The Graduate Student Research Forum is sponsored by the Graduate Student Association of Penn State College of Medicine.
The primary goal of the Forum is to promote interaction and the exchange of ideas between students, faculty and alumni.
The Forum introduces students to the format generally used at scientific meetings, and it provides an opportunity to recognize excellence in graduate research at Penn State College of Medicine.
Forum 2021 will take place March 3, 4 and 5
As part of the Graduate Student Research Forum, student lectures and poster presentations will take place, as well as a keynote presentation by an invited speaker.
The Graduate Student Research Forum Endowed Fund helps to elevate Forum each year through assisting with costs associated with invited keynote speaker travel needs and equipment such as presentation panel rentals.
Anyone can help support Forum through giving.
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More About Forum 2021
Wednesday, March 3, 2021
Virtual event via Zoom from 4 to 7 p.m.: Registration is required
- 4 to 4:10 p.m.: Introduction
- 4:10 to 4:25 p.m.: Live oral presentation by MaryElizabeth Stein
- 4:25 to 4:32 p.m.: Q-and-A with MaryElizabeth Stein
- 4:32 to 4:47 p.m.: Live oral presentation by Ruoheng Zhang
- 4:47 to 4:55 p.m. Q-and-A with Ruoheng Zhang
- 4:55 to 5:10 p.m.: Live oral presentation by Mi Zhou
- 5:10 to 5:17 p.m.: Q-and-A with Mi Zhou
- 5:17 to 5:32 p.m.: Live oral presentation by Kelly Stahl
- 5:32 to 5:40 p.m.: Q-and-A with Kelly Stahl
- 5:40 to 5:47 p.m.: Data blitz presentation by Natella Maglakelidze
- 5:47 to 5:52 p.m.: Q-and-A with Natella Maglakelidze
- 5:52 to 6 p.m.: Data blitz presentation by Jesse White
- 6 to 6:05 p.m.: Q-and-A with Jesse White
- 6:05 to 6:12 p.m.: Data blitz presentation by Kashif Shaikh
- 6:12 to 6:17 p.m.: Q-and-A with Kashif Shaikh
- 6:17 to 6:24 p.m.: Data blitz presentation by Adam Fike
- 6:24 to 6:30 p.m.: Q-and-A with Adam Fike
- 6:30 to 7 p.m.: Closing remarks
Thursday, March 4, 2021
Virtual event via Zoom from 4 to 7 p.m.: Registration is required
- 4 to 4:10 p.m.: Introduction
- 4:10 to 4:25 p.m.: Live oral presentation by Nour Hawila
- 4:25 to 4:32 p.m.: Q-and-A with Nour Hawila
- 4:32 to 4:47 p.m.: Live oral presentation by Angela Snyder
- 4:47 to 4:55 p.m.: Q-and-A with Angela Snyder
- 4:55 to 5:02 p.m.: Data blitz presentation by Mariam Melkumyan
- 5:02 to 5:07 p.m.: Q-and-A with Mariam Melkumyan
- 5:07 to 5:15 p.m.: Data blitz presentation by Megan Olsen
- 5:15 to 5:20 p.m.: Q-and-A with Megan Olsen
- 5:20 to 5:30 p.m.: Keynote speaker introduction
- 5:30 to 6:30 p.m.: Keynote speaker Dr. Tricia Burdo research talk – “HIV in the 21st century: Comorbidities and progress toward a cure”
- 6:30 to 7 p.m.: Q-and-A with Dr. Tricia Burdo and closing
Friday, March 5, 2021
Virtual event and live presentations in Junker Auditorium from 8:45 a.m. to 2 p.m.: Registration is required
- 8:45 to 9 a.m.: Introduction
- 9 to 9:15 a.m.: Live oral presentation by Zachary Nolan
- 9:15 to 9:23 a.m.: Q-and-A with Zachary Nolan
- 9:23 to 9:38 a.m.: Live oral presentation by Kushal Saha
- 9:38 to 9:45 a.m.: Q-and-A with Kushal Saha
- 9:45 to 10 a.m.: Live oral presentation by Elizabeth Lesko
- 10 to 10:09 a.m.: Q-and-A with Elizabeth Lesko
- 10:09 to 10:24 a.m.: Live oral presentation by William Miller
- 10:24 to 10:31 a.m.: Q-and-A with William Miller
- 10:31 to 10:46 a.m.: Live oral presentation by Joseph Cirilo
- 10:46 to 10:53 a.m.: Q-and-A with Joseph Cirilo
- 10:53 to 11:07 a.m.: Live oral presentation by Aflah Hanafiah
- 11:07 to 11:15 a.m.: Q-and-A with Aflah Hanafiah
- 11:15 to 11:30 a.m.: Live oral presentation by Xupeng Hong
- 11:30 to 11:38 a.m.: Q-and-A with Xupeng Hong
- 11:38 to 11:53 a.m.: Live oral resentation by Jordan Chang
- 11:53 a.m. to noon: Q-and-A with Jordan Chang
- Noon to 12:10 p.m.: Keynote speaker introduction
- 12:10 to 1:10 p.m.: Keynote speaker Dr. Tricia Burdo career talk – “Beyond the Scientific Method for Academic Career Success”
- 1:10 to 1:30 p.m.: Q-and-A with Dr. Tricia Burdo
- 1:30 to 1:45 p.m.: Final remarks
Thank you for attending the 33rd annual Graduate Student Research Forum. For many years, Forum has provided an opportunity for graduate students to showcase their research at our institution. With this platform, we are able to learn about all the remarkable work of our friends, colleagues and mentors.
Moreover, we are able to form new connections with alumni who have been through this journey and understand the perseverance needed to achieve our goal. In addition, a keynote speaker in invited every year to not only expose students to new and exciting fields of research, but also provide personal insight about navigating the world of science. This year we are honored to have Dr. Tricia Burdo, a College of Medicine alumnus, as our keynote speaker.
This monumental event would not have been made possible without the support and efforts of many faculty members, students, and staff. We would like to acknowledge and thank our wonderful forum committee (Alison Celigoi, Greg Kincheloe, Gaelyn Lyons, Mariam Melkumyan and Luke Urbanik). Words cannot express how thankful we are for the time and commitment you have all put into making this Graduate Student Research Forum a success. Above all, thank you Rachel Reager and the entire Graduate Education office. You are truly the foundation to the success of Forum and this would not have been possible without your help.
Thank you again for supporting graduate education at the College of Medicine.
Darren Mehay and Claire Werner
The Forum committee sincerely appreciates the support of Dr. Charles Lang, Associate Dean for Graduate Studies; Dr. Leslie Parent, Vice Dean for Research and Graduate Studies; and Dr. Kevin Black, Interim Dean of the College of Medicine, for their commitment to graduate education.
The committee also thanks the following members of the Penn State community who agreed to serve as Forum 2021 evaluators:
- Amy Arnold
- Alistair Barber
- Arthur Berg
- Sarah Bronson
- Kirsteen Browning
- Laura Carrel
- Danielle Covington
- Dhimant Desai
- Zheng-Ming Ding
- Phil Domeier (alumnus)
- Ashwinkumar Subramenium Ganapathy
- Donald Gill
- Sue Grigson
- Andras Hajnal
- Edward Harhaj
- Jianming Hu
- Fumiaki Imamura
- Yuka Imamura
- Jenelle Izer (alumna)
- Shanie Jayasinghe
- Deepkamal Karelia (alumnus)
- Ralph L. Keil
- Yongsoo Kim
- Rinki Kumar
- Taryn Mockus (alumna)
- Amanda Nelson (alumna)
- Meghali Nighot
- Prashant Nighot
- Leslie Parent
- Anirban Paul
- Ira Ropson
- Stephanie Schell (alumna)
- Cara-Lynne Schengrund
- Lisa Shantz
- Ian Simpson
- Thomas Spratt
- Siddharth Sunilkumar
- Guy Townsend
- Vonn Walter
- Joseph Wang
- Christopher Yengo
- Gregory Yochum
Dr. Tricia Burdo received her PhD in cell and molecular biology from Penn State College of Medicine in 2003, followed by a postdoctoral fellowship at The Scripps Research Institute (now Scripps Research) in La Jolla, Calif. In 2007, she became an assistant professor at Boston College and, in 2012, an associate professor. In 2016, she moved to the Lewis Katz School of Medicine at Temple University as an associate professor in the Department of Neuroscience and received tenure in 2020.As the associate chair of education, Burdo is responsible for numerous tasks, including oversight and coordination of departmental educational resources; assisting faculty with training grant and fellowship submissions to the NIH and other agencies; and serving as the liaison for a city-wide neuroAIDS network that integrates training activities, symposia and workshops across Philadelphia.
She lectures on viral immunopathogenesis, vaccines and RNA viruses in numerous professional and graduate courses and is the director of the departmental journal club and Neuroscience Grand Rounds.
Dr. Burdo’s research focuses on the role of ongoing chronic immune activation and the role of monocyte/macrophages in HIV-associated peripheral neuropathy (HIV-PN), HIV-associated neurocognitive disorders (HAND) and cardiovascular disease (CVD). She has pioneered work on soluble CD163 (sCD163) as a marker of chronic immune activation in SIV and HIV infection. She has shown that sCD163 correlated with non-calcified vulnerable plaque and arterial inflammation in HIV-infected patients. Her lab has led key studies on the relationship between monocyte activation and myocardial pathology and neuroAIDS in the rhesus macaque SIV model of AIDS and in pathological cardiac and brain studies from subjects with HIV.
miR-21 regulation of the anti-pathogen response
Authors: Kristen N. Bricker, Adam J. Fike, Sara A. Luckenbill, Nicholas M. Choi, Todd M. Umstead, Zissis C. Chroneos, Nancy J. Olsen, Ziaur S.M. Rahman
Abstract: microRNAs (miRNAs) are short-stranded RNA molecules that bind mRNA transcripts to modulate protein translation. These epigenetic regulators have recently been heavily implicated in influencing immune cell development and function. We used influenza virus as a model to delineate the distinct role that miRNA-21 (miR-21) may play in anti-pathogen responses. We intranasally infected B6.129 (B6) and B6.129.miR-21 -/- (miR-21-/-) mice with a sub-lethal dose of influenza A PR/8/34 (H1N1) and harvested mice at multiple time points. Fourteen days post infection miR-21-/- mice had a two-fold reduction in frequencies of CD4+ and CD8+ T cells in the lung by flow cytometry. These mice also had significantly reduced plasma cell (PC) populations in the draining, mediastinal lymph node (mLN), and mature PCs in the spleen. In addition, miR-21-/- mice had significantly lower numbers and frequencies of germinal center (GC) B cells, effector T cells, and T follicular helper (Tfh) cells in the spleen by flow cytometry. These data were supported by immunofluorescent staining of the spleen which showed significantly reduced GC size and frequency. MiR-21-/- mice also had a reduction in viral specific antibody titers. These data suggest that miR-21 plays a key role in promoting an effective immune response to pathogenic challenge. Dissecting the role of miR-21 in this environment is critical to understand miRNA regulation of the immune system, and more importantly, approaching vaccine generation with novel insights.
Identification of somatic structural variants in B-cell acute ymphoblastic leukemia
Authors: Laura Budurlean, Sinisa Dovat, Scott Chartrand, James Broach
Abstract: Pediatric hematological malignancies comprise 40% of all pediatric cancers. Acute lymphoblastic leukemia is the most common type of childhood cancer with 3,200 new cases/yr. Although mortality of pediatric ALL is steadily decreasing, ALL is the leading cause of death among pediatric cancer, which highlights the unmet medical need for novel treatment for this disease. Children of Hispanic origin have higher incidence of B-ALL as compared to children of Caucasian ancestry. Moreover, mortality rate of children of Hispanic origin with this disease is 39% higher than the mortality of non-Hispanic whites. Understanding the pathogenesis of B-ALL, identifying additional structural variations in B-ALL in Hispanic vs. Caucasian children and developing novel therapies to target specific regulatory mechanisms in this disease will reduce the health disparity in survival for Hispanic children with ALL and advance knowledge of this type of leukemia. Reducing health disparities requires novel integrative platforms to study disease mechanisms and identify therapeutic targets in context of genetic ancestry and specific genetic alterations. We recently developed a new, genome-wide Integrated Framework for Genome Analysis to identify novel genetic alterations in cancer cell lines and the resulting effect on gene expression. The method combines whole genome sequencing with large DNA fragment optical mapping, which identifies and maps structural variants not previously recognizable in both coding and non-coding regions. Our previous application of this method to AML patient samples revealed a number of previously unappreciated cancer related genes that affected patient outcomes. We will use this platform to identify novel somatic structural alterations in pediatric B-ALL from Hispanic versus Caucasian children and determine their effects on genome organization, gene function and clinical outcome. Results of the proposal will provide the first comprehensive and detailed genome-wide analysis of pediatric B-ALL somatic structural variants and their impact on genetic regulation and clinical outcome in Hispanic versus Caucasian children. Results of this proposal will identify new therapeutic targets that will lead to novel targeted treatments to aid in reducing the health disparity in pediatric B-ALL.
Activation of synaptic NMDA receptors on rat dorsal motor nucleus of the vagus neurons in response to acute high fat diet requires gliotransmitter release and activation of purinergic and metabotropic glutamate receptors
Authors: Kaitlin E. Carson and Kirsteen N. Browning
Abstract: Previous work from this lab has demonstrated that, in rats, restoration of caloric intake following acute high fat diet (aHFD) is dependent upon upregulated glutamatergic signaling in the dorsal motor nucleus of the vagus (DMV), which recruits previously silent NMDA receptors. We have further shown that synaptic NMDA receptor activation is driven by the activation of extrasynaptic NMDA receptors in an astrocyte-dependent process, although the exact mechanism involved is still unknown. The current study was designed to test the hypothesis that activation of purinergic and/or metabotropic glutamate receptors (mGluR) by the gliotransmitters ATP and glutamate, respectively, is responsible for activation of extrasynaptic, hence synaptic, NMDA receptors. Whole cell patch clamp recording were made from DMV neurons in thin brainstem slices prepared from male and female Sprague-Dawley rats fed either a control diet (14% kcal from fat) or following acute, 3-5 days aHFD (60% kcal from fat) exposure. The presence of activated synaptic NMDA receptors was assessed by the ability of the antagonist AP5 (25µM) to decrease the frequency of miniature excitatory synaptic currents (mEPSC). The involvement of P2X receptors was investigated using the antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 10µM) and agonist αβ-methyl adenosine triphosphate (αβMeATP; 10µM) while the involvement of group I mGluR was investigated using the antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 300µM) and agonist (RS)-3,5-dihydroxyphenylglycine (DHPG; 20µM). In aHFD DMV neurons, perfusion with AP5 decreased mEPSC frequency in 8/9 neurons (3.18 ± 1.71 vs 2.11 ± 1.12; p<0.05). Application of PPADS blocked the ability of AP5 to decrease mEPSC frequency in 6/8 neurons (2.49 ± 1.63 vs 2.57 ± 2.10; P<0.05 vs AP5 alone). In a similar manner, application of the group 1 mGluR antagonist, AIDA, blocked the ability of AP5 to decrease mEPSC frequency in 7/10 aHFD DMV neurons (2.18 ± 1.42 vs 1.88 ± 1.45; P<0.05 vs AP5 alone). In contrast, in control DMV neurons, the ability of AP5 to decrease mEPSC frequency was uncovered following application of the agonist DHPG in 6/9 neurons (1.90 ± 0.96 vs. 1.35 ± 0.82; P<0.05; P>0.05 compared to aHFD neurons) and following application of the agonist αβMeATP in 3/9 neurons (2.85 ± 0.74 vs. 2.08 ± 0.99; P<0.05; P>0.05 compared to aHFD neurons) suggesting activation of group I mGlu and purinergic P2X receptors are involved in the aHFD-dependent uncovering of synaptic NMDA receptors. Taken together, these results show that purinergic and metabotropic glutamatergic signaling is required for activation of synaptic NMDA receptors on DMV neurons following aHFD exposure. The current study provides a potential mechanism by which caloric balance is restored following aHFD exposure. Specifically, aHFD-dependent release of the brainstem gliotransmitters ATP and glutamate is required for the subsequent activation of DMV extrasynaptic and synaptic NMDA receptors. This upregulates NTS-DMV glutamatergic signaling and alters the excitability of central vagal neurocircuits involved in the modulation of gastrointestinal functions including feeding behavior.
Degree program: Biomedical Sciences
Department: Medicine and Microbiology and Immunology
Committee chair or adviser: Leslie Parent, MD
Other committee members: Nicholas Buchkovich, PhD; Clare Sample, PhD; Zhonghua Gao, PhD; Feng Yue, PhD (Northwestern University)
HIV-1 Gag localizes to euchromatin and associates with viral RNA at transcription sites
Authors: Jordan Chang, Breanna Rice and Leslie Parent
Abstract: Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that attacks the host immune system, depleting host T cell population and leading to the onset of acquired immunodeficiency syndrome (AIDS). Despite the availability of anti-retroviral therapies, HIV continues to be a global epidemic without an effective vaccine or cure. One major contributing factor is its ability to integrate into the host genome and establish a latent reservoir in resting T cells. Although the viral integration site varies between viruses, integration occurs primarily in active gene loci. Furthermore, cells actively expressing HIV-1 viral genes exhibit distinct chromatin markers in the euchromatin. Once active, the virus utilizes host transcription machinery to produce nascent unspliced viral RNA (vRNA) that serves as either an mRNA template for translation or is packaged into new virions as genomic RNA by Gag, the major viral structural polyprotein. Historically, it was thought that the selection of the vRNA occurred in the cytoplasm and at the plasma membrane, although recent results from our lab demonstrated that the HIV-1 Gag protein undergoes nuclear localization and binds to unspliced vRNA at active transcription sites. This finding raises the possibility that genomic RNA incorporation begins in the nucleus with binding to Gag. However, the mechanisms underlying Gag nuclear trafficking, including the timing and subcellular localization of Gag, remain unknown. To further explore this, we used a HeLa cell line that contains a stably-integrated HIV-1 provirus that expresses Gag-CFP in a doxycycline-inducible manner. We performed immunoblot analysis for Gag expression every four hours over a 24-hour period and detected the presence of Gag in both the nuclear and cytoplasmic protein fractions, indicating HIV-1 Gag nuclear trafficking occurs during early stages of expression. We previously demonstrated that full-length Gag proteins associate with host nuclear proteins, such as Mediators, transcription factors, and a subunit of RNA polymerase II. Moreover, cells treated with transcription inhibitors underwent increased Gag-vRNA co-localization in the nucleus, suggesting that Gag accumulates on vRNA made at active transcription sites. Therefore, we asked whether HIV-1 Gag localizes to the euchromatin. To this end, we employed our doxycycline-dependent proviral construct to control the amount of HIV-1 Gag expression by titrating the doxycycline concentration. Chromatin fractionation of low Gag-expressing cells revealed that Gag primarily localizes to the euchromatin compared to the nucleoplasm and heterochromatin. Furthermore, we sought to determine whether shifting the cellular chromatin state to euchromatin would affect Gag localization. To alter the chromatin state, we treated Gag-expressing HeLa cells with romidepsin, a histone deacetylase (HDAC) inhibitor and performed chromatin fractionation followed by immunoblotting. We found that romidepsin treatment not only upregulated Gag expression, but also led to an increase of Gag in the euchromatin fraction. Taken together, we demonstrated that a subset of HIV-1 Gag traffics to the nucleus upon expression and is preferentially found in the euchromatin, supporting our hypothesis that HIV-1 Gag is localizing to active transcription sites where it is able to bind to its vRNA.
MYO3A motor properties are fine-tuned for its role in stereocilia length regulation
Authors: Joseph A. Cirilo Jr. and Christopher M. Yengo
Abstract: The stereocilia of inner ear hair cells are the mechanosensors of sound waves in the inner ear and their structure must be precisely maintained for normal hearing. There are two class III myosins, MYO3A and MYO3B, which are actin-based ATPase motors that are involved in regulating stereocilia length and morphology. Mutations in the motor domain of MYO3A were found to cause a form of delayed onset non-syndromic hearing loss known as DFNB30. We hypothesize that the unique motor properties of these myosins fine tune them for their role in stereocilia maintenance and, as such, changing these properties alters the ability of MYO3A/B to localize to the stereocilia tips and exert a force that elongates them. To investigate this, we created several chimeras of MYO3A that contained an N- terminal GFP, the motor domain and neck region of different myosins (MYO1A, non-muscle MYO2A, MYO5A, MYO7A, MYO10, or MYO15A), and the tail region of MYO3A. Chimeric myosins were transfected into COS-7 cells and assessed for their ability to form filopodia, localize to filopodia tips, regulate filopodial length, and extend filopodia. We found that a high duty ratio, fraction of the ATPase cycle myosin is strongly bound to actin, is important for efficient filopodia tip localization, while extension velocity correlates with motor actin-gliding speed. Overall, our data supports a model whereby high duty ratio motors are able to tip localize in actin-based protrusions and exert a force on the membrane, producing a net protrusion elongation that is correlated with the speed of the motor.
Brain mapping addiction
Authors: Brianna Evans, Sarah Ballard, Kyra Newmaster, Yongsoo Kim and Sue Grigson
Abstract: While we are familiar with brain areas and pathways that are implicated in opioid use disorder (OUD), we do not have a full understanding of the neural circuits activated upon drug exposure. In order to identify areas of the brain most activated by opioids, we ran a pilot study using transgenic cFos-GFP mice that were injected with saline or heroin and examined the brain-wide activity patterns using a quantitative high-resolution mapping method. We observed many brain regions highly activated upon drug exposure. To examine cFos based brain activation in rats, we also ran a pilot study using a tissue clearing and 3D immunolabeling method combined with light sheet microscopy. The different activation patterns are shown.
B cell-intrinsic STAT3 signaling controls the germinal center dark zone
Authors: Adam J. Fike (co-first author), Sathi Babu Chodisetti (co-first author), Kristen N. Bricker, Philip P. Domeier, Nicholas M. Choi, Sara Luckenbill, Steven Ziegler and Ziaur S.M. Rahman
Abstract: B lymphocytes respond to foreign or self-antigen and differentiate via the extrafollicular or germinal center pathways (GC). GCs are specialized immunological niches within secondary lymphoid organs which promote clonal expansion and the evolution of B cells into memory B cells (MBCs) or long-live plasma cells (LLPCs). Canonically, GCs are divided into semi-distinct zones defined as the dark zone (DZ) and light zone (LZ). Within the DZ, GC B cells undergo rounds of proliferation and AID-mediated somatic hypermutation (SHM) in the variable regions of the BCR/Ig, resulting in affinity maturation of the B cell response. Within the LZ, B cells receive T follicular helper cell (Tfh)-mediated selection signals. Tfh driven-signal strength is tightly linked to the GC B cell fate, whereby signals can result in GC-exit, DZ re- entry, or apoptosis. GC B cells undergo rounds of cycling between the LZ and DZ prior to differentiation into MBCs or LLPCs, or undergoing apoptosis. The signals dictating these decisions remain incompletely understood. STAT3 is a transcription factor regulated through the phosphorylation of tyrosine (705) and serine (727) residues which permit the dimerization and translocation to the nucleus to induce transcription of target genes. STAT3 has a known role in the regulation of plasma cell differentiation, but its function within the GC remains elusive. Previous studies have suggested a B cell-intrinsic role of STAT3 in GC maintenance and antibody responses; however, the mechanism by which STAT3 signaling in B cells contributes to the GC reaction is not clear. Using immunization and infection models, we show that STAT3 in B cells regulates the GC reaction by controlling the GC DZ, resulting in decreased high affinity antibody production, but intact SHM within a defined BCR region. STAT3 deletion in B cells also led to defects in LLPC formation and MBC responses. We further confirmed these findings using a post- Class Switch Recombination (CSR) B cell-specific conditional deletion model. Transcriptomic analysis of GC B cells from STAT3 deficient and sufficient mice, reveals alterations in multiple cellular pathways known to be involved in GC DZ maintenance. We further identified that STAT3 phosphorylation occurs dominantly within the LZ of the GC. Surprisingly, while the majority of the STAT3 phospho-Y705 can be found in the nucleus of GC B cells, STAT3 phospho-S727 is largely cytoplasmic, potentially indicating differential functions for tyrosine and serine phosphorylated STAT3 in GC B cells. In conclusion, STAT3 signaling in B cells controls the GC DZ, which, in turn, helps maintain GCs for the generation of LLPCs and MBCs, and high-affinity antibody production.
Apolipoprotein E variant alters cytokine secretion patterns in frontal cortex of aged mice
Authors: Rebecca M. Fleeman, Amanda M. Snyder, Madison K. Kuhn and Elizabeth A. Proctor
Abstract: Alzheimer’s disease (AD) is a neurodegenerative disease characterized by amyloid-β plaques, neurofibrillary tau tangles, and neuroinflammation that currently affects more than 5.6 million Americans over the age of 65. The ε4 variant of apolipoprotein E (APOE) is the strongest and most common genetic risk factor for AD, due to incompletely understood mechanisms. APOE has three common isoforms in the population, ε2, ε3, and ε4, with ε3 being the most common (78% of the population) and conferring average risk for AD. Neuroinflammation has increasingly garnered attention for exacerbating AD pathology, and we hypothesize that APOE genotype affects the neuroinflammation cascade in the brain to increase AD risk. Our goal was to measure whether there are discernable differences in cytokine signaling between the frontal cortex of aged APOE3 and APOE4 mice. Differences that appear with age, that also correspond with pathways known to be upregulated in AD will elucidate the potential AD-promoting mechanism of APOE4. Knowing the mechanism of APOE4 conferring AD risk will allow us to design preventative medicine regiments for APOE4-carrying individuals. Importantly, cytokine signaling is extremely complex, with many interacting pathways, and thus, multivariate analysis is necessary to take into account the covariation and dependence between levels of cytokines. Using partial least squares discriminant analysis, we found that cytokine signatures differ between genotypes in the frontal cortex of APOE3 and APOE4 mice, and these cytokine signature differences are more pronounced in aged mice (12-18 months old) than in younger (3-month-old) mice, indicating that the cytokine signature evolves with age, just as AD does. Specifically, the frontal cortex of APOE4 mice had higher levels of interleukin (IL)-9 and macrophage inflammatory protein (MIP)-1a, which increased in significance as the mice aged, while the frontal cortex cells of APOE3 mice secrete more IL-1a. Current evidence shows IL-9 is a proinflammatory cytokine, MIP-1a activates macrophages, and IL-1a is produced by activated macrophages and is involved in wound healing. Our results highlight the importance of understanding the impact of genetic risk factors on neuroinflammation. Future studies will define the downstream neural mechanisms underlying the protective and detrimental effects of the cytokine signatures in APOE4 vs. APOE3 systems.
The R345W-fibulin-3 mutation increases the sensitivity of the outer retina to damage caused by sodium iodate
Authors: Jasmine S. Geathers, Stephanie Grillo, Jennifer Liao, Weiwei Wang, Alistair J. Barber and Jeffrey M. Sundstrom
Abstract: Malattia Leventinese (ML), an autosomal dominant inherited degenerative eye disease, is caused by a point mutation (R345W) in the fibulin-3 (Fib3) gene (efemp1) that results in hyperreflective foci (detected by optical coherence tomography) in association with sub-retinal pigment epithelium (RPE) drusen and hyper-pigmentation. The purpose this study was to determine the extent of augmentation of hyperreflectivity caused by sodium iodate-induced retinal degeneration in mice carrying the R345W fib3 mutation. Eighteen C57 three-month-old male and female mice were injected with SI dissolved in PBS at 15, 30, 45, or 60 mg/kg. Spectral-domain optic coherence topography (SD-OCT) images were acquired 7 days later (Bioptigen). Frozen vertical retinal cross-sections (10 µm thick) of eyes from wild-type (WT), heterozygous (Fib3+/ki), and homozygous (Fib3ki/ki) mice were processed for H&E staining. Hyperreflective foci (HRF) were detected by SD-OCT in the retinas of WT mice given 45 and 60 mg/kg, but not in those given 15 and 30 mg/kg. H&E sections appeared normal in the control mice, and in the WT mice that received 15 and 30 mg/kg of SI. The retinas of WT mice receiving 45 and 60 mg/kg were thinner than the control (31.2% and 27.1%, p<0.001) , due to the loss of tissue in the photoreceptor layer. Thinning of the ONL and disorganization of the RPE layer was also noted. Hyperreflective foci were evident in Fib3+/ki and Fib3ki/ki mice given SI at 30, 45, and 60 mg/kg but were not apparent in mice given 15mg/kg. H&E images followed a similar trend, with the 15mg/kg dose looking similar to the controls, whereas sections from Fib3+/ki and Fib3ki/ki mice treated with 30, 45, and 60mg/kg presented with pigmented lesions in the neural retina. The results suggest that SI causes HRF and induces pigment migration into the neural retina in WT mice. Furthermore, presence of the fib3 mutation increases sensitivity to SI exposure, leading to more dramatic retinal degeneration and pigment migration. Our results suggest that the R345W mutation increases susceptibility of the outer retina and RPE to chemical or metabolic insult.
Degree program: Biomedical Sciences
Department: Penn State Cancer Institute and Pharmacology
Committee chair or adviser: Jeffrey D. Neighbors, PhD
Other committee members: Raymond J. Hohl, MD, PhD; Wei Li, PhD; Ralph Keil, PhD; Daleela Dodge, MD
A subset of high-risk neuroblastoma displays reduced sensitivity to mevalonate pathway inhibition likely due to an altered differentiation response
Authors: Michelle V. Green, Jessie L. Reed, Raymond J. Hohl and Jeffrey D. Neighbors
Abstract: Neuroblastoma (NB) is one of the deadliest childhood cancers, involving tumors arising from neural crest cells. At diagnosis over 50% of patients present with high-risk (HR) disease that has very poor prognosis and five-year survival rates of only 40%. Despite intensive treatment regimens for patients with HR NB approximately 15% will not respond and about 50% will relapse. Thus, this subset of patients, termed ultra-high-risk (UHR), are in desperate need of novel therapeutics. The mevalonate (MVA) pathway is a potential therapeutic target for UHR NB as it produces key biomolecules including cholesterol and isoprenoid pyrophosphates, which are required for cancer cell growth and proliferation. The goal of this study is to test the potential therapeutic benefit of MVA pathway inhibition with statin, bisphosphonate inhibitors of specific isoprenoid pyrophosphate synthases, and isoprenoid transferase inhibitors in UHR NB models. Assays were done with a cell line that has UHR NB characteristics, SK-N- AS, and the HR SH-SY5Y cell line. The effect of MVA inhibitor treatment on NB cell viability was analyzed by MTT assays and subsequent crystal violet staining, which measure metabolic activity and adherent cell colonies respectively. Treatment induced apoptosis and differentiation was analyzed by microscopy and western blot protein quantification analysis. Neurosphere formation assays were done to investigate the MVA inhibitor effect on differentiation and stemness. The effect of MVA inhibitor treatment on NB cell motility is being analyzed by migration and invasion transwell assays. Treatment induced disruption of the RET-hedgehog signaling axis, which is known to regulate NB differentiation, is actively being investigated through western blots and qRT-PCR. We found that UHR NB cell lines appear less sensitive to treatment with statins than the HR cell line in MTT assays and crystal violet staining. We also found that the UHR cell lines appear to have a slight increase in metabolic activity and crystal violet staining with bisphosphonate treatment. Morphological changes including cell body rounding and neurite outgrowths, are seen in the HR cell line with 24- and 48-hour statin treatments. The UHR cell line displays some cell body rounding and neurite outgrowths with 24-hour statin treatment, however these are not seen with 48-hour treatment. We found that maximum spheroid formation capability is significantly reduced with MVA pathway inhibition in both the HR and UHR cell lines with inhibitor concentrations that did not significantly alter cell viability in vitro. Similarly, the UHR cell line, SK-N-AS, had reduced stem cell frequency with fluvastatin and the zoledronate (bisphosphonate) treatment. Interestingly low-dose zoledronate treatment of the HR cell line SH-SY5Y significantly increased stem cell frequency while high dose zoledronate, statin and geranylgeranyl transferase inhibition decreased stem cell frequency. Inhibition of the MVA pathway may be a viable therapeutic option for HR and UHR neuroblastoma in a subset of patients. The observed decrease in maximum spheroid formation and decreased stem cell frequency in the UHR NB cell line suggests that treatment with MVA pathway inhibitors can target cancer stem cells, which are thought to be responsible for cancer recurrence and treatment failures.
The roles of Pcgf2-containing PRC1 complexes in early neural development
Authors: Aflah Hanafiah, Zhuangzhuang Geng, Qiang Wang and Zhonghua Gao
Abstract: The polycomb-group (PcG) proteins are a family of proteins first discovered in Drosophila melanogaster. PcG proteins form two major protein complexes designated, Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). PRC1 and PRC2 catalyze two repressive chromatin modifications: mono-ubiquitination of histone H2A at lysine-119 (H2AK119ub1) and di- and tri-methylation of histone H3 at lysine-27 (H3K27me2/3), respectively. In mammals, PcG proteins modulate the chromatin structure to maintain gene silencing, being critical for many aspects of mammalian development, including stem cell maintenance and differentiation. Although both PRC1 and PRC2 are generally involved in gene repression, mammalian components of the PRC1 complexes are far more diverse, and are considerably much less understood. Our previous proteomic analysis has led to the identification of six groups of mammalian PRC1 complexes, designated as PRC1.1-1.6, based on their mutually exclusive association with one of the six Polycomb group RING finger (PCGF) proteins. All groups of PRC1 complexes contain RING1A/B, the E3 ligase catalyzing H2AK119ub1, but differ in association with additional specific factors. Interestingly, among all PRC1 groups, only PRC1.2 and PRC1.4 contain factors that are components of the canonical PRC1 (cPRC1) complexes identified from Drosophila. Despite the previous studies showing that these complexes are involved in cellular differentiation, the underlying mechanisms remain elusive. Recently, a study of a cohort of young patients revealed that a heterozygous single missense mutation of the PCGF2 locus, which results in the substitution of the conserved proline at residue 65 with either serine or leucine (P65L/S), is associated with developmental defects of mesoderm- and ectoderm-origin. This finding is interesting as it suggests a broader role of PCGF2 in regulating development. Additionally, to our knowledge, this report is first to associate a cPRC1 component with a disease. Here we aim to understand the roles of PCGF2 in neuronal differentiation by using an in vitro mouse embryonic stem cell (mESC) differentiation model. To achieve this purpose, we have generated mESCs lacking Pcgf2 or with Pcgf2-P65L mutation via the CRISPR/Cas9-mediated gene editing. Transcriptomic analysis revealed that the loss of Pcgf2 leads to the deregulation of neuroectodermal genes upon neuronal differentiation. Additionally, we observed that Pcgf2 could be playing a role in chromatin looping at select gene loci based on the chromatin conformation capture analyses. Using biochemical analysis, we hope to understand the molecular mechanisms for the Pcgf2-containing PRC1 complexes to regulate the neuronal cell fate determination. Furthermore, studies in murine models will inform us of the Pcgf2 functions during the later stages of neuro-ectodermal development. These studies will likely provide important insights into the epigenetic regulation of cell identity as well as therapeutic targets for various diseases.
Assessing the impact of COVID-19 on registered interventional clinical trials
Authors: Nour Hawila and Arthur Berg
Abstract: The COVID-19 pandemic has led to a dramatic impact worldwide and presented unprecedented challenges for clinical and translational medicine. We assess the impact of COVID-19 on submitted and completed interventional clinical trials that have been registered on ClinicalTrials.gov. After classifying over 85% of the registered clinical trials by their source, we carefully model the number of submitted and completed trials before and after March 2020. Overall, we find minimal impact of COVID-19 on the number of submitted clinical trials, though a much more substantial impact is observed for completed clinical trials. We also show that clinical trials with a pharmaceutical sponsor were more successful completing trials during the pandemic compared to the trials with academic/hospital/government sponsors.
Type IV pili identified as a virulence factor in the human pathogen Paenibacillus thiaminolyticus Mbale using comparative proteogenomics
Authors: Christine Hehnly (co-first author), Aiqin Shi (co-first author), Paddy Ssentongo (co-first author), Lijun Zhang, Albert Isaacs, Sarah Morton, Joseph Paulson, Steven J. Schiff, Michael Galperin and James R. Broach
Abstract: Hydrocephalus is a leading indicator of childhood neurosurgery worldwide and is exceptionally high in low- and middle-income (LMIC) countries. Hydrocephalus preceded by an infection is characterized as postinfectious hydrocephalus (PIH) and accounts for 60% of the hydrocephalus cases in LMIC. The outcome of hydrocephalus is often disappointing even with treatment, so it is essential to prevent hydrocephalus. Recently, Paenibacillus thiaminolyticus was identified as a significant contributor to PIH in Uganda, and a clinical isolate, Mbale, was isolated from a patient with PIH. Utilizing our complete nonpathogenic, reference genome of the type strain of P. thiaminolyticus, NRRL B-4156, and the virulent Mbale strain, we use comparative genomics and proteomics to identify potential virulence factors. Virulence factors were validated using CRISPR-Cas9 and a mouse infection model. A functional-based comparison of the two genome annotations identified the type IV pilus as a unique protein in the Mbale strain compared to the B-4156 strain. Proteomic data confirmed its expression and the related type II secretion system in the Mbale strain. The type IV pili impact on pathogenicity was tested by doing a partial operon knockout with CRISPR-Cas9. After infection with mice of the wildtype, the knockout, B- 4156, and vehicle control at similar concentrations, the knockout lost lethality while the wildtype remained lethal to the mice. Comparative proteogenomic analysis successfully identified a virulence factor of P. thiaminolyticus Mbale and, in doing so, identified a potential therapeutic target that could help treat a P. thiaminolyticus Mbale infection and consequently prevent hydrocephalus.
Degree program: Biomedical Sciences
Department: Microbiology and Immunology
Committee chair or adviser: Jianming Hu, MD, MS, PhD
Other committee members: Edward Harhaj, PhD; Craig Meyers, MS, PhD; John Wills, PhD; Yuka Imamura, PhD; Fang Tian, PhD
Roles of N-linked glycosylation sites in precore/core gene on precore and core expression and functions in woodchuck hepatitis virus and hepatitis B virus
Authors: Xupeng Hong, Stephan Menne and Jianming Hu
Abstract: Hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) are two closely related orthohepadnaviruses. Both viruses share the precore/core gene, encoding the viral core and precore proteins via overlapping coding sequences. We recently found that the secretory products of the WHV precore gene, the e and PreC antigens, are N-glycosylated. This is in contrast to the analogous antigens of HBV that lack N-glycosylation. The roles of the N-linked glycosylation sites in viral precore and core functions are unknown. Thus, we exchanged the Thr residues in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) to the corresponding HBV residues (Glu77 or Asn92) to eliminate the motifs, and conversely, these N-glycosylation motifs were introduced into the HBV precore/core gene. We found that N-glycosylation increased the levels of both secreted WHV and HBV precore gene products in cell culture. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, whereas the HBc N92T mutant impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA species responsible for establishing and maintaining viral infection, during de novo infection. Cross-species complementation showed that both HBc and WHc support all steps of intracellular replication of the heterologous virus. Interestingly, WHc could support more efficient cccDNA formation than HBc in the context of either HBV or WHV. However, WHc was unable to interact with HBV envelope proteins for virion secretion. Thus, these results reveal novel determinants of precore secretion levels and core functions. They further suggest that the loss of the N-glycosylation motifs in the HBV precore/core gene was necessitated during viral adaptation to human hepatocytes in order to maintain virion production and infectivity.
Uncovering the role of nucleosome spacing in gene silencing, DNA stability and chromatin higher order structures
Authors: Brianna Kable, Sangita Chakraborty and Sergei Grigoryev
Abstract: In eukaryotes, DNA is supercoiled around histone octamers forming nucleosomes which are connected by linker DNA, creating long chains resembling “beads-on-a-string.” Differential nucleosome packing generates the basis for different functional states in the genome such as the open and active euchromatin and the condensed and repressed heterochromatin. The structural basis and mechanisms underlying this differential nucleosome packing are unknown. We hypothesize that such difference is mediated, at least in part, by the natural variation in linker DNA lengths that generates nucleosome spacings with an integer number of DNA turns (e.g. 10, 20, 30, 40 bp) and with a non-integer number of DNA turns, in which length (L) takes on a 10n+5 length (i.e. L = 5, 15, 25, 35 bp) is predominant. Previously, our lab shown that the differences in nucleosome linker length can impact nucleosome packing in vitro. To study the role of nucleosome linker length in vivo, we have established a circular minichromosome reporter system that allows tracking of a reporter URA3 gene expression in Saccharomyces cerevisiae. In this system arrays of eight clone 601 nucleosome positioning templates containing either 20 bp (10n) or 25 bp (10n+5) linker DNA, separate the URA3 reporter and the yeast mating type locus HML-E silencer element. By studying efficiency of yeast growth either on the media lacking uracil or on that containing 5-fluoroorotic acid (5-FOA), the drug that is lethal for cells expressing URA3, we found that (10n+5) linkers were more effective in conducting silencing compared to (10n). Further we observed that minichromosomes containing (10n) linkers had significantly decreased DNA stability compared to (10n+5) containing minichromosomes. To investigate structural differences in between (10n) and (10n+5), we sought to isolate minichromosomes with preserved topology. Through these efforts we determined that clones containing up to 8 subsequent (10n+5) nucleosomes and residing within a silenced DNA locus may be obtained in more than 10 copies per cell sufficient for chromosome structural studies and EM imaging.
Degree program: Neuroscience
Department: Neural and Behavioral Sciences
Committee chair or adviser: Yuval Silberman, PhD
Other committee members: Kirsteen Browning, PhD; Andras Hajnal, MD, PhD; Nicholas Graziane, PhD
The effects of chronic fluoxetine treatment on alcohol intake and metabolic functioning in binge drinking/eating mouse model
Authors: Bailey N. Keller, Amanda E. Snyder, Mariam Melkumyan, Yuval Silberman
Abstract: Alcohol use disorders (AUDs) are intersectional and causal to many other disease states, including depression, metabolic disorders, and Type II diabetes. Binge eating disorder (BED) is the most prevalent eating disorder and, like AUD, is also co-morbid with depression and metabolic disorders. BED and AUD co-morbidity may increase susceptibility to metabolic dysfunction and Type II diabetes. Our lab has developed a novel mouse model of co-morbid BED+AUD that mimics aspects of binge high-fat diet (HFD) and alcohol (EtOH) intake and presents with hyperglycemia and insulin/glucose intolerance as a result of their binging behaviors. Fluoxetine has shown some effectiveness in the treatment of AUD and is used clinically for BED; however, the mechanisms involved in these fluoxetine effects remain elusive. We aim to directly examine the effects of chronic fluoxetine treatment on EtOH intake behaviors and metabolic functioning in our BED+AUD mouse model. Adult male C57Bl/6J mice received 15-20 mg/kg fluoxetine p.o. or water for two weeks before undergoing our BED+AUD time course followed by metabolic testing. During the BED+AUD time course, all mice were exposed to an increasing percentage of EtOH through limited access two-bottle choice and received either HFD (1 day per week with control chow the remaining days) or control chow for 8 weeks. Glucose tolerance tests were performed at the end of the study. Our preliminary data indicate that chronic fluoxetine treatment reduces binge intake behaviors toward both HFD and EtOH, resulting in improvements in glucose tolerance in these mice. Further elucidation of fluoxetine’s therapeutic effects in our BED+AUD mouse model may lead to improved targeted treatments of BED and AUD.
Dissecting the role of TCF7L1 in colorectal cancer
Authors: Carli King, Melanie Eshelman, Stephen Matthews, Walter Koltun and Gregory Yochum
Abstract: Deregulation of the canonical Wnt/β-catenin signaling pathway drives colorectal cancer (CRC) through altering expression of downstream target genes. Target gene expression is regulated by T-cell factor (TCF) transcription factors that associate with Wnt responsive DNA elements (WREs) through their high mobility group (HMG) DNA binding domains. Of the four TCFs, TCF7L1 functions predominantly as a transcriptional repressor of gene expression and patients with increased expression of TCF7L1 have a significantly worse prognosis. TCF7L1 functions as an oncogene in CRC, but the underlying mechanism remains poorly understood. Interestingly, data suggests that TCF7L1 might operate outside canonical downstream Wnt/β-catenin targets, suggesting a potential novel mode of transcriptional regulation by TCF factors. Using a luciferase assay of a known TCF7L1 target gene, we confirm that TCF7L1 functions as a transcriptional repressor in CRC. Microarray and RNA-sequencing analyses indicate that a Wnt target gene signature is absent amongst TCF7L1 regulated genes. We hypothesize that TCF7L1 operates outside the canonical pathway to promote CRC. To test this hypothesis, we are studying CRC cells depleted of endogenous TCF7L1 and then engineered to express WT TCF7L1 or a TCF7L1 containing blocking mutations in the DNA binding domain. Preliminary RNA-sequencing analysis of these cells supported the hypothesis that TCF7L1 may promote CRC tumorigenesis independently of Wnt/β-catenin pathway by modulating genes associated with development, invasion and metastasis, and angiogenesis.
Chemically-induced cutaneous neoplasms spontaneously regress in mice lacking autoimmune regulator
Authors: Elizabeth M. Lesko, Ting Gao, Robert P. Feehan and Ryan P. Hobbs
Abstract: Cutaneous squamous cell carcinomas (cSCCs) frequently arise from precancerous lesions known as actinic keratoses (AKs). Factors that determine whether an AK will progress to a cSCC, remain stable, or regress to normal tissue are poorly defined. In AK and cSCC mouse models upregulation of autoimmune regulator (Aire) is associated with skin inflammation and tumor onset and the genetic lack of Aire attenuates the early stages of skin tumorigenesis. Here, to better assess the role of Aire in the onset and promotion of AK- and cSCC-like lesions, we subjected germline Aire null mice (Aire-/-; FVB/NJ or C57BL/6J backgrounds) to a classic two-step chemical carcinogenesis protocol [1x 7,12- dimethylbenz[a]anthracene (DMBA), 2x/wk 12-O-Tetradecanoylphorbol-13-acetate (TPA)], which resulted in papilloma formation in 92% of all mice (n=36/39). Aire deficiency resulted in reduced papilloma burden and smaller sized papillomas compared to Aire+/+ control mice. Unexpectedly, we observed that all papillomas that formed in 12 out of 22 Aire-/- mice rapidly and synchronously regressed despite continued tumor promotion with TPA. Histopathology indicated that regressed papilloma tissue from Aire-/- mice more closely resembled normal mouse skin architecture than non- regressed papilloma tissue from Aire-/- mice or from Aire+/+ papilloma tissue. After 15 weeks of TPA treatment, CD8+ cells consistent with cytotoxic T cell infiltrate were observed in the stroma surrounding the hyperproliferative epithelium in non-regressed papilloma tissue from Aire-/- mice or Aire+/+ mice. However, these cells were markedly absent from regressed papilloma tissue in Aire-/- mice, suggesting that an absence of functional Aire may improve the anti-tumoral immune response during the early stages of skin tumorigenesis. Altogether, this study indicates that Aire contributes in multiple ways to the development of skin neoplasms (onset, promotion, and regression) and suggests that the targeting of Aire function may provide a therapeutic benefit to patients at risk for skin cancers.
Exercise training during chemotherapy treatment may not alter response of chemokines to chemotherapy among breast cancer patients
Authors: Dan Lin and Kathleen Sturgeon
Abstract: Serum chemokine biomarkers are altered in breast cancer patients compared to healthy controls and circulating chemokine levels differ between breast cancer patients with and without a complete clinical response following neoadjuvant chemotherapy. Exercise training has a significant impact on the immune system and alters chemokine levels. The purpose of this exploratory analysis was to characterize the change in chemokine levels during chemotherapy treatment and to assess exercise- induced differences in circulating chemokine levels in breast cancer patients randomized to an exercise intervention during chemotherapy treatment. Methods: 19 breast cancer patients receiving neoadjuvant chemotherapy were randomly assigned to the intervention or control arm for the duration of chemotherapy (16-24 weeks). Patients performed a submaximal exercise test to predict VO2max prior to (baseline), and following completion of (follow up), chemotherapy. Blood was collected at baseline, prior to cycle 4 infusion (mid-point), and follow up. The intervention group completed a phased, exercise prescription (introduction, intermediate, maintenance) with the goal of one aerobic exercise session at 80-90% VO2max for 25 minutes/week and 65%-75% VO2max for ≥50 minutes/week. Participants in the control group maintained their usual level of physical activity during study participation. Chemokines (Eotaxin, Eotaxin-3, IL-8, IP-10, MCP-1, MCP-4, MDC, MIP1a, MIP1b, and TARC) plasma levels were measured by multiplex using Meso Scale Discovery. Women in the intervention group maintained their fitness capacity compared to the control group (VO2max: 22.4 vs
19.1 ml/kg/min, respectively; P<0.01). Age positively correlated with Eotaxin-3 (r=0.69; P<0.01) and negatively correlated with MCP-4 at baseline (r=-0.59; P<0.05). At mid-point, IP-10 levels were significantly (P<0.05) elevated in patients treated with anthracycline + cyclophosphamide chemotherapy compared to women receiving Taxotere + carboplatin + Herceptin. Eotaxin and TARC levels increased significantly over the course of chemotherapy treatment independent of intervention group. Further, independent of intervention group, Eotaxin level was significantly (P ≤ 0.05) higher in patients with a pathological complete response compared to patients with residual disease. There was a trend (P=0.052) for increased MCP-1 levels in the intervention group compared to control. No significant interaction effect (intervention group x time) was observed for chemokine levels. Conclusions: The study exercise intervention mitigated the chemotherapy-induced decrease in fitness capacity. Exploratory analysis of chemokine response to chemotherapy treatment with and without exercise was underpowered. However, we detected potential chemokines that are responsive to chemotherapy treatment and may have potential as biomarkers of clinical response. We also detected differences in chemokine levels by age, chemotherapy drug treatment, time course of chemotherapy progression, and tumor response. These differences will be important to consider for randomization criteria and covariate inclusion in statistical models of future studies. Lastly, while a home-based, remotely delivered, aerobic exercise intervention was efficacious in improving fitness capacity among breast cancer patients receiving chemotherapy, we did not observe any significant exercise-induced differences in chemokine response to chemotherapy treatment.
Genetic ablation of autoimmune regulator (Aire) results in spontaneous alopecia
Authors: Natella Maglakelidze, Ting Gao, Robert P. Feehan and Ryan Hobbs
Abstract: Autoimmune regulator, Aire, is a transcriptional regulator best known for eliminating autoreactive T cells in the thymus. Aire is also expressed in follicular keratinocytes, and patients with loss-of-function mutations in AIRE are 15x more likely to develop alopecia areata (AA) than the general population. We report here that adult C57BL/6J female mice genetically null for Aire (Aire-/-) spontaneously developed patchy non-scarring hair loss as early as three months of age that macroscopically resembled AA (n=35/73; 48%). Our data indicate this is a novel mouse model of AA. Histopathology of alopecic Aire-/- skin, relative to age- and body site-matched Aire+/+ skin, revealed several AA hallmarks including miniaturized hair follicles (HFs), decreased hair shaft diameter (55.1% reduction, p<0.0001), and increased inflammatory infiltrate at the anagen hair bulb (191% increase in perifollicular CD8+ T cell density, p=0.0546; 61% increase in mast cell density, p=0.0004). It is widely accepted that anagen HFs are targeted by autoreactive T cells in AA due to a collapse in HF immune privilege (IP), a phenomenon characterized in part by abnormal overexpression of major histocompatibility complex proteins (MHC class I and II) and upregulation of Janus kinase-signal transducer and activator of transcription (JAK- STAT) signaling. Using RT-qPCR we observed a 3-fold and 3.2-fold increase in MHC class I and MHC class II expression, respectively, as well as a 15.6-fold increase in interferon-γ expression and an upregulation of JAK-STAT signaling components in Aire-/- lesions (n=7) compared to controls (n=3). Additionally, immunohistology indicated significant upregulation of MHC class I expression along anagen HFs in Aire-/- lesions relative to controls (95.5% increase, p=0.0084). This discovery provides a basis for why AA is more prevalent in patients with AIRE mutations, is likely to offer novel insight into AA etiology, and highlights a new role for Aire in HF biology and IP regulation.
Degree program: Biomedical Sciences
Committee chair or adviser: Raymond J. Hohl, MD, PhD
Other committee members: Kent Vrana, PhD; Barbara Miller, MD; Jeffrey D. Neighbors, PhD; Elizabeth Proctor, PhD
Effects of schweinfurthins on malignant plasma cells
Authors: Barbara Manfredi and Raymond J. Hohl
Abstract: Multiple myeloma (MM), a bone marrow neoplasm is currently incurable and effective alternative therapies are needed. Schweinfurthin-3114 (S-3114) purified from a plant extract, inhibits growth of established MM cell lines. The mechanism(s) for this effect are not fully characterized. To advance understanding, MM cell lines RPMI-8226 and MM.1s were incubated with increasing concentrations of S-3114 for up to 48 hours. S-3114 decreased cell viability (trypan blue assay) and mitochondrial activity (MTT assay) in a concentration and time-dependent manner (p≤0.05). RPMI-8226 cells were more sensitive (EC50 2.36 nM) than MM.1s (EC50 11.1 nM) at 48 hours. Flow cytometry analyses revealed increased apoptosis in a concentration and time-dependent manner in both cell lines (64.0% in RPMI- 8226 with 20 nM S-3114; 86.3% in MM.1s with 270 nM S-3114, both at 48 hours). S-3114 also induced cell cycle arrest at G2/M. In RPMI-8226, the percent of cells arrested in G2/M increased from 45.9 to
66.1 with 3.5 nM S-3114 at 24 and 48 hours, respectively. In RPMI-8226, 20 nM S-3114 for 24 and 48 hours caused a 100% G2/M arrest. In both cell lines, S-3114 reduced protein kinase B (Akt) activation (Western blot) in a concentration and time-dependent manner. In MM.1s the apoptotic extrinsic pathway (caspase 8), and not the intrinsic pathway (caspase 9), is highly involved. In fact, S-3114 (20 nM for 48 hours, and 270 nM for 24 and 48 hours) caused a major cleavage for both 43 and 18 KDa caspase 8 fragments. Also cleaved Poly ADP-Ribose Polymerase 1 (PARP-1) increased at S-3114 concentrations of 20 and 270 nM at 24 and 48 hours. S-3114 influence on Golgi-mediated secretion was evaluated by measuring the soluble chemokine Macrophage Inflammatory Protein-1 alpha (MIP-1α), which is secreted by the Golgi and it is involved in the progression of MM disease. S-3114 treatment for 48 hours stimulated the MIP-1α negative cell line (RPMI-8226) to significantly increase MIP-1α RNA levels and to secrete MIP-1α at low concentrations. On the contrary, despite that prolonged S-3114 treatment significantly increased MIP-1α RNA levels, its secretion is decreased in the MIP-1α positive cell line (MM.1s). In aggregate, these in vitro studies demonstrate that S-3114 impairs the Phosphoinositide 3- Kinase (PI3K)/Akt signaling pathway resulting in cell death and/or cell cycle arrest. Data also shows an increased gene expression of the pro-survival MIP-1α by S-3114. Our ongoing studies are focused on schweinfurthin effects on MIP-1 α and Golgi secretion. We postulate that the cytotoxic impact of S-3114 requires disruption of MIP-1α signaling to regulate MPCs cell cycle, survival, and impairment of Golgi architecture and secretion. In aggregate these results reveal mechanism(s) for S-3114 effects and support further work advancing S-3114 as a novel therapy for MM.
Degree program: Biomedical Sciences
Department: Microbiology and Immunology
Committee chair or adviser: Jianming Hu, MD, MS, PhD
Other committee members: Clare Sample, PhD; Gregory Yochum, PhD; Ziaur Rahman, MD, PhD
Role of cellular endosomes and viral capsid in hepatitis B virus entry
Authors: Megan McGinley, Xupeng Hong, Jun Luo and Jianming Hu
Abstract: Hepatitis B virus (HBV) is a DNA virus belonging to the hepadnavirus family and is responsible for chronically infecting over 250 million individuals worldwide, resulting in about a million deaths yearly due to severe liver diseases including cirrhosis and hepatocellular carcinoma. The HBV nuclear episome, the covalently closed circular DNA (cccDNA), is the template for viral transcription and subsequent translation of all viral products, and thus is the molecular basis that establish and sustain HBV replication. Much is known about the HBV replication events following cccDNA formation, however little is known about the intracellular viral trafficking steps that are required for the initial cccDNA production to establish productive infection, which is thought to depend on receptor-mediated endocytosis. We have identified a number of HBV capsid mutants that have a unique phenotype, in that they fail to form cccDNA during infection but can form cccDNA from viral DNA produced in the cytoplasm, which bypasses the viral entry steps including endosomal trafficking during infection. These results led us to the hypothesis that these mutant viruses have a specific defect(s) in one or more viral trafficking steps during infection, possibly within the endosomal compartments. To further examine the pertinent steps for HBV endosomal trafficking, we have tested the effects of several endosomal inhibitors on HBV infection. Chloroquine, which prevents the acidification in the lysosomal compartments, increased cccDNA during infection, while EGA, which prevents early endosomal maturation, decreased cccDNA during infection. We found that the infection-deficient capsid mutants accumulated novel DNA species during the entry process which were absent from the WT and could be increased by proteosome inhibitors, suggesting that the capsid mutants underwent abnormal viral DNA processing, possibly as a result of mis-directed viral trafficking during entry, and an involvement of the host cell proteasome in the process. In conclusion, we have identified a role of early endosomal maturation, but not low pH, in productive HBV infection. Moreover, our results have revealed an important role of the viral capsid in directing viral trafficking for successful infection.
Degree program: Anatomy
Department: Neural and Behavioral Sciences
Committee chair or adviser: Amy Arnold, PhD
Other committee members: Patricia McLaughlin, MS, DEd; Kirsteen Browning, PhD; Andras Hajnal, MD, PhD; Scot Kimball, PhD
Chronic high fat diet disrupts angiotensin-(1-7) mas receptor localization in the arcuate nucleus of the hypothalamus
Authors: Darren Mehay, Sarah S. Bingaman, Yuval Silberman and Amy C. Arnold
Abstract: The control of energy balance involves communication of peripheral hormones with brain regions controlling food intake and energy expenditure such as the arcuate nucleus of the hypothalamus (ARC). Within the ARC, two primary neuronal subpopulations control energy balance: proopiomelanocortin (POMC) neurons, which reduce food intake and increase energy expenditure; and agouti-related protein (AgRP) neurons, which inhibit POMC neurons and conversely increase food intake and suppress energy expenditure. These circuits are typically disrupted by high fat diet (HFD) leading to a chronic state of energy imbalance and obesity. Accumulating evidence suggests that HFD-induced obesity is associated with deficiency of angiotensin (Ang)-(1-7), a protective renin-angiotensin system hormone. Our recent data show that systemically administered Ang-(1-7) induces adipose thermogenesis to enhance energy expenditure and promote weight loss. We propose that effects of Ang-(1-7) on energy balance involve activation of ARC neurocircuits, but this has not been tested. Additionally, the localization and neuronal subpopulations expressing Ang-(1-7) mas receptors (MasR) in the ARC is unknown. In this study, we hypothesized that: Ang-(1-7) activates ARC neurons; MasR are expressed in the ARC and are primarily colocalized with POMC neurons; and the ability of Ang-(1-7) to activate ARC neurons as well as co-localization of MasR with POMC neurons is disrupted following chronic HFD. Methods: Male C57Bl/6J mice were fed a 60% HFD or matched control diet ad libitum for 12 weeks. Mice then received subcutaneous injection of Ang-(1-7) [2 mg/kg] to induce neuronal activation in the ARC, as measured by c-fos gene expression (n=4-6/group). In a second cohort of mice, RNAscope in situ hybridization was performed on coronal ARC sections to determine co-localization of MasR mRNA within POMC versus AgRP neurons (n=5/group). We found that Ang-(1-7) increases the number of c-fos positive cells in the ARC (39±6 vs. 19±3 saline; p=0.022) in control diet mice. Ang-(1- 7)-mediated activation of ARC neurons was attenuated in HFD mice (34±3 vs. 23±4 saline; p=0.185). The rostral-medial-caudal distribution of ARC MasR was similar between control diet and HFD mice, with no difference in percentage of MasR positive neurons between groups (18±1 and 15±5%, respectively; p=0.733). MasR were more highly co-localized to POMC versus AgRP neurons, with HFD tending to reduce these co-localizations (MasR/POMC: 49±10 control vs. 33±5% HFD, p=0.199; MasR/AgRP: 36±11 control vs.16±7% HFD, p=0.209). These findings suggest that chronic HFD reduces the ability of Ang-(1-7) to acutely activate neurons in the ARC. Further, HFD disrupts co-localization of MasR with POMC and AgRP neurons in the ARC indicating disconnect in the endogenous neurocircuitry controlling energy balance. Further studies are needed to explore the importance of MasR in these neuronal subpopulations for energy balance, to determine the potential for targeting of Ang-(1-7) as an innovative pharmacological strategy for obesity treatment.
Degree program: Neuroscience
Department: Neural and Behavioral Sciences
Committee chair or adviser: Yuval Silberman, PhD
Other committee members: Salvatore Stella, PhD; Mohamed Trebak, PhD; Aron Lukacher, MD, PhD; Nicholas Graziane, PhD
Neuroimmune cells play a critical role in mediating the effect of acute alcohol on central amygdala glutamatergic transmission
Authors: Mariam Melkumyan, Angela Snyder and Yuval Silberman
Abstract: Alcohol Use Disorder (AUD) affects around 15 million individuals annually in the United States. The central amygdala (CeA) has been implicated in AUD, with alcohol (EtOH) exposure inducing neuroadaptive changes resulting in increased neuronal excitability and enhancing further EtOH intake. EtOH also increases neuroinflammation in this brain region which may be causative to AUD, but whether neuroinflammatory signaling contributes to enhanced CeA neuronal activity by EtOH has not been fully elucidated. To that end, we sought to test the hypothesis that acute EtOH exposure may increase excitatory neurotransmission in the CeA via modulation of neuroimmune cells. Whole cell patch clamp recordings of spontaneous excitatory postsynaptic currents in CeA neurons (lateral subdivision) from adult male and female C57Bl6/J mice showed that 10 minutes of bath applied 20mM EtOH significantly increased glutamatergic transmission in the CeA. This effect was significantly attenuated by 100uM fluorocitrate, an astrocyte inhibitor, or by 100uM minocycline, a microglia inhibitor, suggesting that EtOH increases glutamatergic transmission in the CeA by acting on microglia and astrocytes. Effects of 100mM EtOH on glutamatergic transmission were only blocked by fluorocitrate but not minocycline, suggesting that astrocytes may be the primary target of higher EtOH concentrations. Bath application of 500ng/mL lipopolysaccharide (LPS), the endotoxin portion of the gram-negative bacterial cell wall, also significantly enhanced CeA glutamate transmission, an effect that was blocked by fluorocitrate or minocycline pretreatment. These findings suggest that EtOH induces neuroinflammation through a similar pathway as LPS. We next utilized chemogenetics to selectively inhibit CeA astrocyte function and found that the effect of EtOH on CeA glutamatergic transmission was blocked, further confirming that astrocytes are a primary mediator of EtOH enhancement of glutamatergic transmission in the CeA. The exact mechanism of acute EtOH effect on astrocytes and the role that microglia may have in this pathway in the CeA needs to be further explored, and the effect of chronic vs acute EtOH exposure needs to be examined to provide insight into potential therapeutics for treatment of AUD.
Degree program: Biomedical Sciences
Department: Cellular and Molecular Physiology
Committee chair or adviser: Michael Dennis, PhD
Other committee members: Scot Kimball, PhD; David Waning, PhD; Alistair Barber, PhD; Jeffrey Sundstrom, MD, PhD
Oxidative stress prevents REDD1 degradation by chaperone-mediated autophagy in models of diabetic retinopathy
Authors: William P. Miller, Siddharth Sunilkumar, Allyson L. Toro, Joseph F. Giordano and Michael D. Dennis
Abstract: Accumulation of the stress response protein regulated in development and DNA damage (REDD1) has been associated with diabetes-induced retinal defects in preclinical models and diabetic patients. In fact, clinical trials involving intravitreal administration of siRNA targeting REDD1 have shown some efficacy in treating patients with diabetic macular edema. Currently, the mechanism behind the increase in REDD1 protein as a consequence of diabetes has yet to be established. Herein, we investigated the hypothesis that diabetes promotes REDD1 expression by inhibiting its degradation. In the retina of streptozotocin- induced diabetic mice, REDD1 protein was increased in the absence of any change in REDD1 mRNA. Administration of antioxidants to these mice prevented any increase in both oxidative stress and REDD1 protein. In human MIO-M1 Müller cells, REDD1 degradation was reduced under conditions of hyperglycemia-induced oxidative stress and addition of antioxidants to the culture medium prevented both the increase in oxidative stress and reduction in REDD1 degradation. Surprisingly, the suppressive effect of oxidative stress on REDD1 degradation was independent of the ubiquitin-proteasome pathway. Instead it was determined that REDD1 was rapidly degraded upon activation of chaperone-mediated autophagy (CMA) and exposure to hyperglycemic conditions prevented the suppressive effect of CMA activation on REDD1 expression. Overall, these findings identify new potential therapeutic targets for preventing the diabetes-induced retinal defects caused by increased REDD1.
Degree program: Biomedical Sciences
Committee chair or adviser: Kent Vrana, PhD
Other committee members: Patricia “Sue” Grigson-Kennedy, MS, PhD; Erika F.H. Saunders, MD; Kirsteen Browning, PhD; Andras Hajnal, MD, PhD
Therapeutic properties of Cannabigerol (CBG)
Authors: Rahul Nachnani, Greer McKendrick, Diana Sepulveda, Nicholas M. Graziane, Sarah S. Bingaman, Amy C. Arnold, Natalia Loktionova, Richard B. Mailman, Wesley M. Raup-Konsavage and Kent E. Vrana
Abstract: Objective Cannabigerol (CBG), is the precursor molecule to most cannabinoids including delta 9- tetrahydrocannabinol (THC) and cannabidiol (CBD). Non-psychoactive cannabinoids, such as CBD and CBG, show promise for attenuating substance seeking behavior along with anti-inflammatory, analgesic, and anti-epileptic activity. CBG, in particular, displays potency at distinct non-cannabinoid receptors: alpha-2 adrenoceptors, PPARα/ɣ, and serotonin 5-HT1A receptor. We propose CBG as a novel therapeutic in the cannabinoid class of drugs with potential for pain, inflammation, and psychiatric disorders (substance abuse, ADHD, etc.). Methods To study the potential effects of CBG, we performed an array of biochemical, molecular, and radiotelemetric studies to better characterize its molecular mechanism and potential hazards. Mouse models were used to assess analgesic applications of CBG and radiotelemetry sensors were used to monitor blood pressure. Receptor binding characteristics have been investigated for CBG, CBD, and THC. Results Data suggest that CBG acutely lowers blood pressure in conscious, freely-moving mice as measured by radiotelemetry. Moreover, it shows a non-significant trend for acute pain reduction in mice, and further study is needed. CBG displays a fundamentally different pharmacological profile from CBD and THC. Conclusion Novel compounds of the Cannabis sativa plant show promise as new pharmacotherapeutics; however, most still do not yet have thorough characterization of therapeutic uses and adverse effects. CBG provides new opportunities and hazards as its popularity increases among cannabinoid supplement users. Our findings suggest that CBG has unique pharmacological potential for pain and blood pressure regulation, presumably due to its affinity for alpha-2 adrenoceptors. Translational and clinical research must be prioritized to reduce harm from pharmacological interactions and investigate CBG’s ability to modulate diseases as a therapeutic.
Quantitative mapping of spontaneous activity and synaptic plasticity in postnatal mouse brain
Authors: Kyra Newmaster, Yuan Ting Wu and Yongsoo Kim
Abstract: Neurodevelopmental disorders including ADHD and ASD impact at least 1 in 6 Americans, yet our ability to help individuals with developmental delays is limited by our lack of knowledge about brain changes during formative years. One phenomenon that appears to control a number of processes including synaptic plasticity is the presence of spontaneous activity throughout the brain. Most work in this area investigated selected regions using calcium sensors or electrophysiology allowing us to only obtain region specific snapshots. Using whole brain immunostaining and light sheet microscopy imaging to analyze spontaneous activity via an immediate early gene, cfos, we are investigating a system wide snap shot of spontaneous activity during postnatal development. Recent research indicates that oxytocin (OT) system modulate spontaneous activity and synaptic development via specific spatiotemporal patterns throughout postnatal neurodevelopment. However, how OT signaling in specific cell type affect postnatal brain development is unclear. Here, I used conditional oxytocin receptor knockout mice crossed with either excitatory (vGlut2-Cre) or inhibitory (Gad2-Cre) neuronal Cre drivers to examine developmental role of OT signaling in a cell type specific manner. Specifically, I am testing my hypothesis that lack of OTR in inhibitory neurons will significantly delay the maturation of spontaneous firing leading to increased spontaneous activity and increased excitatory-inhibitory balance throughout the brain. My preliminary result with whole brain cFos mapping showed decreased spontaneous activity during the second week of development. Furthermore, we will use high resolution tiled confocal images to visualize and quantify excitatory and inhibitory puncta jn order to evaluate synaptic changes upon cell type specific OTR deletion. In summary, this project can provide insight on the role of oxytocin signaling in resting state brain activity and synaptic development from postnatally developing mouse brains.
Degree program: Biomedical Sciences
Committee chair or adviser: Amanda Nelson, PhD
Other committee members: Charles Lang, MS, PhD; Elizabeth Proctor, PhD; Vonn Walter, PhD; Xiang Zhan, PhD
Isotretinoin disrupts skin microbiome composition and metabolic function after 20 weeks of therapy
Authors: Zachary Nolan, Kalins Banerjee, Zhaoyuan Cong, Samantha Gettle, Amy Longenecker, Xiang Zhan, Yuka Imamura, Andrea Zaenglein, Diane Thiboutot and Amanda Nelson
Abstract: Despite being the gold standard treatment for severe acne for nearly 40 years, how isotretinoin influences the entire skin microbiome, the strain level composition of C. acnes, and the metabolic function of the microbiome throughout treatment is still not clear. To investigate these questions, we collected cyanoacrylate glue follicular casts from the cheek of acne patients (14 M, 4 F; ages 14-29 yrs.) throughout isotretinoin treatment (0wk, 1wk, 4wks, 8wks, 20wks and 6 months after cessation) followed by whole genome sequencing to determine how the skin microbiome and strain composition of C. acnes is influenced by isotretinoin. As expected, isotretinoin significantly decreased sebum levels by 4wks of treatment, while disease severity scores significantly decreased by 8wks of treatment, and lesion counts were significantly decreased by 20 weeks of treatment. The relative abundance of C. acnes significantly decreased after 20 weeks of treatment (p=0.03); although, neither -diversity (Shannon Diversity Index) nor -diversity (Bray-Curtis dissimilarity) of the species were altered by isotretinoin throughout the course of treatment. However, populations of C. acnes strains were significantly impacted. By building a database of all single nucleotide variants in published C. acnes genomes, we determined the relative abundance of each strain for each patient. Strains were grouped by phylogenetically similarity using the Single Locus Sequence Typing (SLST) model of C. acnes identification. Specifically, phylogenetically similar strains in SLST cluster A revealed that SLST A strains were significantly decreased in -diversity after 4 weeks (q=0.0488) and 20 weeks (q=0.0244) of treatment when compared to baseline. No significant differences in -diversity were found for C. acnes strains. Using a computational approach to examine the functional consequences of this altered skin microbiome, we identified 989 KEGG orthology (KO) terms that were enriched at 20wks compared to baseline (0wks). Pathway analyses revealed significant decreases in 3 pathways: amino acid biosynthesis (q=.00055), peptidoglycan biosynthesis (q=.0143), and folate biosynthesis (q=.05). Down regulation of these three pathways likely reflects the change in energy sources (decreased sebum) and decreased levels of C. acnes. Six months after isotretinoin, sebum and C. acnes levels, microbial composition and metabolic pathways returned to pretreatment levels, indicating that isotretinoin does not permanently alter the skin microbiome. In sum, 20 weeks of isotretinoin treatment is necessary to induce simultaneous changes in the critical factors of acne pathogenesis.
Degree program: Anatomy
Department: Neural and Behavioral Sciences
Committee chair or adviser: Patricia “Sue” Grigson-Kennedy, MS, PhD
Other committee members: Jennifer Nyland, PhD; Nicholas Graziane, PhD; Patricia McLaughlin, MS, DEd; Scott Bunce, PhD, MA
Exendin-4 pretreatment decreases aversive taste reactivity to cue-elicited conditioned opioid withdrawal in rats
Authors: Megan N. Olsen, C. Fields, Patricia “Sue” Grigson
Abstract: In drug-induced reward devaluation models, access to a natural reward cue precedes drug access and subsequently becomes a predictor of drug administration. We have hypothesized that avoidance of such a drug-paired taste cue depends not only on devaluation of the natural reward (i.e., anhedonia) but also on the onset of a conditioned aversive state of withdrawal. If the withdrawal hypothesis is correct, then neural correlates associated with avoidance of a drug-paired cue should mimic those associated with a conditioned state of withdrawal. To begin to test this hypothesis, we first established a model of conditioned withdrawal. We then used this model to assess aversive taste reactivity behavior (i.e., gapes) in response to intraoral delivery of a gustatory cue paired with opioid withdrawal and the effectiveness of a glucagon-like peptide-1 receptor (GLP-1R) agonist in blockade of this conditioned state of withdrawal. Rats were given an intraperitoneal (ip) injection of either saline or 15 mg/kg morphine. Twenty-two h later, saccharin was delivered, one infusion/min/15 min to all rats via intraoral (I.O.) cannulas. Immediately thereafter, each rat was ip injected with either saline or 3 mg/kg NLX. There were three such pairings. A test was conducted the following day during which rats were pretreated with either saline or 2.4 mg/kg exendin-4 twenty min. prior to I.O. infusions. Taste reactivity behavior was recorded and analyzed. Results. Rats pretreated with morphine, and to a lesser extent, saline gaped significantly more when the saccharin taste cue was paired with naloxone than when it was paired with vehicle. The average number of gapes to saccharin paired with naloxone significantly decreased when the morphine treated rats received an exendin-4 injection prior to I.O. delivery. These data show that rats with a history of morphine exposure exhibit aversive taste reactivity (i.e., gapes) during cue-elicited opioid withdrawal and that this aversive behavior can be blocked by pretreatment with the satiety agent, exendin-4. Future studies will examine the neurochemical profile associated with this aversive state of conditioned withdrawal and will determine how this profile is changed by pretreatment with exendin-4. Strides towards understanding and mediating the aversive state of withdrawal is important for the prevention of opioid relapse. Supported by: PA DOH, SAP #4100079742; DA009815.
β-endorphin and opioid growth factor as biomarkers of physical ability in multiple sclerosis
Authors: Chirag L. Patel, Gary Thomas, Naseem Zomorodi, Ian S. Zagon and Patricia J. McLaughlin
Abstract: Multiple sclerosis (MS) is an autoimmune mediated degenerative disease of the central nervous system with an estimated prevalence 2.5 million worldwide. Localized neuro-inflammation presents as demyelinating plaques within white matter monitored with magnetic resonance imaging. With the increasing MS-related healthcare costs, the need to validate minimally invasive biomarkers is imperative. In this study, conducted between 2019 and 2020 at the Penn State Health Neurology Clinic, 60 male and female relapsing-remitting MS patients on disease modifying therapies were consented to provide blood samples for analysis of serum cytokines (TNFα, IL6, and IL-17A) and endogenous opioid peptides (OGF and β-endorphin), as well as to complete the MSQOL-54 survey. Vitamin D levels were obtained from patient records. Patient age ranged from 23-78 years, with length of disease ~ 14 years. Mean serum OGF levels in all MS patients were elevated (p<0.01) and ranged from 193 -393 pg/ml in comparison to age matched controls (~98 pg/ml). The increased interaction between OGF and its receptor was hypothesized to facilitate a modulation of cytokines TNFα and IL-17A. Elevated OGF levels corresponded to increased levels of TNFα (r = 0.78) and IL-17A (r = 0.81). Analyses of MS-QOL data showed no significant differences in physical or mental composite scores between treatment groups. However, β-endorphin levels correlated with physical health composite score (r = 0.70), physical function score (r = 0.68), and social function (r = 0.68). Social function scores were indirectly correlated with increases in TNFα (r = 0.63) and IL6 (r = 0.63). Although the correlations do not demonstrate causality of disease, the data provide strong associations suggesting that serum levels of OGF, β- endorphin, and Vitamin D are potential biomarkers of patient perception of their disease.
Sex differences of ocular surface complications associated with type 1 diabetes
Authors: Indira Purushothaman, J.W. Sassani, Ian S. Zagon and Patricia J. McLaughlin
Abstract: Approximately 4.6 million women in the United States have symptomatology associated with ocular complications resulting from diabetes. Dry eye, delayed corneal epithelial repair, and surface sensitivity are evident, and can be reversed, in male rats with type 1 diabetes (T1D) following naltrexone blockade of the opioid growth factor (OGF) – OGF receptor (OGFr) pathway. Research of this pathway is limited in T1D female models. Hypothesis is the OGF-OGFr pathway is involved in the temporal course and magnitude of corneal complications in female rats with T1D. Female Sprague-Dawley rats were rendered hyperglycemic with streptozotocin (STZ), and a subset received insulin minipumps (T1D-INS) to establish controlled hyperglycemia; non-diabetic rats were controls (Normals). Body weights, blood glucose levels, corneal surface sensitivity (Cochet-Bonnet aesthesiometer), and dry eye (Schirmer 1 test) were monitored over an eight-week period. The rate of corneal epithelial wound closure was recorded at 6 weeks post STZ and correlated with levels of OGF in corneal tissue and serum. Female T1D rats had elevated glucose levels and depressed body weights throughout the 8 weeks in comparison to Normals and T1D-INS rats that had comparable values. However, corneal sensitivity was significantly abnormal in both groups being decreased 2.5-fold by 5 weeks in T1D rats. Dry eye was evident in both T1D and T1D-INS rats with tear production being significantly decreased as early as 2 weeks after STZ, and corneal epithelial repair was delayed. Levels of the inhibitory peptide OGF were elevated in both diabetic groups, along with expression in corneal tissue, relative to Normals. Dysregulation of OGF was associated with dry eye, increased sensitivity, and delayed repair in female rats with diabetes, and are the first data to systematically monitor the onset and magnitude of corneal epithelial complications in female rats with uncontrolled or insulin-controlled T1D.
Maturational trajectories of slow wave and sleep spindle activity in youth with psychiatric/behavioral disorders
Authors: Anna Ricci, Fan He, Susan L. Calhoun, Jidong Fang, Alexandros N. Vgontzas, Duanping Liao, Edward O. Bixler and Julio Fernandez-Mendoza
Abstract: Slow wave activity (SWA) in the delta frequency range (0.4-4.0Hz) declines, while sleep spindle activity in the sigma frequency range (11.0-16.0Hz) follows distinct maturational trajectories in typically developing (TD) children as they transition to adolescence. However, it remains elusive whether the maturational trajectories of NREM SWA and sleep spindles differ between TD youth and those with psychiatric/behavioral disorders. Methods: We analyzed the sleep EEG of 664 subjects (6-21y, 46.8% female, 24.7% racial/ethnic minority) from The Penn State Child Cohort, of whom 449 were TD, 123 had psychiatric/behavioral disorders and were un-medicated and an additional 92 were medicated with stimulants, anti-depressants, anxiolytics, and/or anti-psychotics. Multivariable regression models adjusting for sex, race/ethnicity, BMI, AHI and PSG system tested age-related trajectories of NREM SWA power and spindle density, frequency and power within each diagnostic group. Results: SWA power in TD and un-medicated youth showed cubic age-related trajectories (both p<0.05). In TD youth, SWA power was highest at age 6.6 and lowest at age 19.9, while in un-medicated youth it was highest at age 8.9 and lowest at age 18.6. The decreasing slope in SWA power was 39.7% steeper in un-medicated youth than TD youth. SWA power in medicated youth showed a distinct linearly decreasing trajectory (p<0.01) from age 6 (highest) to 21 (lowest). Spindle density showed a quadratic trajectory from age 6 (lowest) to 21 with a peak at age 16.7 in TD youth (p<0.05), but not in un-medicated (p-linear=0.64) or medicated (p-linear=0.49) youth. Highest spindle density occurred at age 6.0 and lowest at age 21.0 in un-medicated youth, while the opposite was observed in medicated youth (lowest density at age 6.0 and highest at age 21.0). Spindle frequency in TD (p<0.01) and un-medicated (p<0.05) youth showed a linear increase from age 6 (lowest) to 21 (highest), while spindle power a linear decline in both groups (p<0.05 and p<0.01, respectively) from age 6 (highest) to 21 (lowest). In medicated youth, the trajectories for spindle frequency (p-linear=0.24) and power (p-linear=0.35) did not reach statistical significance. Conclusions: TD and un-medicated youth with psychiatric/behavioral disorders show SWA trajectories typical of brain maturation biomarkers (e.g., gray matter volume) characterized by a decline at the onset of puberty that reaches its nadir by late adolescence. Also, both TD and un-medicated youth experience age-related increasing trajectories in spindle frequency and decreasing trajectories in spindle power. However, SWA in un-medicated youth peaks two years later and reaches its nadir one year earlier than in TD youth, which indicates not only a neurodevelopmental delay but also a faster decline in SWA throughout adolescence. Additionally, un-medicated youth experience abnormally higher spindle density in childhood compared to TD youth. In medicated youth, SWA is dampened in early childhood with its slope linearly decreasing with age, and sleep spindle metrics show maturational trajectories that deviate from both TD and un-medicated youth. These data suggest that psychotropic medications may potentially impact neurodevelopmental processes that occur in adolescence (e.g., synaptic pruning) and the maturational trajectories of SWA and sleep spindles. Future studies should examine these developmental trajectories in youth with specific diagnoses.
Degree program: Biomedical Sciences
Department: Medicine (Division of Gastroenterology and Hepatology)
Committee chair or adviser: Prashant Nighot, MVSc, PhD
Other committee members: Gregory Yochum, PhD; Thomas Ma, MD, PhD; Ralph Keil, PhD; Hong-Gang Wang, PhD
Autophagy enhances intestinal tight junction barrier by sorting occludin out of constitutive degradation pathway
Authors: Kushal Saha, Ashwinkumar Subramenium Ganapathy, Eric Suchanec, Thomas Y. Ma and Prashant Nighot
Abstract: Loss of paracellular tight junction (TJ) barrier function of the gut epithelium is associated with inflammatory bowel disease (IBD). Defects in autophagy, an evolutionarily conserved cell survival mechanism that degrades and recycles cytosolic contents, are associated with IBD. Our previous studies showed that autophagy reduces TJ permeability by targeting pore-forming claudin-2 to lysosomal degradation. However, the role of autophagy in regulation of the barrier forming protein occludin remains unknown. We investigated the role of the autophagy pathway in regulation of the barrier-forming TJ protein occludin. Pharmacologic and genetic tools were used to study the effect of autophagy on occludin levels and localization, and the role of the mitogen-activated protein kinase (MAPK) pathway in this process. Induction of autophagy by nutrient starvation or pharmacological inhibition of mTOR with rapamycin significantly increased trans-epithelial resistance and decreased paracellular flux of inulin in human intestinal Caco-2 as well as T84 and MDCKII cells. Autophagy induction increased total occludin levels as well as Thr phosphorylation of occludin which enhances localization of occludin to TJs, while decreasing occludin Tyr phosphorylation which destabilizes the association of occludin with TJs. Confocal immunofluorescence showed increased localization of occludin to TJs upon autophagy induction. Autophagy induction inhibited basal caveolar endocytosis and lysosomal degradation of occludin by reducing cytoplasmic co-localization of occludin with caveolin-1 and lysosomes. Nutrient starvation also enhanced the half-life of occludin. Pharmacological and genetic inhibition of autophagy with bafilomycin A1 or ATG7 siRNA inhibited the starvation-induced increase in total occludin levels. In view of changes in occludin phosphorylation, we investigated the role of the Ser/Thr MAPK pathway on autophagy-mediated occludin upregulation. Phosphorylation of the MAPK protein ERK-1/2 increased upon induction of autophagy. Pharmacological inhibition of MAPK using U0126 or PD 98059 in Caco-2 cells inhibited starvation-induced autophagic flux and the associated TJ barrier enhancement. Confocal imaging showed that MAPK inhibition prevented the autophagy-induced increased membrane localization of occludin. In vivo autophagy induction with rapamycin increased occludin levels in mouse proximal colon. Additionally, acute Atg7 knockout in adult mice decreased intestinal occludin levels. Our data suggest a novel role of autophagy in sorting the barrier-forming TJ protein occludin out of constitutive degradation pathway. Consistent with its role as a cell survival mechanism, autophagy promotes TJ barrier function by reducing degradation and increasing membrane localization of occludin at the TJs, in a MAPK-dependent manner.
ZFAND6 is a negative regulator of the innate immune response to a virus infection
Authors: Kashif A. Shaikh, Sarah McCormic, Mellisa Bowman and Edward W. Harhaj
Abstract: ZFAND6 is a zinc finger protein with an A20-like zinc finger (ZF) which mediates ubiquitin binding. A20 is a ubiquitin-editing enzyme and an important negative regulator of NF-kB signaling and inflammation. While the roles of A20 in regulating innate immune signal transduction pathways have been well studied, the physiological roles of other members of the A20-ZF family, including ZFAND6, are not well understood. Here, we show that knockout of mouse ZFAND6 imparts strong resistance to both DNA and RNA virus infections. Zfand6–/– mice develop normally and do not exhibit impairment of immune cell development or function. Surprisingly, Zfand6–/– mice were more susceptible to mortality at sublethal doses of mouse adapted influenza A virus as compared to Zfand6+/+ mice. RNA-Seq analysis revealed elevated interferon stimulated genes (ISGs) at basal levels in Zfand6–/– bone marrow-derived macrophages (BMDMs), which likely promotes the increased virus resistance in vitro. Inhibition of STING, an endoplasmic reticulum localized adaptor in the cGAS DNA sensing pathway, with a small molecule inhibitor increased the sensitivity of Zfand6–/– murine embryonic fibroblasts (MEFs) to RNA virus infection. Furthermore, STING protein, but not mRNA, was upregulated at basal levels in Zfand6–/– MEFs, and STING was more stable in Zfand6–/– MEFs as shown by a cycloheximide chase assay. These data suggest a potential role of ZFAND6 in the targeted degradation of STING. Furthermore, depletion of mitochondrial DNA with a low dose treatment of ethidium bromide diminished the high basal levels of ISGs in Zfand6–/– MEFs suggesting that leakage of mitochondrial DNA may be responsible for the activation of STING in knockout cells. Overall, these data establish a novel role of ZFAND6 in negatively regulating innate immune signaling by promoting basal STING degradation and furthermore implicate ZFAND6 in maintaining mitochondrial homeostasis.
Degree program: Neuroscience
Department: Neural and Behavioral Sciences
Committee chair or adviser: Yuval Silberman, PhD
Other committee members: Kirsteen Browning, PhD; Blaise Peterson, PhD; Nicholas Graziane, PhD; R. Alberto Travagli, PhD
Acute stress upregulates functional beta2-adrenergic receptors in ventral BNST CRF neurons likely involved in post-abstinence stress-enhanced alcohol consumption
Authors: Angela E. Snyder and Yuval Silberman
Abstract: According to SAMHSA, ~14.5 million Americans suffer from Alcohol Use Disorder (AUD), with 16 million using alcohol heavily. Stress significantly contributes to alcohol drinking, therefore investigating mechanisms underlying stress-influenced increases in alcohol consumption is vital for preventing harmful drinking and escalation into AUD. The bed nucleus of the stria terminalis (BNST) is a limbic structure critical for stress regulation and AUD. Norepinephrine and corticotropin releasing factor (CRF) are keystone neuromodulators involved in BNST regulation of stress-induced reinstatement of drug-seeking. The BNST is divided into subregions that may differentially influence stress and alcohol-related behaviors. In the dorsal BNST, CRF neurons enhance excitatory neurotransmission after acute stress or withdrawal from chronic intermittent ethanol via the beta1-adrenergic receptor. In the ventral BNST (vBNST), beta2-adrenergic receptors (beta2-ARs) predominate in mechanisms of stress-induced drug- seeking behavior, but how beta2-ARs influence CRF and excitatory vBNST neurotransmission modulating post-stress alcohol intake is unknown. We hypothesize that stress enhances beta2-AR control over vBNST excitatory transmission via modulation of CRF signaling to enhance alcohol intake. Utilizing our newly-developed mouse model incorporating 2-Bottle Choice with Forced Abstinence and acute Stress Exposure (2BC-FASE), we observed control mice significantly decreased EtOH intake after abstinence, but forced swim stress (FSS)-exposed mice did not, in males (control-before abstinence: 12.29±0.78g/kg, after: 9.81±0.86g/kg, p=0.009; FSS-before: 12.53±0.86g/kg, after: 11.43±1.15g/kg, p=0.303; two-way repeated-measures ANOVA) and females (control-before: 27.22±0.82g/kg, after: 24.56±0.78g/kg, p=0.016; FSS-before: 26.96±0.76g/kg, after: 25.90±0.87g/kg, p=0.415). Using whole-cell patch-clamp electrophysiology, we uncovered a stress-dependent beta2-AR-mediated increase in vBNST excitatory signaling. In non-stressed animals, 10uM clenbuterol (selective beta2-AR agonist) had no significant effect on sEPSC frequency (n=6 cells, 114.4±7.24% of baseline, p=0.104; unpaired t-test), but significantly increased sEPSC frequency after FSS or restraint stress (RS) (FSS: n=5 cells, 125.7±9.04%, p=0.0468; RS: n=11 cells, 138.4±10.85% of baseline, p=.0057; unpaired t-tests). CRFR1 antagonist pretreatment blocked clenbuterol’s effects (n=4 cells, 118.5±11.14% of baseline, p=0.196), suggesting beta2-AR enhancement of vBNST sEPSCs requires CRF signaling. Preliminary RNAscope data also suggest that this beta2-AR upregulation occurs primarily in CRF neurons. Further exploring these newly- identified vBNST neurocircuitry changes occurring in response to stress will be helpful for further understanding how each contribute to harmful alcohol use and may uncover novel AUD prevention or treatment targets.
Benefits of surgical treatment in stage IV breast cancer in patients with known hormone receptor and HER2 status
Authors: Kelly Stahl, Daleela Dodge, Ashton Brooks, Christopher McLaughlin, Elizabeth Olecki, Joseph Lewcun, Kristina Newport, Monali Vasekar and Chan Shen
Abstract: Of the 5% to 10% of breast cancer patients diagnosed at stage IV, systemic therapy is the cornerstone of treatment with an unclear role for surgery. Limited evidence exists delineating treatment modalities when it comes to hormone receptor and HER2 status. Methods. The National Cancer Database was used to identify 12,838 stage IV breast cancer patients with known hormone receptor and HER2 status from 2010-2016. Chi-square tests examined subgroup differences between the treatment modality received. Using the Kaplan Meir method 5-year overall survival (OS) was assessed. Multivariate Cox proportional hazard models examined factors associated with survival. A survival advantage was noted in patients who received either systemic therapy and surgery (ST+Surg) or systemic therapy, surgery, and radiation (Trimodality) compared to systemic therapy alone (ST) (ST+Surg HR 0.723, 95% CI 0.671-0.779; Trimodality HR 0.640. 95% CI 0.591-0.694, both p<0.0001). HER2+ patients who received trimodality or ST+Surg, had an improved 5-year OS rate when compared to ST (Trimodality 48%, ST+Surg 41%, ST 29%, p<0.0001). The timing of chemotherapy in relation to surgery was significant, with the greatest survival advantage being noted with the receipt of neoadjuvant chemotherapy (NAC) compared to adjuvant chemotherapy in patients with positive hormone receptor and HER2 status (HER2+ NAC HR 0.477, ER+ NAC HR 0.453, PR+ NAC HR 0.448, all p<0.0001). Surgery in addition to ST has a survival benefit in stage IV breast cancer patients with known hormone receptor and HER2 status, and should be considered after NAC in patients with ER+, PR+ or HER2+ disease.
Discovering genome-wide responses in pre-cancerous, chronically inflamed tissues of ulcerative colitis patients
Authors: MaryElizabeth Stein, Bryan Tsao, Suzanne Hile, Fengming Chen, Lianna Goetz, Yongjun Liu, Francesca Ruggiero, Walter Koltun, Gregory Yochum and Kristin Eckert
Abstract: Ulcerative colitis (UC) is a lifetime disease marked by chronic inflammation of the colon and an elevated risk of colorectal cancer. Chronic inflammation is a hallmark of cancer and a multifaceted relationship exists between inflammation and genome instability. However, the mechanisms underlying cancer progression in UC patients remain poorly understood. In this discovery phase study, we hypothesize that a colonic environment shaped by chronic inflammation results in differential gene expression, including dysregulation of DNA damage response genes, generating a tolerable environment for increased mutational burden. We examined precancerous formalin-fixed, paraffin embedded colon tissues from the following groups: (1) UC with high grade dysplasia/cancer (Progressors), (2) UC only (Nonprogressors), (3) Slow transit/trauma (Controls). UC patients were similar in covariates such as UC duration, smoking status, BMI, and gender. Tissues from three colon regions (left, transverse, and right) of each patient were assessed in two ways. Morphological changes were evaluated using the Geboes histological index, which measures disease activity based on the presence of neutrophils and eosinophils, as well as chronic inflammation indicators (e.g., ulceration, inflammatory infiltrate). RNA extracted from each tissue was subject to RNAseq for evaluating gene expression genome wide. Histological analysis of control tissues showed chronic inactive disease, as expected. We found a statistically significant difference in disease activity between Progressor and Nonprogressor tissues. Specifically, more Progressor tissues had chronic inactive disease, compared to Nonprogressor tissues, particularly at the left colon. These data suggest a difference in inflammatory response between the two groups. External factors (e.g., disease duration, age at diagnosis) were not statistically significant contributors to this difference. Additionally, a thorough medical chart review showed Progressors and Nonprogressors were treated similarly and there was no statistically significant difference in the types or duration of medications taken between the two groups. Disease activity differences between the two groups also do not appear to be due to medication. Our RNAseq analysis detected differentially expressed genes (DEGs) in the left colon tissues only, consistent with histological differences observed at the left colon. An in-depth analysis of left colon tissues of UC patients compared to controls showed elevated expression of genes related to immune signaling, lipid metabolism, and mitochondrial function. ATM, a critical DNA damage response regulator, was found to be highly expressed in some UC patients. Assessment of gene expression based on ATM status (high vs. low) revealed upregulation of immune signaling genes and downregulation of metabolic and mitochondrial genes in UC samples with high ATM expression. Additionally, UC samples with high ATM expression had altered DNA repair gene and DNA polymerase gene expression. Taken together, these data underscore previous findings of altered mitochondrial and metabolic function in UC, which may be associated with changes in ATM expression. Our study provides a foundation for our long-term goal, to identify novel biomarkers in precancerous tissues and create novel diagnostic tools for precision medicine using genetic signatures to improve management and outcomes for UC patients.
Preliminary assessment of the effects of GLP-1 agonism and buprenorphine on cue- and drug-induced fentanyl seeking in rats
Authors: Luke Urbanik, Nick Acharya and Patricia “Sue” Grigson
Abstract: Opioid use disorder (OUD) is a chronic relapsing disorder that has a negative impact on the individual, the family, and the community at large. Using animal models, the literature shows that rats readily self-administer heroin and, following an extinction period, reinstate drug-seeking behavior when presented with cues for drug, the drug itself, or stress. Furthermore, we have shown that treatment with the glucagon-like peptide-1 receptor (GLP- 1R) agonist, liraglutide, can reduce heroin seeking behavior in rats. That said, most opioid users, either intentionally or unintentionally, are exposed to the much more potent opioid, fentanyl. It is, then, critical that we test whether treatment with the GLP-1 receptor agonist also can reduce fentanyl seeking in fentanyl experienced rats. In the present study, adult male Sprague-Dawley rats were implanted with intravenous catheters and trained to self-administer 2.5 ug/kg fentanyl intravenously (iv) over 14 daily 6 h sessions by licking an active empty spout operant. The rats were matched on the number of infusions earned/6 h across the final two days of acquisition, divided into high and low drug takers, and assigned to one of three treatment conditions: 0.3 mg/kg liraglutide (Lir) sc, 1.0 mg/kg buprenorphine (BUP) iv, or saline matched controls. On the test day (day 15), rats were injected with Lir 6 h prior to test, BUP 30 min prior to test, or vehicle (saline). Rats were then placed in the self-administration chamber under extinction conditions (i.e., no drug was delivered) and fentanyl seeking was measured across a three h period. Thereafter, rats received a single iv infusion of fentanyl (2.5 ug/kg) and drug-induced reinstatement of fentanyl seeking was examined for 1 h. The results showed that some rats self- administered more fentanyl (high drug takers) than others (low drug takers) and that the acute administration of LIR attenuated cue-induced seeking and blocked drug-induced reinstatement of fentanyl seeking behavior. Pretreatment with the opioid agonist, BUP, fully blocked both cue and drug induced seeking of fentanyl. This work was supported by UG3DA050325vand R37DA009815.
Colonic neuromuscular transmission failure in female rats after spinal cord injury
Authors: Claire M. Werner and Gregory M. Holmes
Abstract: Along with lost mobility, spinal cord injury (SCI) impacts visceral control of the autonomic nervous system; eliciting critical challenges to quality of life. Gastrointestinal (GI) complaints in the SCI population include decreased colonic motility and subsequent disruptions in evacuation. Although SCI damages the central nervous system, the enteric nervous system located intrinsic to the GI tract is not damaged by the initial trauma. The enteric neuromuscular control of colonic slow-wave propagation is a balance of excitatory and inhibitory inputs to smooth muscle cells that are recorded electrophysiologically as junction potentials (EJPs and IJPs, respectively). IJPs consist of a fast (purinergic; fIJP) and subsequent slow (nitrergic; sIJP) component. Our previous research has demonstrated diminished EJPs and sIJPs in male SCI rats. The higher prevalence of GI abnormalities in females underscores the differential control of motility in health and disease. This study aims to expand our mechanistic understanding of colonic dysmotility in female rat models of acute SCI. Female rats received a surgical control or severe T3 contusion SCI. After 3 days recovery, ex vivo distal colon samples of the circular and longitudinal smooth muscle were dissected for electrophysiological recording and immunohistochemical analysis. For electrophysiology experiments, smooth muscle cells were recorded in response to pharmacologic treatments that isolate the EJPs and IJP components. Our data reveals that, in contrast to comparable regions of male rats’ distal colon, female rats have minimal sIJP (i.e., nitrergic) responses. With no significant difference between acute control and SCI responses, this suggests that female neuromuscular transmission is less dependent on nitrergic signaling. Control female sIJPs are significantly smaller than control male rats and resemble the sIJPs in SCI male rats. The resting membrane potential and fIJP component appears unchanged by SCI, with values consistent to male rats. Conclusion: All of the current data illustrates sex-differences in colonic physiology and pathophysiology after SCI. Addressing gender differences would improve clinical post- injury care for these individuals. Significance: To date, the mechanism for colon dysmotility after SCI remains uncertain and this project provides novel insight to female neurogenic bowel. The colonic neuromuscular junction is the final common pathway for proper motor function and abnormalities have implications for peripheral and central neurostimulation therapies. Our observed differences from neuromuscular transmission in the male rat, reinforces the importance of developing gender specific treatment. Future studies will evaluate female EJP dysfunction and specific agonist pharmacodynamics.
Phosphorylation of the selective autophagy receptor TAX1BP1 by canonical and noncanonical IB kinases promotes its lysosomal localization and clearance of MAVS aggregates
Authors: Young Bong Choi, Jiawen Zhang, Mai Tram Vo, Jesse White, Chaoxia He and Edward W. Harhaj
Abstract: During RNA virus infection, cytosolic RIG-I-like receptors (RLRs) recognize 5’-triphosphate RNA derived from viral genomes and activate the downstream mitochondrial signaling adaptor molecule MAVS. Upon activation, MAVS forms functional prion-like aggregates that recruit TRAFs, TBK1 and IKK signaling proteins to induce the production of type I interferon and pro-inflammatory cytokines through activation of the transcription factors Interferon Regulatory Factors 3 and 7 (IRF3/7) and Nuclear Factor Kappa, respectively. However, it remains unclear how MAVS aggregates are cleared to downregulate signaling and restore homeostasis after virus infections are resolved. Here, we show that the selective autophagy receptor TAX1BP1 negatively regulates RLR signaling by clearing functional MAVS aggregates through autophagy in a phosphorylation-dependent manner. Mass spectrometry was used to identify 13 putative phosphorylation sites, of which Serines 254, 593 and 666 played the most critical roles in TAX1BP1 autophagy function. The noncanonical IB kinases TBK1 and IKKi were found to phosphorylate TAX1BP1 and regulate its basal turnover, while the canonical IB kinase IKK was required for RNA virus-induced degradation of TAX1BP1. MAVS aggregates formed spontaneously and were found at higher levels during virus infection in TAX1BP1-deficient cells. Together, our data suggest a model whereby IB kinases regulate TAX1BP1 autophagosomal degradation of MAVS to inhibit RLR signaling.
Degree program: Biomedical Sciences
Committee chair or adviser: Raymond J. Hohl, MD, PhD
Other committee members: Jeffrey D. Neighbors, PhD; Todd Schell, PhD; Hong Zheng, MD, PhD; Jong Yun, PhD
Schweinfurthins induce calreticulin exposure independent of ER stress
Authors: Ruoheng Zhang, Jeffrey D. Neighbors and Raymond J. Hohl
Abstract: Our previous study demonstrated that schweinfurthin analogs improve anti-PD-1 immunotherapy in a murine melanoma model by inducing sustainable in vivo anti-tumor immunity.1 We speculate that the induction of Immunogenic Cell Death (ICD) contributes to these effects. The cell surface exposure of calreticulin (CRT) initiates the first step during ICD as it stimulates tumor cell phagocytosis by dendritic cells and further activates anti-cancer T cells. Green florescence labeled B16F10 cells were incubated with or without 100nM schweinfurthin (TTI-3114) for 24 hours, then co-cultured with Bone Marrow- Derived Dendritic Cells (BMDCs) cells stained CD11c-APC for 5 hours. ImageStream flow cytometer with fluorescent detection demonstrates that BMDCs phagocytosed 1.8-fold more TTI-3114 treated tumor cells as compared to control. TTI-3114 also induced a time- and concentration-dependent CRT exposure on B16F10 cells. CRT exposure was first detected at 3 hours and the maximum exposure of 6-fold increase occurred at 24 hours. Endoplasmic Reticulum (ER) stress pathway and essential proteins on this pathway, e.g., PERK and eIF2α are considered necessary for CRT exposure.  However, Western Blotting demonstrated that even TTI-3114 caused PERK phosphorylation but did not activate ER stress pathway. Interestingly, TTI-3114-induced CRT exposure was not blocked by the pharmacological inhibitor of ER stress, TUDCA. The PERK inactivator, GSK2606414 inhibited PERK phosphorylation induced by TTI-3114, but paradoxically enhanced CRT exposure. Unexpectedly, TTI-3114 induced CRT exposure in PERK knockdown cells. These finding suggest that TTI-3114 induced CRT exposure in a non-canonical pathway independent of ER stress and PERK. Ongoing studies are exploring the mechanism underlying TTI-3114- induced CRT exposure.
Degree program: Biomedical Sciences
Committee chair or adviser: Jeffrey Sundstrom, MD, PhD
Other committee members: John Wills, PhD; Xuemei Huang, MD, PhD; Patricia “Sue” Grigson-Kennedy, MS, PhD; James Connor, MS, PhD; Nicholas Buchkovich, PhD
Extracellular vesicles from R345W-Fibulin-3 retinal pigment epithelial cells induce epithelial-mesenchymal transition in recipient cells
Authors: Mi Zhou, Yuanjun Zhao, Sarah R. Weber, Han Chen, Alistair J. Barber, Stephanie L. Grillo and Jeffrey M. Sundstrom
Abstract: Previous studies in our lab found that the expression of R345W-Fibulin-3 induces retinal pigment epithelial (RPE) cells to undergo epithelial-mesenchymal transition (EMT). The purpose of the current study was to investigate the size, cargo and function of extracellular vesicles (EVs) derived from RPE cells expressing the R345W-Fibulin-3 mutation, and to determine the role of these EVs in regulating RPE cell dysfunction. ARPE-19 cells were infected with luciferase-tagged wild-type Fibulin-3 (WT)- or luciferase-tagged R345W-Fibulin-3 (mutant) using lentivirus. EVs were isolated from the media of ARPE-19 cells by either conventional ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo-EM) were performed to study EV morphology. The amount and size distribution of EVs were determined by Nanoparticle Tracking Analysis (NTA). EV protein concentrations were quantified using the DCTM Protein Assay (Bio-Rad). EV cargo were analyzed by unbiased proteomics using LC-MS/MS with subsequent pathway analysis (Advaita). The EV-associated transforming growth factor beta 1 (TGF-β1) protein was measured by Enzyme-linked immunosorbent assay (ELISA). The migration ability of ARPE-19 cells, in the absence and presence of mutant EVs, was evaluated by using scratch assays. TEM and cryo-EM imaging revealed vesicles that were concave-appearing and spherical, respectively, that had two subpopulations of EVs: a small group with diameters around 30nm and a large group with diameters around 100nm. Imaging also indicated a greater number of small EVs (~30 nm) in the mutant group compared to the WT group. This result was further confirmed by NTA showing that, in the mutant group, the particle size distributions were smaller than those of the WT EVs. There were no significant differences in EV protein concentrations per EV between WT and mutant groups. Proteomic studies showed that EVs derived from ARPE-19 cells expressing WT-Fibulin-3 contain critical members of sonic hedgehog signaling (SHH) signaling and ciliary tip components. Whereas, EVs derived from RPE cells expressing R345W-Fibulin-3 contain EMT mediators, including TGF-β-induced protein (TGFBI), vimentin, and mothers against decapentaplegic homolog 4 (SMAD4). This suggests that the EV cargo may reflect the phenotypic status of their parental cells. Subsequent studies have shown that enhanced TGF-β1 activity is associated with mutant EVs compared to WT EVs. Furthermore, mutant RPE cell-derived EVs elevated EMT marker expression and enhanced migration ability in naïve, recipient RPE cells. The size, cargo and autocrine function is altered in EVs from RPE cells expressing R345W-Fibulin-3. Additionally, these mutant EVs are sufficient to induce EMT in naïve RPE cells.