesis that Scl-Ab ameliorate the deleterious effects of IR on trabecular bone and BMAT in a mouse IR model. This suggests further research into cellular function modulation after IR insult are warranted.
1065. Uncovering Candida albicans factors that modulate the host phagocyte response
Graduate Presentation. Author(s): Bailey Blair, Emma Bragdon, Lena Stasiak, Robert Wheeler. Mentor(s): Robert Wheeler.
Abstract: The ability of C. albicans to evade the host immune system helps it to be a successful pathogen, but little is known about how C. albicans achieves this. Previous work suggests that the ability of C. albicans to switch to hyphal growth may help limit the recruitment of phagocytes to the infection site, but the mechanism responsible for this is not yet understood. In order to further understand this, we are using the larval zebrafish infection model to screen a large number C. albicans mutants for virulence. Virulence in this model has previously been observed to be linked to the phagocyte response early in infection. This is the first screen of this scale to look at the virulence of C. albicans mutant strains in a live host. This has led to a number of mutants that show a virulence defect, many that have not previously been characterized for virulence. Transgenic zebrafish are now being used to investigate the phagocyte response to hypovirulent mutants. This allows us to identify important genes and pathways for limiting the host phagocyte response. Furthermore, we can identify factors that limit different stages of the phagocyte response such as recruitment and engulfment. Preliminary data with one hypovirulent mutant suggests that deletion of a single fungal cell wall protein enhances uptake by host macrophages in the zebrafish. This work will allow us to gain valuable insight into C. albicans factors that influence the host phagocyte response and evade immune attack.
1066.* The Meninges and Choroid Plexus are Prominent mTert-Expressing Adult Stem Cell Niches in the Mouse Brain
Graduate Presentation. Author(s): Gabriel Jensen, Kristy Townsend. Mentor(s): Kristy Townsend.
Abstract: Adult neurogenesis persists throughout life in the adult mammalian brain in discrete regions, including the subventricular zone (SVZ) of the lateral ventricles and the hypothalamus. Within these ventricular niches, the choroid plexus produces cerebrospinal fluid (CSF) which distributes various growth factors that modulate this process. Changes in CSF composition affect the neurogenic and migratory capabilities of adult neural progenitor cells that reside within the SVZ. The choroid plexus and meninges also contain stem-like cells that that form neurospheres and differentiate into neurons and glia in vitro. We have identified a population of cells within the adult murine choroid plexus, SVZ, hypothalamus, and meninges that express telomerase reverse transcriptase (Tert). Tert is expressed by regenerative, slowly cycling stem cells in many adult organs. A population of Tert+ cells in the brain express the proliferation marker Ki67, but do not express progenitor or mature neuron markers. Lineage tracing of Tert+ cells in adult mice shows drastically increased numbers of labeled cells within the choroid plexus, SVZ, hypothalamus, and meninges when compared to direct reporter mouse lines. The presence of proliferative Tert+ cells that repopulate major neurogenic niches and lack expression of neural progenitor cell markers or mature neuron markers leads us to conclude that Tert+ cells are a previously undiscovered population of adult neural stem cells in the adult mammalian brain.
1067.* Drawing the road map to JC Polyomavirus infection in the brain: Using bioinformatics to elucidate the cell signaling pathways activated upon virus infection
Graduate Presentation. Author(s): Michael Wilczek, Francesca Armstrong, Remi Geohegan, Melissa Maginnis. Mentor(s): Melissa Maginnis.
Abstract: Bioinformatics is revolutionizing the way we approach biomedical research. The human cell has ~20,000 protein-coding genes, which are important for cell survival and growth. Viruses upon infection, commander the regulation of many genes to hijack the cell, replicate inside it, and thus result in human disease. Conventional biomedical approaches would delay discoveries, but bioinformatic approaches allows the researcher to determine the significance of each gene during virus infection and elucidate therapeutic targets towards human disease. Most of the population is infected with JC polyomavirus (JCPyV), which can cause a deadly infection in the brain. In healthy individuals, the virus persists in the kidneys as a symptomless infection. During immunosuppression, the virus can reactivate and spread into the brain, infecting two main cell types. The destruction of these cells leads to the fatal disease, progressive multifocal leukoencephalopathy (PML). Currently, there are no effective treatments for PML, and it is fatal. To determine the genes influenced during JCPyV infection, bioinformatic techniques can be used to understand how the virus hijacks the cell. Currently, we have discovered the cellular pathways important in JCPyV infection, including the mitogen-activated protein kinases (MAPK), a pathway critical in cellular proliferation and survival. Furthermore, using infectivity assays, siRNA, and quantitative immunofluorescence assays, we validated the importance of the MAPK pathway and other cellular pathways involved in JCPyV infection of NHAs. Using bioinformatics and experimental validation, we will enhance our understanding of PML pathogenesis and define the role of astrocytes in this deadly disease.
1068.* Defining Localization Dynamics of 5-HT2 Receptor Subtypes in JCPyV Entry Using Super-Resolution Localization Microscopy
Graduate Presentation. Author(s): Kashif Mehmood, Jeanne DuShane, Matthew Parent,Michael Wilczek,Colleen Mayberry,Francois Levasseur. Mentor(s): Melissa Maginnis.
Abstract: Among human-specific viruses, JC polyomavirus (JCPyV) asymptomatically infects most of the adult population and remains controlled by the immune system in healthy individuals. JCPyV can reactivate in immunocompromised individuals and relocate to the brain, leading to the fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML). Patients receiving immune-modifying drugs or suffering from immune-mediated disorders such as HIV/AIDS remain at high risk for PML development, and unfortunately the disease remains incurable. Studying the viral attachment and entry mechanisms in host cells is crucial for understanding viral infectivity and disease pathogenesis. Previous studies demonstrate that JCPyV requires α2,6-linked sialic acid receptors for attachment, and viral entry is mediated by 5-hydroxytryptamine 2 (5-HT2) receptor subtypes. However, JCPyV interactions with 5-HT2 receptors is not well understood due to limitations of resolution in conventional fluorescence microscopy techniques. Super-resolution microscopy techniques provide an innovative method to characterize the spatial and temporal dynamics of 5-HT2 receptors during JCPyV infection. We utilized fluorescence photoactivatable localization microscopy (FPALM) to demonstrate that JCPyV localizes with 5-HT2 receptor subtypes that are expressed in photoactivatable Dendra2 constructs within transfected cells. Our findings indicate that viral localization varies between the different 5-HT2 receptor subtypes. These data improve our knowledge and understanding of JCPyV interactions with 5-HT2 receptors for entry into host cells. Using super resolution microscopy and image analysis, we can better understand the spatial and temporal patterning of virus-receptor interactions. These findings can be applied to the future development of an antiviral therapy for JCPyV infection and prevention of PML.
1070.* Murine Myeloma Model Demonstrates Correlation Between Body Composition and Tumor burden
Graduate Presentation. Author(s): Sasha Weller, Heather Campbell, Michaela Reagan. Mentor(s): Michaela Reagan.
1071.* DGAT Activity is Dispensable for Myeloma Cell Proliferation
Graduate Presentation. Author(s): Connor Murphy. Mentor(s): Michaela Reagan.
Abstract: Multiple myeloma (MM) is a blood cancer defined by the clonal expansion of malignant plasma cells in the bone marrow (BM). MM remains incurable due to the development of drug resistance; therefore, it is paramount to investigate novel treatments and the mechanisms of drug resistance in myeloma cells (MCs). Changes in lipid metabolism have been shown to support the proliferation and the development of drug resistance in other cancers. However, the role of lipid metabolism in MCs has been understudied. Therefore, we hypothesized that lipid metabolism contributes to MC proliferation. The role of global lipid metabolism in MC proliferation was tested by treating MCs with Triacsin C (TriC), an inhibitor of both catabolic and anabolic lipid metabolism. TriC treatment severely and specifically decreased MC proliferation and increased apoptosis in a dose-dependent manner. To understand the mechanism of TriC’s toxicity, we explored anabolic lipid metabolism and observed that MCs accumulate lipid droplets (LDs), express LD-related transcripts and use LDs as an energy source. Interestingly, treatment with inhibitors against the rate-limiting enzymes responsible for the formation of LDs did not alter the proliferation of MCs. Our data suggests that global lipid metabolism is critical for myeloma cell proliferation while lipid droplet formation is dispensable. Future directions will explore the role of catabolic lipid metabolism with respect to MC proliferation and drug resistance.
1073.* Engineering Zebrafish Constructs to Test Whether Muscle-Disease Associated MYLPF Variants are Sufficient to Impair Sarcomere Assembly
Graduate Presentation. Author(s): Teresa Easterbrooks, Jared Talbot. Mentor(s): Jared Talbot.
Abstract: Distal Arthrogryposis (DA) is a congenital, genetically-linked disease of muscle development characterized by muscle weakness and contracture of limbs. A recently discovered DA gene, MYLPF, has a zebrafish homolog, mylpfa, that when knocked out leads to a similar phenotype in fish to the DA phenotype (muscle weakness and limb paralysis). These symptoms are explained by sarcomere assembly defects that we find in the mylpfa mutant. These findings suggest that loss of Mylpf function causes DA in humans and DA-like symptoms in zebrafish. However, it remained unclear whether the specific gene variants linked to human DA (residues Gly163 and Cys157) affect protein function. We designed a method for expressing human MYLPF protein (wild-type and disease-associated variants) in place of the zebrafish Mylpfa to investigate variants’ impacts on sarcomere formation in muscle development. Briefly, we will produce a plasmid containing human MYLPF cDNA with polyadenylation sequences and use CRISPR to insert this plasmid into the second intron of zebrafish mylpfa; the inserted human sequences will replace over 80% of the native mylpfa coding sequence. Over the course of the rotation, I designed CRISPR guide RNAs targeting this mylpfa intron, tested their activity in fish, optimized dosage, and in the end found a viable guide RNA for use in line construction. In case new guide RNAs need to be designed, I also characterized polymorphisms in mylpfa intronic sequences. My findings lay the groundwork towards generating zebrafish lines that will test whether the Gly163 and Cys157 protein variants are sufficient to impair sarcomere assembly.
1074.* Rab27a in Exosome Function in Perivascular Adipose Tissue
Graduate Presentation. Author(s): Ashley Soucy, Larisa Ryzhova, Samantha White, Benjamin Tero, Bethany Fortier, Anne Harrington. Mentor(s): Lucy Liaw.
Abstract: Perivascular adipose tissue (PVAT), an adipose depot, surrounds the vasculature of humans and mice. In healthy and non-obese mice, PVAT exhibits a thermogenic phenotype and confers vascular health. Conversely, in obese mice, PVAT exhibits a lipid storage phenotype which is detrimental to vasculature. When comparing endogenous protein expression between healthy and obese mice, we found a significant increase in Rab27a. A member of the Ras-related brain protein family, Rab27a is a trafficking protein that regulates the formation and secretion of exosomes. Misregulation of exosome secretion has been linked to various diseases including type II diabetes and increased heart muscle deterioration. However, the role of Rab27a has yet to be defined in PVAT regarding exosome secretion and cardiovascular disease (CVD). We hypothesized that increased Rab27a expression in PVAT during obesity alters the function of secreted exosomes. To test our hypothesis, we developed a novel Rab27a global knockout mouse, characterized by an ashen coat color that mimics Griscelli’s syndrome in humans. This mouse strain also shows resistance to weight gain compared to wild type mice on a high fat diet. Immunoblot comparison of endogenous exosome markers between different adipose depots from wild type mice suggest that PVAT has a unique capability to secrete exosomes. We have also begun characterizing exosomes secreted from our PVAT APCs utilizing Tunable Resistive Pulse Sensing. With this technique, we discovered that upon differentiation, PVAT exosome secretion increases substantially. This suggests that PVAT exosome secretion is dependent on the cell’s metabolic state.
1075.* Rab27a Expression in Perivascular Adipose Tissue Changes Upon Differentiation
Graduate Presentation. Author(s): Katie Stieber, Joshua Boucher, Jacqueline Turner, Lucy Liaw. Mentor(s): Lucy Liaw.
Abstract: Introduction: Obesity is one of the strongest risk factors for cardiovascular disease. Cellular interactions between adipose tissue and the vasculature may explain this link. Perivascular adipose tissue (PVAT) surrounds most vessels and exerts a paracrine effect on the underlying blood vessel. Rab27a regulates protein and trafficking. We hypothesize that Rab27a in PVAT mediates local paracrine signaling to the vessel wall during cardiovascular disease. Hypothesis: We assessed the hypothesis that Rab27a is dynamically regulated in murine PVAT during diet-induced obesity and differentiation. Methods: C57BL/6J male mice were fed a 60% high fat diet for 12 weeks starting at 8 weeks of age. Body weight and fat and lean mass were determined. Immunoblot was used to assess Rab27a levels. Using primary pre-adipocytes derived from murine PVAT, 3D adipospheres were generated for in vitro study. Adipospheres were differentiated, and probed for lipid markers and Rab27a. Results: High fat diet resulted in increased expression of Rab27a in PVAT in vivo. Adipospheres showed specific staining for lipid markers and Rab27a. Rab27a expression is more localized as differentiation time increases. Conclusions: High fat diet leads to increased Rab27a levels in PVAT, which will be studied further to examine Rab27a control of adipogenesis. We demonstrated the feasibility of adapting 3D cultures for murine PVAT, the ability to differentiate spheroids using standard adipogenesis differentiation conditions, and have shown temporal changes of Rab27a expression with differentiation in murine adipospheres. We are currently using the model to test the expression of RAB27a during differentiation in human derived PVAT adipose progenitor cells.
1077.* Sending Signals: Adipose Sensory Nerves may Communicate to the Brain via Lipid Metabolites
Graduate Presentation. Author(s): Cory Johnson, Jake Willows, Kristy Townsend. Mentor(s): Kristy Townsend.
Abstract: Obesity and its related co-morbidities, such as type II diabetes, have reached pandemic proportions affecting millions worldwide. Current research aims to identify molecular mechanisms that may provide treatment and/or prevention options for these diseases. Diet is one contributor to energy balance. If energy intake exceeds energy output, individuals become susceptible to metabolic disease. The type of energy intake is crucial and certain dietary fats, such as n-3/n-6 polyunsaturated fatty acids, may promote beneficial metabolic activity. Through unbiased lipidomics, in which white adipose tissue (WAT) compositions were compared, we uncovered that dietary intake of specific fatty acids alters WAT lipid composition. Interestingly, mice fed an n-6 enriched diet produced higher concentrations of pro- and anti-inflammatory signaling lipid metabolites in subcutaneous WAT (scWAT), compared to n-3 or saturated fat enriched diets. A robust increase of a specific octadecanoid, 13-HODE, was observed in scWAT following the n-6 enriched diet. Some octadecanoids are known to activate the calcium permeable transient receptor potential (TRP) channels located on sensory nerves. We have shown that the TRP channel TRPV1 is expressed on sensory nerves in mouse scWAT. Additionally, the lipid 13-HODE is capable of activating TRPV1 channels, thereby uptaking intracellular calcium. We have used intravital imaging of calcium uptake in mouse scWAT sensory nerves using a fluorescent calcium indicator upon 13-HODE delivery. Currently we are investigating the metabolic contributions of 13-HODE activation of TRPV1 expressing sensory nerves by assessing adipose, sensory ganglia, and hypothalamus following 13-HODE administration to scWAT.
1078. The Mechanisms of TAp63-dependent and -independent DNA Damage Response in Meiotically Arrested Oocytes
Graduate Presentation. Author(s): Monique Mills, Chihiro Emori, Zachary Boucher, Ewelina Bolcun-Filas. Mentor(s): Ewelina Bolcun-Filas.
Abstract: Development of aggressive cancer treatments increases patient survival rates, however the genotoxicity of these lifesaving treatments leave female cancer survivors facing risk of premature ovarian failure. Cancer treatments induce DNA double strand breaks (DSBs) preferentially killing cancer cells, but can kill healthy cells. In somatic cells, DSBs activate the ataxia-telangiectasia-mutated (ATM) DNA damage response (DDR) pathway, leading to CHK2 kinase activation of p53. Active p53 induces senescence, cell cycle arrest, or apoptosis. Arrested oocytes largely utilize the same DDR; however CHK2 activates TAp63. TAp63 is exclusively expressed in oocytes and activation by low levels of DSBs induce apoptosis of oocytes before DNA repair occurs. At low-dose irradiation (IR), female mice are depleted of arrested oocytes and become infertile, while TAp63-deficient females remain fertile. This suggests that TAp63 plays a major role in oocyte apoptosis after DNA damage. Conversely, at high-dose IR the majority of TAp63-deficient oocytes are eliminated. This suggests elimination of oocytes at higher levels of DNA damage involves a TAp63-independent mechanism. We hypothesize the TAp63-independent mechanism requires p53 activity and our goal is to resolve relative contribution of these pathways in oocytes. Preliminary data supports this hypothesis as TAp63-/- p53-/- females receiving high-doses IR retain oocytes and a larger from of p53 was expressed in IR purified oocytes but not in purified somatic cells. Defining such oocyte-specific responses to DNA damage will allow us to target unique DDR pathways to prevent oocyte loss, infertility, and premature ovarian failure in patients receiving cancer treatments.