Poster Session Abstracts
Incorporation of Implicit Bias Training to Decrease Care Avoidance at Believe Shelter for the Homeless: A Pilot Program
Jessica A. Dudley ’14, Doctor of Nursing Practice Program, University of Illinois at Chicago, Chicago, IL
Implicit biases involve associations outside conscious awareness that lead to a negative evaluation of a person on the basis of characteristics, like race or gender. There is a growing body of evidence that suggests that these implicit biases have a negative effect on providers’ care in the medical setting. A needs assessment at Believe shelter indicated a disconnect between shelter staff and residents. Care avoidance may result as a defense mechanism against this judgement. Implicit bias and cultural sensitivity training may serve as a resolution for this disconnect. This project aims to pilot implicit bias training on homeless shelter staff in order to assess its effectiveness in reducing care avoidance in homeless shelter residents. A six-point framework will be used to develop and deliver an implicit bias training at Believe Shelter, a 50 bed homeless shelter in Chicago. The Implicit Associations Test (IAT) will be used at baseline to measure the strengths of shelter staff associations between race and evaluations/ stereotypes. Baseline data on perceptions of trust or judgement from shelter residents will also be recorded. Checkpoints will be used to evaluate effectiveness of the training in reducing care avoidance in shelter residents. The growing body of evidence of the negative impact of implicit biases on decision making in medical settings highlight the need for healthcare professions and allied staff to address the role of implicit biases in disparities in healthcare. As such measures need to be taken to raise awareness of potential bias and conflict in order to standardize and commit to patient equity.
Why do attractive males take more risks when searching for mates? Testing the “live fast, die young” hypothesis
Chris Edomwande ’19 and Flavia Barbosa, Biology Department, Lake Forest College, Lake Forest, IL
Mating signals are often conspicuous and can be eavesdropped on by predators. Therefore, we expect individuals to attend to predator cues when signaling to mates. Males of the lesser waxmoth sing to attract females, but their song also attracts bat predators. Singing males respond to bat calls by freezing, but this response varies among individuals. Interestingly, attractive males take more risks. We investigate why by testing the hypothesis that attractive males have shorter lifespans. Attractive males should then benefit from taking risks that favor current reproduction versus being cautious to favor future mating opportunities.
Local synaptic connectivity of subicular pyramidal neurons sorted by multivariate analysis and its impact on population activity
Michael P. Fiske ’101, M. Anstötz1, G. Maccaferri2. 1Northwestern University Feinberg School of Medicine, Chicago, Illinois; 2Department of Physiology, Northwestern University, Chicago, IL
The subiculum is a key area involved in temporal lobe epilepsy, and its local excitatory circuits have been suggested to be involved in the generation of various forms of epileptiform activity. Although it has now established by many studies that subicular pyramidal cells exhibit distinct intrinsic excitability, to our knowledge only one study has examined the local connectivity between the distinct types of subicular pyramidal neurons classified as “regular firing” or “bursters” (Böhm et al., 2015). We have re-addressed pyramidal cell diversity with direct patch clamp recordings from subicular slices, classified by a combination of principal component analysis and Gaussian mixture models. Out of n=995 recorded cells, we observed variable proportions of what we term “type 1” and “type 2” cells (51.1% and 48.9%, respectively). When the intercellular relationships were examined, we found functional evidence for both homotypic (type 1 to type 1, n=29 and type 2 to type 2, n=34 synapses) and heterotypic synaptic connectivity (type 1 to type 2, n=25, and type 2 to type 1, n=7 synapses). Overall, the probability of finding a connection was roughly symmetrical for homotypic connections (type 1 to type 1, p=0.046, type 2 to type 2, p=0.052), whereas it was highly asymmetrical in heterotypical connections (type 1 to type 2, p=0.088, type 2 to type 1, p=0.025). The properties of the unitary excitatory postsynaptic potentials were also similar across connections (amplitude 0.54 mV), without obvious differences. In n=6 experiments we verified that uEPSPs were glutamatergic and fully blocked by the AMPA receptor antagonist NBQX (20 µM). When the anatomy of the connected pairs was examined at the light microscopy level, we observed putative contact sites that ranged in numbers between 1 and 9, and appeared consistently biased towards the basal dendrites of the postsynaptic cell (85.3% basal vs 14.7% apical). Lastly, we tested the influence of these local excitatory connections on population activity by recording from pairs of cells in slices surgically isolated from extra-subicular inputs and exposed to gabazine (12.5 µM) and CGP55845 (5 µM). In n=11 recordings, we found that blockade of GABAergic circuits allowed the development of synchronous epileptiform bursts (number of action potentials/burst=29.5, n=27 cells). In conclusion, our data suggest a complex cell type-specific connectivity of intrinsically diverse pyramidal neurons that can sustain, in the absence of GABAergic inhibition, epileptic-like hypersynchronous activity. Supported by NIH grant NS096092
The role of subcortical hippocampal inputs in contextual memory formation
Viktoriya S Grayson1, Yuan Han1,2, Anita L. Guedea1, Vladimir Jovasevic1 , Can Gao2, Apkar V Apkarian3, Jelena Radulovic1. 1Department of Psychiatry and Behavioral Sciences, Northwestern University, Chicago, IL; 2Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, China; 3Department of Physiology, Northwestern University, Chicago, IL
The role of cortical afferents to the hippocampus in the formation of episodic-like memory is well established, however, less is known about the contribution of subcortical memory circuits to memory. In the present study, we studied the roles of several subcortical inputs into the dorsal hippocampus in mouse models of contextualfear conditioning, extinction, and reinstatement. Fear conditioning was induced by a single exposure of mice to a context followed by foot shock. Subsequently, mice were exposed to daily extinction trials. After significant reduction of freezing, indicating successful extinction, mice were exposed to a brief reminder shock and re-tested in the conditioning context. Circuit manipulations were performed by chemogenetic silencing with the inhibitory designer receptor exclusively activated by designer drugs (DREADD) hM4(Gi) or targeted cholinergic depletion induced by 192 IgG-saporin, at different stages of fear conditioning, extinction, and reinstatement. We identified projection- and neurotransmitter-specific roles of discrete circuits, indicating complex regulation of fear-inducing memories by subcortical afferents.
Insight into Parkinson’s Disease from Yeasts: Combined Impact of Covalent Modifications and Familial Mutations on α-Synuclein
Yoan Ganev, Rosemary Thomas, Alexandra Roman, Thea Grauer, Chisomo Mwale, Paul Jones, Ariane Balaram, Estella Tcaturian, & Shubhik DebBurman, Neuroscience Program and Biology Department, Lake Forest College, Lake Forest, IL 60045
Parkinson’s Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are composed of misfolded α-synuclein protein. α-Synuclein is a highly post-translationally modified protein, in healthy and diseased states. While phosphorylation and nitration of α-synuclein are well-studied as contributors to PD pathology, less is known about SUMOylation, acetylation, and glycation. Also, the combined effects of these modifications remain largely unclear, on both wildtype α-synuclein (linked with sporadic PD) and the six mutant forms (A30P, E46K, H50Q, G51D, A53T, A53E) linked with early-onset familial PD. We first evaluated the effects of blocking SUMOylation on α-synuclein in the well-established budding yeast model for PD and found that α-synuclein becomes more toxic and aggregated, losing its membrane localization. Second, we found that SUMOylation and phosphorylation counteract each other in toxicity and localization. Third, we expanded our investigation to two newly reported modifications – acetylation and glycation. We found that acetylation is protective and glycation is harmful. When we combined acetylation or glycation manipulations with SUMOylation and phosphorylation alterations on the α-synuclein level, we found that the effects of these modifications are not additive – the impacts of acetylation and glycation depends on phosphorylation status. Finally, we investigated the effects of the familial mutants in tandem with altered acetylation or glycation. We report two familial mutant-specific effects: H50Q is surprisingly sensitive and non-toxic in a hyper-acetylation environment, while A53E is highly toxic with hypo-glycation. These studies show the relevance of covalent modifications in sporadic and familial PD, and their in-tandem effects that underlie toxicity mechanisms.
Examining Attention as a Moderator of Verbal and Visual Encoding
Kristina Johnson ’12, Julia Thomas, Kristen M. Klipfel, Elizabeth Geary, Leslie Guidotti Breting, Jerry J. Sweet. NorthShore University Health System (An affiliate of the University of Chicago, Pritzker School of Medicine), Chicago, IL
One goal of neuropsychological evaluations is to accurately measure an individual’s memory ability. The purpose of this study is to examine the possible role of simple and complex auditory attention on initial encoding. Scores on visual and verbal learning tasks, measured by Wechsler Memory Scale 4th Edition (WMS-IV) Visual Reproduction I (VR I) subtest and WMS-IV Logical Memory I (LM I) subtests were analyzed to examine if auditory attention, measured by Wechsler Adult Intelligence Scale – Fourth Edition (WAIS-IV) Digit Span subtests, moderated performances. Our sample included 388 patients (Mage = 53.85, SD = 22.84) who completed VR I, LM I, and Digit Span subtests. Multiple regression analyses were conducted. In the first block, age was entered. In the second block, scores on Digits Forward, Backward, and Sequencing were entered as measures of simple and complex attention. Results indicated that attention significantly moderated the relationship between age and VR I (R2 = .20, F (4, 383) = 25.50, p <.000). Attention also significantly moderated the relationship between age and LM I (R2 = .16, F (4,383) = 19.45, p<.000). In both visual and verbal models, Sequencing was the only significant predictor of learning, suggesting that complex attention matters over and above simple attention.
Understanding Medicine Through an Artistic lens
Morgan N. Marshall ’16, Master of Science in Biomedical Visualization Program, University of Illinois at Chicago, Chicago, Illinois
Learning complex anatomy and science is difficult for anyone in school. Weather this be individuals in undergraduate degrees, or people in various grad school course. However, despite of this difficulty using visuals to help aid in understanding topics can allow for the learning experience to be more comprehensive and easier. Medical illustration is a highly intensive field and study for scientists well equipped with concise visual skills that help make difficult topics easier to understand for every student. A Master of Science in Biomedical visualization includes learning how to work with both traditional and 3D media to convey anatomy and scientific concepts. An example that will be shown today will be an in-depth interactive 3D didactic of how various viruses work in the human body. Along with illustrations done for the Field Museum, medical legal malpractice cases, and general physiology topics.
Effects of prolonged social isolation in adolescence: Sex differences in anxiety and sociability behavior in rats
Eliska Mrackova ’19, J. Amiel Rosenkranz, Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science
Early life stress such as prolonged social isolation is regarded as a risk factor for the development of depression and anxiety in adulthood. Extensive research has found that sex differences in anxiety-like behaviors emerge during adolescence, with females more at risk than males. Despite the rising social isolation and loneliness across the world, the causes and effects of social isolation in adolescence remain still poorly understood. The purpose of this study was to determine whether the housing condition of rats (social isolation vs group housing), social isolation duration (1-week vs 4-week continuous isolation), or sex (male vs. female) would affect sociability behavior as well as the development of anxiety-like behaviors. Four behavior tests (open field, conditioned place preference, social interaction, and appetitive operant conditioning) were administered at postnatal days 53-69 to determine the effects of social isolation on hyperactivity, thigmotaxic behavior, social behavior, and reward seeking. Overall, we found that 4 wk social isolation in both males and females increased thigmotaxic behavior and in female rats decreased social interaction. Furthermore, 1 wk social isolation in male rats resulted in an increased social place preference compared to a novel object. Lastly, while 4 wk social isolation did not significantly impact conditioned appetitive sucrose seeking behaviors, it increased sensitivity of conditioned seeking to a bright light anxiogenic stimulus. Overall, this research adds new insight into the understanding of sex differences associated with prolonged social isolation and subsequent anxiety-like behaviors. Future research will focus on immunohistochemistry staining of brain areas implicated in anxiety.
Investigation of the Neuroinflammatory Response in Hippocampal Subfields After a Single Mild Traumatic Brain Injury (mTBI) in Rats.
D. J. Sychowski ’19, J. Love, R. Greene, and D. A. Peterson. Rosalind Franklin University of Medicine and Science, North Chicago, IL
Abstract: Mild forms of traumatic brain injury (mTBI), such as concussions from falls or sports, are often associated with memory deficits. We recently described a clinically relevant closed-head model for mTBI in rats, and are using this model to evaluate mechanisms of neuroinflammation and explore possible therapeutic strategies. Rat brains were immunostained to detect mature neurons, astrocytes, and microglia. These sections were imaged and analyzed using Stereo-Investigator software to evaluate neuronal loss and inflammation in hippocampal subfields. Establishing these effects after mTBI would provide a model for investigating regenerative approaches to replacing neurons through reprogramming in the hippocampal CA subfields.
Quantitative determination of cell proliferation in Prdm16 and Mecom null mutant palate shelves and Meckle’s Cartilage
Cosmo Vivirito’151, Bryan C. Bjork2, 1Dept. of Biochemistry, College of Graduate Studies, 2College of Dental Medicine, Midwestern University, Downers Grove, Illinois
Human orofacial clefting is one of the most common and debilitating congenital disorders. A multitude of genetic and environmental susceptibility factors have been identified yet the etiology of this multifactorial condition is still not fully understood. Pierre Robin sequence (PRS) is one of the most common syndromic causes of cleft palate (CP) in which CP arises due to palate-extrinsic mechanisms secondary to micrognathia and glossoptosis. Prdm16 null mutant mice exhibit PRS-like cleft palate, consistent with its robust craniofacial expression. Its paralog, Mecom, also shows strong craniofacial expression. We have demonstrated that Mecom null mutant mice also exhibit cleft palate; however, palatogenesis is perturbed through a different mechanism than Prdm16 mutants that is consistent with a palate-intrinsic role for Mecom. Both genes encode transcription factors that regulate gene expression through DNA-binding as well as DNA-independent binding mechanisms. The conserved structures of their gene products and partially overlapping expression suggest the possibility of compensatory mechanism with the loss of either gene’s expression. Previous analysis of the mid-palate region of Prdm16 mutants identified reduced cell proliferation in palate shelves and also MC in agreement with its smaller size. Surrounding MC perichondrium showed expansion and increased cell proliferation. We hypothesized that similar cell proliferation differences will be evident in anterior and posterior palate regions in Prdm16 mutants and that since loss of Mecom causes CP due to palate-intrinsic mechanisms a more profound reduction in cell proliferation within Mecom mutant palate shelves will be observed. We performed immunofluorescence using BrdU labeling to evaluate cell proliferation in anterior, middle and posterior palate and Meckel’s cartilage (MC) of E13.5 wt (n=2) and Prdm16 (Prdm16cGT) null mutants (n=3). BrdU-positive cells will be visualized via fluorescent microscopy, and digital images will be analyzed for cell proliferation rate using ImageJ. The ratio of BrdU-positive cells among total cell DAPI-stained cells in palate shelves or MC from 5 consecutive serial sections for each embryo was averaged and compared between genotypes using an unpaired students T-test. Reduced cell proliferation in Prdm16 mutant palate shelves suggests some palate-intrinsic requirement for Prdm16. In addition, perichondrium was expanded and cell proliferation elevated which suggests impaired differentiation of these cells. We have prepared comparable slides containing BrdU-labeled Prdm16 mutant and wt coronal head sections with which to carry out BrdU detection and determination of cell proliferation rates. In addition, Mecom mutant and wt embryos have been harvested from BrdU-injected Mecom heterozygous female mice for the comparative analysis. We expect to observe decreased cell proliferation within palate shelves of both Prdm16 and Mecom mutants while MC proliferates will be altered in only Prdm16 mutants.
Current Student Abstracts
Investigating inspiratory rhythmogenic neurons in the preBötzinger complex
Nia Alfaro ’22, Kaiwen Kam, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Respiratory rhythmic generation can be isolated in the ventrolateral medulla, in a nucleus called the preBötzinger complex (preBötC). This research project aimed to investigate the types of neurons that operate within the preBötC. We hypothesize that the preBötzinger complex consists of several distinct sets of neurons, involved in regulating inspiratory rhythmic generation, that distinguish the boundaries of the complex through neuronal patterns. To test our hypothesis, we performed perfusions on several litters of mice. Brain slices were cut and immunohistochemistry was performed (IHC). Confocal microscopy was used to image the preBötC of each slice. Neurons were identified as glyT2 or Dbx1 by DAPI, GFP, and tdtomato fluorescence. CellProfiler showed that there are about 150+ total cells and 20 glyT2 neurons in the preBötC area of each slice. Cell counts were difficult in respect to Dbx1 neurons due to broad tdtomato expression. Additionally, the CellProfiler program used may have occasionally missed or overcounted cells. Based on the data, no significant neuronal counts or patterns arose that may have distinguished the preBötC from the VRC, more data is needed to draw significant conclusions. The results derived from this research adds to the vast amounts of neurons that have already been counted and identified. Our data could be added to these past results to draw potential patterns that may categorize neuronal components of the preBötC.
The Possibility of Reprogramming Oligodendrocyte Progenitor Cells into Functional Neurons
Omar Alrawashdeh, D. A. Peterson, E. Reisenbigler, and M. Thaqi, DePaul University, Chicago, IL 60614; Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Neurodegenerative diseases are of a great concern for the ongoing scientific research. Most of the neurodegenerative diseases share one specific characteristic in between them. The death or depletion of neuronal cells. Scientists have proposed many ideas for how we can either prevent the death of brain neurons, or stimulate the growth of new ones. The advent of Induced Pluripotent Stem Cells research has opened the door for scientific research into reversing the growth cycle of some of our different body cells, and engineer them back into their pluripotent state. By doing so scientists could bypass many of the ethical dilemmas related to stem cell research. But neurons are more complex and specific to their environment from other body cells. In order for a neuron to function properly within its true circuit it would have to grow within its specific environment. To overcome this predicament, the possibility of recruiting Oligodendrocyte progenitor cells into functional neurons prevailed. Using specific transcription factors, it might be possible to guide the ever regenerating, brain originating Oligodendrocyte progenitor cells to grow into neurons rather than glial cells. The hippocampus is known as an active site of glial cells regeneration, and that was taken as an advantage in this research and the progenitor cells were employed as a subject of the research. This specific research was focused on two specific aims: the possibility of using the transcription factors to convert hippocampal glial cells into neurons into neurons, and the viability of the recently converted neurons within their true circuits. And to evaluate the longevity of those neurons after 7, 14, and 21 days.
Developing a new model organism system with Berghia Nudibranch
W. Frost, E. Hill, D. Berninzoni, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Not much is known about the map of an organism’s brain and neurons. Berghia nudibranch, a sea slug is a small organism with just a few thousand neurons in the brain, making it an ideal candidate for an entire mapping of the brain for transgenic models. Not much is known about the Berghia nudibranch, but with four other labs, the Berghia Nudibranch will be genetically modified for certain genotypes and phenotypes. Before doing this, the behavior of the berghia must be explored and understood. Berghia are nocturnal animals, leaving them more active at night. However, the precise attitude and behavior of the berghia is not understood. To understand the motor behavior of the berghia nudibranch, several trials were run to determine at what ideal conditions the berghia must be at before continuing with further behavioral trials. Data found that berghia took several hours to calm down before being comfortable in a testing environment and that they were more comfortable in darker lighting than brighter lighting. Continuing work uses serotonin to explore the motor behavior of berghia, causing lops and turns in their normal movement. Indicating that the serotonin pathways could potentially be the key to berghia motor behavior. Continuing research intends to create genetically modified berghia with the help of four other labs in the upcoming ten years.
Role of plasma membrane citrate transporter (PMCT) in metabolic diseases
Which methodology effectively inhibits PMCT activity?
Ruqia Bibi, Roni Klein, June Mayor, Robert Marr, Shivaputra Patil, & Ronald Kaplan, Lake Forest College; Lake Forest, IL 60045; Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Citrate is a key intermediate in both catabolism and anabolism and thus occupies a prominent position in eukaryotic energy metabolism. One source of intracellular citrate originates in the blood. The human plasma membrane citrate transporter plays a central role in the sodium driven active transport of citrate from the hepatic circulation inwardly across the plasma membrane to the cytoplasm of hepatocytes. Upregulation of PMCT expression has been observed in obese, nonalcoholic fatty liver disease patients, high-fat diet-treated rhesus monkeys, and xenobiotic-treated human and rat hepatocytes. Despite the emerging importance of PMCT in energy homeostasis, reduction of PMCT activity in the pathological state is unknown. We hypothesize that design of specific human PMCT inhibitors may potentially permit the pharmacological amelioration of metabolic disorders such as obesity, atherosclerosis, cancer and diabetes resulting from the synthesis of excess lipid, cholesterol, and glucose. In the present study, we aim to knockdown human PMCT activity via two pathways: small molecule inhibition of transporter function versus RNA interference of transporter expression. In the first instance, the human PMCT is overexpressed via the mammalian expression system HEK293T (human embryonic kidney cells). Upon transfection of the cultured cells, the recombinant transporter is tested for inhibition of activity by various compounds. In the second case, antisense oligonucleotides (ASOs) directed at intronic and/or exonic regions of the PMCT RNA are designed. Upon ASO transfection of HepG2, a hepatocarcinoma cell line with a characteristic high rate of lipogenesis, the innate transport protein produced in the HepG2 cells is tested for a reduction (knockdown) or elimination (knockout) of activity. We found a potential inhibitory compound exhibiting > 45% inhibition at C=5µM, and a promising ASO that successfully knocked down PMCT cellular activity, although we are still replicating ASO experiments.
Effects of upregulated Nucleolin on Breast Cancer Tissue
C.R. Boatfield, N. Walia, M. Repak, O. Powrozek, Lake Forest College, Lake Forest, IL 60614; Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Inflammatory breast cancer (IBC) is cancer that forms in the cells of the breast tissue, characterized by inflammation and redness of the skin localized by breast tissue. The disease is extremely variant and contains many subtypes, each with their own risk to the patient. Additionally, previous studies have shown a correlation between nucleolin and a myriad of cell signalling pathways such as AKT, mitogen activated protein kinases (MAPSs) and mammalian target of rampamycin (mTOR) in multiple types of cancer, including breast cancer. Nucleolin is a multifunctional phosphoprotein distributed in the nucleolus, nucleus and cytoplasm of the cell. Nucleolin in animals is involved in the regulation of RNA polymerase II-dependent gene expression. Its levels are correlated with the rate of functional activity of the nucleolus in exponentially growing cells. In our lab’s previous research, we have investigated the relationship between Nucleolin and Breast Cancer, showing a relationship between the increase of unmitigated cancer growth and presence of nucleolin in cancerous cells. Knowing that nucleolin contributes to cell growth in cancerous tissue, we hypothesized that there must be a relationship between the amount of Nucleolin found in a cell in comparison to both normal cells and other local cells which remained uninfected. In our experiment, we discovered that there was a detectable difference in the amount of Nucleolin in breast cancer tissue cells. Both expression and activation of cellular signaling pathways saw an upregulation in IBC cell lines SUM149PT and SUM190PT when compared to the control cell line of HMEC cells. With this knowledge, the understanding that an upregulated Nucleolin count in a cancerous cell can be information used to target effective therapies to curtail breast cancer growth by limiting the overproduction of Nucleolin.
Insight into Parkinson’s Disease from Yeasts: Combined impact of covalent modifications & familial mutations on α-synuclein
Carris Borland, Yoan Ganev, Thea Grauer, Rosemary Thomas, Chisomo Mwale, & Shubhik DebBurman, Neuroscience Program and Biology Department, Lake Forest College, Lake Forest, IL 60045
Parkinson’s Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are composed of misfolded α-synuclein protein. α-Synuclein is a highly post-translationally modified protein, in healthy and diseased states. These modifications include phosphorylation, nitration, SUMOylation, acetylation, and glycation. While phosphorylation is well-studied as a contributor to PD pathology, less is known about SUMOylation, glycation, and nitration. Particularly, the combined effects of these modifications remain largely unclear, on both wildtype α-synuclein (linked with sporadic PD) and the six mutant forms (A30P, E46K, H50Q, G51D, A53T, A53E) linked with early-onset familial PD. Using a budding yeast model for α-synuclein toxicity in PD, we first evaluated α-synuclein properties in yeast with global hypo- or hyper-SUMOylation and found that excess SUMOylation encourages protection and reduces toxicity in wild-type and several familial mutants (H50Q, G51D and A53E); deficient SUMOylation has the opposite effect. Next, we used yeast strains with reduced global glycation and found that for α-synuclein mutants altered for SUMOylation and phosphorylation, the reduction of glycation was protective. Finally, we investigated the effects of too much or too little nitration on the SUMOylation- and phosphorylation-altering mutants and on the familial mutants. We found that too much nitration exacerbates toxicity of the familial mutants relative to low nitration. Furthermore, decreased nitration protects against toxicity and reduces alpha-syn aggregation in the mutants modifying SUMOylation and phosphorylation. These studies show the relevance of covalent modifications in sporadic and familial PD, and their in-tandem effects that underlie toxicity mechanisms.
Characterizing Telomere Length in Aspergillus nidulans
Brett Palmero’20, Alexsandra Dejneka’21, Jessica Day’22, Graeme Witte’22, Allison Akins’22, & Karen Kirk, Biology Department, Lake Forest College; Lake Forest, IL, 60045
Telomeres are made of a series of repeated nucleotides found at the ends of eukaryotic chromosomes, protecting the important coding DNA. Cell division progressively shortens telomeres because of the end replication problem, where telomeres can be lost without a specific method to continually lengthen telomeres. The enzyme telomerase lengthens telomeres by extending the DNA. It is difficult to study telomerase in mammalian cells because most cells have little to no telomerase activity. However, our lab studies Aspergillus nidulans, a filamentous fungus that has both a uninucleate and multinucleate state that would need to consistently lengthen its telomeres in order to survive. Specific genes can be manipulated and knocked out. This can cause formation of either 1) heterokaryons, which are multinucleate cells with genetically different nuclei, or 2) diploids, when the cell’s survival is threatened. We investigated how the lengths of telomeres in these two cell types differed over five passages, and if they differed from each other. We predict that the heterokaryon cell type will show either progressively shorter or varied telomere length due to the components needed to form telomerase being in separate nuclei and needing to be shared in the cytoplasm. In order to measure the telomere length of these transformant cell types, we implemented Telomere Anchored PCR, a technique established previously in our lab. We observed the heterokaryon lengths were variable, from almost no telomeres to longer telomeres, while diploid telomere length had less variation. These preliminary results indicate potentially different mechanisms of telomerase assembly in diploids and heterokaryons, raising the question of whether more or less nuclei in the cell will affect telomere length.
Protective Capabilities of Caspase-11 and Caspase-1 Against Burkholderia pseudomallei and B. thailandensis:
Casey Duel, Kelly Deobald, Carlos Barbosa, Fabio Re, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Resistance to infection with Burkholderia pseudomallei or B. thailandensis is dependent on the Nlrp3 and Nlrc4 inflammasomes and the non-canonical caspase-11 inflammasome. Nlrc4 mediates protection through induction of pyroptosis in the early phase of infection. As the infection progresses and as IL-18-dependent IFNγ production increases, caspase-11 dependent pyroptosis acquires a preponderant protective role. Production of IL-1β and IL-18 during infection is primarily mediated by Nlrp3. IL-18 is essential for survival because of its ability to induce IFNγ production, which in turn activates macrophage microbicidal functions and primes for caspase-11 expression. Therefore, as supported by previous work, mice lacking either caspase-1 or caspase-11 were expected to be significantly more susceptible than wild type mice to intranasal infection with B. pseudomallei and B. thailandensis. Based on the preliminary work that was supposed to confer the same results, that was not what was observed. Instead, mice with caspase-1 or caspase-11 gene knockouts exhibited less susceptibility than wild type mice when intranasally infected with B. pseudomallei and B. thailandensis.
Bias of Hair Textures in Hiring Practices
Sydni Foley, Emma Remish, Greg Lammers, & Naomi Wentworth, Department of Psychology and Neuroscience Program, Lake Forest College; Lake Forest, IL 60045
High emphasis is placed on the physical appearance of individuals seeking employment. Does the hair texture of a job applicant change their perceived qualification and suggested starting salary? We tried to answer this question by asking participants to imagine that they are a hiring manager looking to fill the position of Executive Assistant. Participants viewed identical job applications with the exception of the applicant’s hair texture. Afterwards, participants assessed perceived qualification for the position and suggested a starting salary. We hypothesized that hair texture would influence looking behavior and candidate evaluations. Eye-tracking data was analyzed in order to examine looking behavior. Questionnaire data was analyzed to examine perceived qualification and suggested starting salary.
Male mating preferences and fitness benefits in a bean beetle.
Elise Grossman, Flavia Barbosa, Department of Biology, Lake Forest College; Lake Forest, IL 60045
Sexual selection theory usually predicts females to have the most costs associated with mating, and thus, to have the strongest mating preferences. However, mating can come along with costs for males, which may result in males also having mating preferences. I examined the mating costs incurred by males in the bean beetle, Callosobruchus maculatus, and whether males have mating preferences as a result of these costs. Bean beetles mate multiple times, and males perform a behavior where they tap the female with their antennae before copulation. This antennation behavior is likely a form of copulatory courtship. In my first experiment, I explored whether males adjust their antennation behavior as a response to variation in female fecundity. If males express mating preferences, we predict they will increase antennation behavior when mating with more fecund females. In my second experiment, I examined how manipulating a male’s ability to antennate affects female preference. We predict that increased antennation behavior will result in higher fitness for males through increased female preference.
Gait Manipulation using Visual Feedback and its influence on Insole Reaction Forces
Gabrielle Gutierrez, Raj Patel, Sai Yalla PhD, Center for Lower Extremity Ambulatory Research (CLEAR), Scholl College of Podiatric Medicine at Rosalind Franklin University of Medicine and Science
Having a regular exercise routine be it walking, light jogging and other non-weight barring exercises can improve the lifestyle of people with type two diabetes. The risk for type 2 diabetics is when they venture into physical activity improvements without proper guidance their risk for foot ulceration and re-ulceration’s increase significantly. Hence, the study aims to validate a feedback (VFB) program that can guide participants to reduce ground reaction forces (GRF) while walking and jogging using wearable sensor technology. Initially, we hypothesize that by reducing tibial acceleration (Tacc) through a visual feedback program participant should be able to reduce GRF during gait and jogging. Since this is the first time a feedback program is introduced, we are validating the program in healthy participants. Participants were required to wear 2 tibial accelerometers at their shin which were used to give them visual feedback. A pressure sensing insole system (Pedar) was used to track their foot pressure distribution along with a metabolic oxygen sensing system (Oxycon) for tracking their energy expenditure. Once all the sensors were placed and calibrated, participants were placed on an instrumented treadmill (Bertec corp) to measure GRF and asked to walk until they reached a steady state in oxygen consumption. 20 seconds of walking data was collected after which visual feedback was provided. The feedback was in terms of a noisy signal that would be flat only if they reduced their shin sensor acceleration by 25%. The same test was repeated for jogging. Results showed that that during visual feedback there was a significant 16% (p<0.05) decrease in peak pressure (PP) when walking when compared to the baseline PP without feedback at forefoot area where ulceration is most common. During jogging forefoot PP decreased by 6% but was not significant. Results did show that during visual feedback there was a significant 36.8% (p<0.05) reduction in Tacc when walking and a 26.6% (p<0.05) reduction on average in Tacc when jogging, when compared to the baseline Tacc without feedback. Participants implemented longer stride to reduce tibial acceleration and in-turn GRF without effecting energy expenditure. Initial results show that the visual feedback could be used safely for diabetics in order to carry out gait manipulation for walking and jogging. Future studies will be aimed to replicate the study with a larger sample size in diabetic population.
Evaluating Direct Reprogramming of Human Fibroblasts Into Neurons as New Model For Alzheimer’s Disease
Aiden Houcek, Sean Schrank, Beth Stutzmann, Robert Marr, Rosalind Franklin University, North Chicago, IL 60045
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder of unknown etiology. While there is substantial investment in AD research, therapies such as memantine and Aricept are limited in their efficacy and simply slow disease progression. While animal models have been valuable for understanding AD pathogenesis, multiple failures of phase 3 clinical trials suggest that new approaches for modeling AD are urgently needed. With the advent of human induced neuron (HiN) technology, we are now able to model AD in human cells. Here, we used a direct reprogramming (DR) method of conversion where AD and Non-AD patient fibroblasts are directly converted into HiNs without the generation of induced pluripotent stem cells (IPSCs). This method of conversion maintains the epigenetic characteristics of patient fibroblasts as well as the state of disease pathology. Interestingly, no single method of DR has dominated the field of transdifferentiation, suggesting that methods with high conversion efficiency and complete differentiation need to be developed. We set out to both optimize the DR method as well as investigate how characteristics of AD and non-AD patient fibroblasts are expressed following reprogramming. This optimization of the DR method will both increase its reliability and provide for a more accurate representation of AD pathology in humans.
Measuring Caffeine-Induced Calcium Responses in Wild-Type and Mutant HEK-RyR2 Cells
Augustana Houcek, Sean Schrank, Dr. Robert Marr, and Dr. Grace Stutzmann, Rosalind Franklin University, North Chicago, IL 60045
Alzheimer’s disease, accounting for 60 to 80 percent of all dementia cases, is an ultimately fatal neurodegenerative disease. This disease is associated with memory loss, changes in mood, poor judgement, difficulty completing tasks, ultimately ending in death from complications of the disease. Age is the best known risk factor for AD; however, there are increasingly more risk factors identified as contributors to the disease. APOE, APP, presenilin 1, and presenilin 2 have all been identified as genetic contributors to AD. Neurofibrillary tangles and amyloid-beta plaques have been identified as some of the key physical markers of AD as well. Though these markers have been identified, there is still little known about their exact origins. Thus far, studies in AD have shown dysregulations in calcium signalling pathways, often involving sustained-upregulation of calcium into the cytosol, appearing to identify pathophysiological consequences long before the physical markers are detected. The regulation of calcium into the cytosol is mediated by an intracellular calcium channel known as the ryanodine receptor, which has previously been identified as a key component of AD pathogenesis. Furthermore, caffeine is widely used agonist of these receptors, specifically the RyR2 receptor. Here, we use a CCD imaging system to measure caffeine-induced calcium responses in wild-type HEK-RyR2 cells and mutant HEK-RyR2 cells, in order to determine differences in responses of the two cell types.
Comparative Evaluation of a, b, and g-Synucleins For Neurodegenerative Potential in Yeast Models
Bilal Khan, Kealey Humphrey, Zubair Mohammed, Yoan Ganev, Ananta Srivastava, Pranav Anand, Rosemary Thomas, and Shubhik DebBurman, Biology Department and Neuroscience Program, Lake Forest College, Lake Forest, IL 60045.
Parkinson’s Disease (PD) is a major hypokinetic neurodegenerative disorder marked by the loss of midbrain dopaminergic neurons. This cell death is linked with Lewy bodies, which are composed of misfolding and aggregation of a lipid-binding protein called αlpha-synuclein. Our lab has successfully developed yeast models to gain new insights into PD pathology linked with alpha-synuclein. Specifically, we have characterized the toxic properties of wild-type and familial mutants of alpha-synuclein, assessed its three domains (N, M, C), and discovered the regulation of alpha- synuclein’s toxicity by covalent modifiers. We have also studied αlpha-synuclein’s disruptions of endocytosis, its effect on oxidative and nitrative stress, on mitochondrial dysfunction, and protein degradative pathways, but have yet to evaluate regulation by lipid metabolic pathways. Alpha-Synuclein belongs to a family of proteins with two other closely related members: Beta- and Gamma-synuclein. While the link between alpha-synuclein and PD has been firmly established, the extent to which beta- and gamma-synuclein contribute to any neurodegenerative disease is less stringent; only one direct genetic connection between beta-synuclein and neurodegeneration is known. Here, we tested the hypothesis that beta- and gamma- synuclein will also have pathology-related properties, but likely to degrees lesser than and with mechanisms different from αlpha-synuclein, and they would have αlpha-synuclein-like domains. We also tested the hypothesis that alpha, beta, and gamma synuclein as well as familial αlpha-synuclein mutants would be differentially toxic to lipids, especially fatty acids. We report that, unlike αlpha-synuclein, neither beta- nor gamma-synuclein bound membranes or aggregated in yeast, instead both remained diffuse in the cytoplasm. However, in a yeast strain with elevated diglyceride levels (dgk1D), beta-synuclein became membrane-bound and also aggregated, while gamma-synuclein remained unaffected. Furthermore, the A30P mutant of alpha-synuclein, which is non-toxic and cytoplasmically diffuse in many yeast models, including ours, also became membrane-associated in this same strain. Our preliminary findings unveil a surprising level of regulation by fatty acids on the member of the synuclein protein family and familial mutants linked with genetic forms of PD.
Hoodwinking Tuberculosis with a Cancer Therapy
Esther Kim, William Conrad, Lake Forest College, IL 60045
Tuberculosis (TB) is a serious, pandemic bacterial infection that targets vulnerable populations. Currently, there is no effective preventative measure against TB. Our goal is to translate CAR-T cancer therapy to create a new TB prophylactic. We will achieve this goal by inserting genes that produce the antibiotic d-cycloserine (DCS) into human cells. My goal is to take DCS genes from the bacterium S.lavendulae and prepare them to go into human cells. In the future, we will insert the DCS genes into human cells and measure their killing ability functionally (measuring bacterial killing) and chemically (direct measure of DCS).
Don’t Take Your Rat to the Beach: An Exploration of Stimulus Materials in the Rodent Attentional Set-Shifting Task
Nathaniel Kregar, Yeshi Tshering, and Jean-Marie Maddux, Department of Psychology and Neuroscience Program, Lake Forest College, IL 60045
Shifting attention in an ever-changing world allows organisms to adapt and survive in their environment. A common way to measure this type of cognitive flexibility is through an attentional set-shifting task (ASST). The goal of this study was to test various digging media and odors as stimuli in a rodent ASST. Rats dug through scented digging media that were distributed across seven different discriminations, to find a food reward buried at the bottom of a bowl. Sand and cat litter were found to be aversive materials for rats, as they refused to dig through these media. These media were eliminated and replaced. Reversal learning, in which a previously rewarded stimulus was no longer rewarded and a previously non-rewarded stimulus was rewarded, was more difficult than preceding discriminations, as evidenced by more incorrect trials on reversal. This finding validates certain aspects of this task, although the lack of a shift cost when shifting attention from one stimulus dimension to another (e.g., odor to media or media to odor) remains problematic for full task utilization. This study highlights the importance of stimulus parameters in the ASST. Once perfected, this task could test the effects of brain lesions and/or pharmaceutical compounds on attention.
The Relationship Between Personality Traits and P300 Amplitudes
Aleksandra Kulesza, Marissa Burch, Ramona Gyorfi, and Dr. Naomi Wentworth, Lake Forest College, IL 60045
Extraversion is a type of personality trait commonly used to describe the level of preference that an individual has for social activity and interaction with other people. The five-factor model states that a person who scores high for extraversion is outgoing and enjoys being with people. On the other hand, it states that someone who scores low for extraversion, or high for introversion, focuses more on internal thoughts and spending time alone. To examine the relationship between extraversion and brain activity, we created a self-report personality questionnaire based on Myers-Briggs Personality tests that provide extraversion scores ranging from 0 (low extraversion) to 56 (high extraversion). To record brain activity, we used electroencephalography (EEG), which visually traces electrical activity by using electrodes on various locations of the scalp. An event-related potential (ERP) is a measurement of electrical activity from an EEG which directly results from a specific sensory, cognitive or motor event. More specifically, we examined a miniscule portion of ERPs called the P300 amplitude, which plays a significant role in attention and memory and is usually elicited in oddball paradigms such as this study. After safely applying the EEG equipment on the scalp, we had participants view a PowerPoint which was split into four blocks, each containing images of nonsocial stimuli (flowers) and social stimuli (male or female faces). In blocks one and three, the oddball stimulus, the one that showed up less frequently, was the social stimulus; the oddball stimulus in blocks two and four was the nonsocial stimulus. We hypothesized that P300 amplitudes would be higher during social stimuli for those with higher levels of extraversion and lower for those with low extraversion scores.
Cloning Syndecan-4 to determine its role in pathogenic Th17 cells
Courtney P. Levitt ’21,1,2 Kathryne E. Marks,1 Joseph M. Reynolds, PhD1 1Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, 2Lake Forest College; Lake Forest, IL 60045
Autoimmune diseases, like Multiple Sclerosis and Lupus, are characterized by the damaging inflammation of tissue. These diseases are often driven by a subset of CD4+ T cells known as Th17 cells. Th17 cells can become pathogenic in the context of autoimmune disease through the activation of Toll-Like Receptor 2 (TLR2) or in response to IL-23 and IL-1β. Pathogenic Th17s are known to be highly inflammatory and are more recently known to produce high levels of IL-17, as well as GM-CSF and IFNg. This leads to the irreversible damage of tissue and may be a target for treatment of autoimmune diseases. TLR2 recognizes pathogenic patterns expressed in infectious agents and mediates the production of cytokines for development of immunity. Activation of TLR2 is thought to induce a change in the transcriptional program of Th17. However, the role of TLR2 signaling in Th17 pathogenicity is poorly examined. Thus, we examined TLR2 activation in Th17 cells in vitro utilizing Pam3CSK4 (PAM), a synthetic ligand used to activate TLR2 signaling. Syndecan-4 (Sdc4) was found to be downregulated in Th17 cells following the addition of PAM and may be implicated in autoimmune diseases. Sdc4 was cloned using a pGEM®-T easy vector system. Restriction enzymes AgeI-HF and BstBI were used to cut the insert. Bacteria was grown with ampicillin (Amp) resistance and was screened with the blue-white method to ensure the proper recombinant plasmid of Amp-resistant Sdc4 in pGEM®-T easy. Sequencing then confirmed the correct sequence of insert properly isolated in pGEM®-T easy. The insert was then isolated and extracted from gel to ligate with pCMMP-IRES-GFP. After transforming, Sdc4 was confirmed to be present in the pCMMP vector. Work in progress includes utilizing a Plat-E cell line with Sdc4-pCMMP-IRES-GFP to produce retrovirus in order to overexpress Sdc4 in Th17 cells in vitro. These experiments will allow us to determine the role of Sdc4 in pathogenic Th17 cells.
Neuropeptide Y (NPY) and Reproductive Experience Modulates Stress Resilience in Female Rats
Dana Midani, Gina DeJoseph, Dr. Janice Urban, Rosalind Franklin University of School and Medicine, North Chicago, IL 60064, in association with Lake Forest College; Lake Forest, IL 60045
With the increasing prevalence of anxiety disorders, understanding the neural mechanisms of stress resilience is becoming even more important. Neuropeptide Y (NPY) is an endogenous peptide that has anti-anxiety effects by modulating H currents carried out by the hyperpolarization-activated cyclic nucleotide-gated channel subunit 1 (HCN1) in the amygdala. It has already been demonstrated that repeated injections of NPY into the BLA of male rats increases resilience behavior and a decrease in expression of HCN1. Stress resilience was assessed by first stressing the animal with restraint stress (RS), then measuring social interaction (SI). Then, stress was also assessed through immunohistochemistry (IHC) on brain tissue collected from the rats. In these previous studies, it was found that NPY did not affect HCN1 channels, SI, or stress resilience in female rats. HCN1 channels in males and females are the same, so it is unclear why the results were different for female rats. It is possible that female rats might therefore have different mechanism for stress resilience. To test stress resilience in female rats, two main groups of female rats were tested on. In one group, a model of a reproductive experience (RE) was used, which has been shown to buffer stress reaction. Perhaps the RE excites or engages mechanisms of stress resilience. The other group of female rats were nulliparous. RS and SI was conducted on the rats on postpartum (PP) day 14 and day 28. SI was not increased in RE rats compared to the nulliparous controls. SI after RS was significantly decreased in the nulliparous controls, but not the RE rats. Next, brain tissue was taken for assessment of cFos expression via immunohistochemistry (IHC). cFos is a transcription factor activated by Ca2+. Expression of cFos indicates neuronal activity. Therefore, an increase in expression of cFos in the BLA would indicate increased BLA activity, a sign of increased anxiety. Work in progress includes quantifying cFos expression in the brain slices. These results so far indicate a sexually dimorphic mechanism of stress resilience in response to RS and intra-NPY injections.
Evolutionary Trade-Offs in The Bean Beetle: The influence of density on sexually selected traits
Desire Uwera Nalukwago, Samuel Gascoigne ’20, & Flavia Barbosa, Biology Department, Lake Forest College; Lake Forest, IL 60045
Sexual selection is one of the most crucial driving forces of evolutionary change. However, sexually selected traits are costly since they require a lot of energy to develop and maintain. Organisms must therefore be strategic on how they allocate their resources to such traits to maximize their fitness. The bean beetle, Callosobruchus maculatus has two distinct morphs that maximize fitness at specific environments: a flightless morph and a dispersal morph. These morphs are defined by either the presence or absence of functional wings. However, the exact mechanism by which the morphs come about is unknown. One factor that could contribute to these morphs is density in terms of larval competition. We manipulated density as number of eggs developing per bean and hypothesized that higher density eggs would produce dispersal morphs. Due to limited resources, individuals who allocate more resources to dispersal traits would then allocate less to reproduction. We predict that individuals reared in high densities would have larger wings and smaller testes, allowing them to leave crowded environments in search of those with more resources. These individuals are also predicted to invest less in other sexually selected traits such as courtship behavior, and to have longer copulation times, to maximize sperm transfer. In contrast, we predict low density beans to produce flightless morphs, which should have larger testes, and spend more time on courtship behaviors and have shorter copulations. Morphological traits like wing size and testes size were also measured as a comparison between individuals from different density groups to determine overall relationships between behavioral and morphological traits.
The Synthesis of Antimalarial Natural Products—The Chaetoxanthones
Amber Lennon ’20, Sara Tallen ’20, Nhu Quach ’22, Sadiq Dabire ’22, & Paul Gladen, Chemistry Department, Lake Forest College; Lake Forest, IL, 60045
Malaria is a disease caused by Plasmodium protozoal parasites. These protozoans are transmitted and spread among people through the bite of infected Anopheles mosquitoes. Although malaria is now preventable and curable, it is still one of the world’s top causes of death in low-income countries. Indeed, in a 2018 report, the World Health Organization (WHO) reveals that Plasmodium falciparum, the most common malarial parasites, have developed resistance to nearly all current antimalarials. In other words, we need to study more about the antimalarial properties of the substances besides the current cure to battle the disease more efficiently and economically. In preliminary tests, Chaetoxanthones, a small family of bioactive natural products isolated from a marine-derived fungus found near Santorini, Greece, was found to be capable of killing not only malaria, but also leishmaniasis, and Chagas’ disease parasites. However, due to the difficulties in extracting this group of compounds from its natural sources, our project is focusing on developing an appropriate pathway to efficiently synthesize these products from simple and commercially available materials. Therefore, the success in synthesizing the Chaetoxanthones will allow further examination of its antimalarial properties.
K63 Ubiquitination during LMP1 Signaling
T. Rosenhagen and D. Everly; Lake Forest College, Lake Forest, IL, 60045, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Epstein-Barr Virus (EBV) is the causative agent of infectious mononucleosis as well as a contributor to a number of human malignancies. EBV’s reservoir is human memory B-cells and can switch between latency and lytic replication. Constitutive NF -kB activation is a well-established consequence of latent EBV latent infection. This is in part due to the viral latent membrane protein 1’s (LMP1) interaction with ubiquitin ligases, as well as post translational modification of cellular proteins by ubiquitination. There are eight different ubiquitin modifications, each with distinct functions that determine not only the modified protein’s fate but also changes in the cellular environment. Linear-linked and lysine 63 (K63)-linked ubiquitin chains are two well-studied types of ubiquitin chains found to be critical regulators of the NF-kB signaling. The NF-kB essential modulator (NEMO) is part of the inhibitor of Ik-B Kinase (IKK) complex which phosphorylates IkBa. With IkBa phosphorylated, it gets degraded, allowing NF-kB to translocate to the nucleus and activate gene expression. Linear ubiquitin chains activate NEMO, which can itself be modified by ubiquitination; however, proteins modified by linear ubiquitin and K63 linkages upstream of NEMO during LMP1 signaling are unclear. The Npl4 zinc finger (NZF) domain of TAB2 binds specifically to K63 ubiquitin chains in cells. Therefore, we developed tandem repeats of the NZF domain with a glutathione S-transferase (GST)-tag. Several different tandems that contained 1, 2, 3, or 4 copies of the TAB2-NZF domain, 1x, 2x, 3x, or 4x, respectively, were expressed in bacteria and checked for solubility. Two different versions of the NZF domain that were truncated at amino acids 585 or 663 were also tested. Soluble proteins were bound to glutathione beads and used for in vitro pull-downs with different ubiquitin chains, including K63, linear, and K48 ubiquitin chains. Binding between the GST-TAB2-NZF tandems and ubiquitin chains were analyzed by western blotting. As expected, the GST-TAB2-NZF tandems specifically bound to K63 but not linear or K48 ubiquitin chains. Structurally, K63 and linear ubiquitin chains are nearly identical, so the specificity of the tandems was vital to understand the specific role of K63 chains in ubiquitin-dependent signaling by LMP1. These studies suggest that the GST-TAB2-NZF tandems will be useful to identify proteins that are modified by K63 ubiquitination using pull-downs from cells expressing LMP1. Identifying K63-ubiquitinated proteins downstream of LMP1 during signaling will identify new pharmacological targets that could be blocked to interrupt viral signaling. Ultimately these studies hope to provide insight into EBV pathogenesis and open new ways to prevent EBV-associated disease progression.
Implications of PLD2 signaling in Kaposi’s sarcoma-associated herpesvirus (KSHV)
Samantha Russell, O. Powrozek, and N. Sharma-Walia, Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, Chicago, IL
The gamma-2 herpesvirus Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) is etiologically associated with Kaposi’s sarcoma (KS), and lymphoproliferative disorder called primary effusion lymphoma (PEL) or body cavity-based B-cell lymphoma (BCBL). KSHV infection is very common in sub-Saharan Africa, moderately prevalent in Mediterranean countries but much less common (<10%) in most of Europe, Asia, and the US. KS and PEL are usually present in immunocompromised patients such as organ transplant patients, HIV patients, and older populations. PEL is a form of highly aggressive B-cell non-Hodgkin’s lymphoma that usually occurs in body cavities (pleural space or pericardium) without tumor masses. Due to the aggressiveness of PEL, it often has a poor prognosis and has a survival rate of less than six months after diagnosis. KS is a highly vascular cancer that is identified by round red lesions that occur in areas of the body that contain soft tissue. KS and PEL are treated with radiation, chemotherapy, antiviral drugs, and surgery. More specialized and targeted antiviral and anticancer treatment is needed to treat this aggressive form of cancer. Here, we focus on the role of phospholipase D (PLD) signaling during KSHV infection. PLD, an enzyme that promotes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidate (PA) and choline, regulates mitogenic signals upstream of the mammalian target of rapamycin (mTOR). Two major PLD isozymes in mammals are PLD1 and PLD2. PLD2 enzyme located in the membrane and is linked to signals that mediate the processes involved in cell proliferation, tumorigenesis, and metastasis. We hypothesize that KSHV is utilizing PLD2 signaling for infected cell proliferation and tumorigenesis. We analyzed the skin tissue sections of healthy subjects and KS patients obtained from ACSR for the presence of PLD2 with anti- PLD2 antibody. Normal healthy control tissue sections showed membrane, whereas KS skin tissue showed intense, dispersed cytoplasmic staining of PLD2. KSHV+/EBV-; BCBL-1 cells expressed a higher level of phosphor-PLD2 as compared to non-infected KSHV-/EBV-; BJAB cells. Normal healthy control tissue sections showed membrane, whereas KS skin tissue showed intense, dispersed cytoplasmic staining of PLD2.In our preliminary studies, we found that PLD2 inhibitor treatment was selectively toxic to BCBL-1 cells as compared to BJAB cells. We are currently working to elucidate the mechanism of PLD2 activation and its role in oxidative stress, KSHV infected cell proliferation, and migration.
Decitabine Treatment Demethylates Vast Majority of High-Confidence Differentially Methylated Regions In HCT-116 Colorectal Cancer Cells.
Anna Sandler, Jonathan Anderson, Ariane Balaram, Yoan Ganev, Samuel Gascoigne, Christina Gimondo, Brett Palmero, Ayesha Quraishi, Alejandro Rodriguez, Said Omer Sadat, William H Conrad, Lake Forest College, Lake Forest, IL 60045
Colorectal cancer (CRC) is a common, and often incurable, form of cancer. Gene silencing by CpG island hypermethylation often plays a role in CRC progression. Certain regions of the genome, called high confidence differentially-methylated regions (DMRs), are consistently hypermethylated across numerous patient samples. In this study, we used bisulfite PCR sequencing to investigate methylation levels at DMRs in the promoter region of CDKN2A, DKK3, EN1, MiR34b, SPG20, and TLX1 in HCT-116 CRC cells. We observed that for all DMRs except CDKN2A, the demethylating agent decitabine significantly reduced CpG methylation. Using ENCODE project data, we observed that transcriptional activator binding inversely correlates with DNA methylation at all of these sites across diverse cancers and cell types. Our data increase resolution of the methylation status at the above DMRs, show the reversibility of methylation at these sites by decitabine, and the likely role of hypermethylation at these sites in gene silencing. In the future, we plan to test if DMR any specific gene silencing protects HCT116 cells.
Sensing turbulent waters: connecting biomechanics with sensory systems
STINSON, HANNAH M1; MORGAN, KJ1; TYTELL, ED2; SCHWALBE, MAB1,2
1Lake Forest College
Fish encounter complex hydrodynamic environments while swimming, such as horizontal vortices that may come from waterfalls or over the top of rocks. These turbulent currents can knock a fish off course. Fish probably rely on multiple sensory systems to remain stable while swimming in these environments. It is unclear how the lateral line and vision contribute to a fish’s ability to compensate for these unsteady flows. Preliminary experiments showed that bluegill sunfish (Lepomis macrochirus) were stable in horizontal vortices with and without their lateral line and visual systems. Here, we challenged fish in a flow tank to more frequent horizontal vortices generated by a custom-made flapper and faster flow rates to continue testing the relative importance of these sensory systems in fish swimming. During each trial, a fish was positioned behind the flapper (frequencies = 1, 2, or 3 Hz) while swimming at one of three speeds and was recorded with three high-speed cameras to obtain the fish’s position relative to the flapper over time. Fish were filmed under regular or infrared light to test vision and were treated with cobalt chloride to deactivate this sensory modality to test the lateral line system. Overall, fish recovered quickly from the flapper action and differences in swimming movements were observed under the various flow and sensory conditions. Therefore, we continue to support that bluegill sunfish are relatively stable in horizontal vortices. This is likely due to passible properties – the vertically tall and thin shape – of their bodies that allow the horizontal vortices to pass around them, but sensory input from the lateral line and visual systems contribute to regaining stability after being knocked off course from the initial exposure to turbulence.
Role of Mental Health Diagnosis and Current Symptoms on Substance Abuse, Sleep, and Daily Hassles in College Students
Ellen Szostak & Rachel Greenley, PhD, Lake Forest College, Rosalind Franklin University of Medicine & Science
Mental health has become a common topic of conversation on college campuses across America. About one fifth of all college students have been diagnosed with either anxiety or depression. With these diagnoses being so prevalent, we want to see what behaviors could be causing this increase as well as the effects these diagnoses are having on college students. In this study, we surveyed students at a Midwestern university in numerous areas: mood and anxiety symptoms, physical activity, drug use, alcohol use, sleep quality, and daily hassles. Using their responses, we were able to use the students who have either reported an anxiety or depression diagnosis or symptoms to see what other behaviors they engage in. We looked at both mental health symptoms and diagnoses in order to have one category that was self-reported and one that was verified by a medical professional. Those with a diagnosis and those who have common mental health symptoms will engage in different behaviors so we did not want these people to be in the same group. After performing a multiple regression on selected data, we saw that anxiety and depression diagnoses are correlated with higher alcohol consumption and substance abuse. There were also some significant relationships found between the increase of mood and anxiety symptoms and sleep quality and daily hassles. Those who only experience symptoms without a diagnosis experience a decrease in sleep quality and an increase in daily hassles. Knowing these relationships can help college and university wellness centers aid their students with the management of anxiety and depression symptoms and diagnoses.
Alternative splicing of BACE1 mRNA due to common polymorphism in Alzheimer’s Disease (AD)
Estella Tcaturian, Francine M. Jodelka, and Michelle L. Hastings, Center of Genetic Diseases, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Alzheimer’s disease (AD) is the most common neurodegenerative disease of our time. A hallmark of AD is the presence of amyloid plaques, primarily in the hippocampus and entorhinal cortex, though other regions of the brain are also affected as the disease progresses. Amyloid plaques are comprised of amyloid beta (Ab) peptides, which are a cleavage product of the amyloid precursor protein (APP). APP is cleaved by a number of peptidases to produce Ab, one of which is beta-secretase (BACE1). The cause of AD and why some people develop the disease while others are spared is not clear. It is possible that there are a number of genetic risk factors that could contribute to AD risk. We hypothesize that BACE1 abundance or activity affects the production of Ab and thereby, the formation of Ab plaques. One factor that can affect BACE1 activity is the production of alternative isoforms of the protein by alternative pre-mRNA splicing. Here, we tested the hypothesis that a common polymorphism at position +5 on the intron 5 (IVS5) in BACE 1, weakens splicing of exon 5 leading to a decrease in full-length BACE1 mRNA and an increase in BACE1 lacking exon 5. BACE1 mRNA lacking exon 5 would lead to a frameshift and the production of a truncated BACE1 protein that would be unable to cleave APP to form Ab. To test our hypothesis, we first identified the SNP status in a number of human cell lines and then assessed exon 5 splicing to establish a correlation between the SNP predicted to weaken splicing and the skipping of exon 5. We isolated DNA from the cells and performed polymerayse chain reaction (PCR) followed by sequencing to identify the SNP and we isolated RNA and performed reverse transcription (RT)-PCR to assess splicing. Our results suggest that a BACE1 polymorphism may be associated with skipping of exon 5 or exon 6 of BACE1. Work is still in progress to confirm these results. Additional work was also done to compile a database of other SNPs and disease-associated mutations in the +5 position of the 5’ splice site to better understand the role of this position in efficient splicing.
Why Do I Still Hate Thee? Misperceptions of Opposing Political Attitudes
Are Americans truly as polarized as the media makes it seem?
Belle Tseitlin & Vivian Ta, PhD; Psychology Program, Lake Forest College, Lake Forest, IL 60045
It has been argued that the US has increasingly become more polarized over recent years. Is there evidence to support this, or has this been largely exaggerated? To examine this, we replicated a study by Chambers and Melnyk (2006) which found that members of partisan social groups tend to exaggerate how much their own opinions differ from those of their rivals. In the current study, we sought to replicate Chambers and Melnyk (2006) to determine the degree to which members of partisan social groups misperceive disagreement and the extent to which this has changed since 2006. We recruited 314 participants via MTurk. Participants rated their own positions on the topic of abortion and estimated the position of the average member from the opposing political party about various pro-choice and pro-life issues (N = 210 pro-choice participants; N = 104 pro-life participants). Our results replicated Chambers and Melnyk (2006) such that perceived disagreement was greater than actual disagreement, and participants perceived more disagreement regarding values central to their own ideology than values central to the opposition’s. Overtime, perceived disagreement increased for some values and decreased for others, and actual disagreement has consistently increased with regards to pro-life values. Importantly, although actual disagreement has increased in some values, it is still consistently lower than perceived disagreement.
Sex differences in the rat basolateral amygdala projections to the bed nucleus stria terminalis and prolonged cued fear conditioning responses
Yesenia Uribe ’21, Jaime Vantrease, Brittany Avonts, Janice Urban, and Amiel Rosenkranz, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Anxiety disorders are the most common psychiatric disorders in America with 264 million people diagnosed worldwide.1 Within those patients, it has been found that females are twice as likely to be diagnosed.2 For this reason, understanding the sex differences underlying anxiety disorders can help develop sex specific therapeutics.3 The amygdala plays a critical role in emotional learning and memory, and sex differences have been found in the formation and projection of specific nuclei.4 The basolateral nucleus of the amygdala (BLA) is an important structure in the development of emotion-related behaviors by integrating highly processed cues from the enviornment.5 The bed nucleus of the stria terminalis (BNST) or extended amygdala, is associated with generalized anxiety-like behaviors, is hyperactive in patients with anxiety and is modulated by input from the BLA.6-7 Since sex differences have been observed in the BLA, and female rats express less generalized anxiety-like behavior, the goal of this research was to determine if there were sex differences between the projection from the BLA to BNST (BLA-BNST) between male and female rats. We hypothesized that inhibition of BLA-BNST neurons would decrease generalized anxiety-like behaviors and that this effect would be greater in males compared to females. In order to test this hypothesis, we utilized a chemogenic approach via DREADD (designer receptor exclusively activated by designer drug) inhibition and social interaction (SI) as the behavioral test. DREADD is a mutated G protein-coupled receptor which has the advantage of having a nanomolecular potency for selective activation by the small molecular ligand clozapine-N-oxide (CNO).8 When CNO binds DREADDs used here, membrane hyperpolarization occurs via decreased cAMP signaling and increased activation of potassium channels resulting in temporary suppression of neuronal activity.9 In our studies, we performed bilateral injections of viral vectors containing Cre-depedent DREADDs that require Cre-recombinase to be expressed into the BLA, and Cre-recombinase into the BNST. Therefore, CNO administration would selectively reduce BLA-BNST neuronal activity and allow us to determine changes in generalized anxiety-like behavior in BLA-BNST mediated tasks. Determination of rat cyclicity was done through daily vaginal lavage and analyzed through cell cytology. This 4-day estrous cycle contains phases of high estrogen (proestrus) and low estrogen (met-diestrus). Following viral injections, baseline social interaction (SI) was recorded for both female and male rats. Total time spent interacting, classified by the total time a rat’s nose touched a novel rat’s body, was recorded in an open field for 10 mins. It was observed that male rats had a higher SI time than female rats (62.30s vs 50.21s). Within female rats, there appeared to be an estrous cycle effect with low estrogen females interacting more than high estrogen females (39.84s vs 82.57s). This suggests that estrogen may contribute to decreased SI when no treatments have been given. After their baseline was collected, rats were randomly given either saline or CNO (1mg/kg; i.p) 45 mins prior to the experiment and SI was recorded as before. Four days later, when females were back in the same phase of the estrous cycle, rats were given the opposite treatment and SI was recorded. Female rats given CNO displayed comparable SI times with female rats given saline (56.81s vs 55.28s). Proestrous female rats showed comparable SI times between CNO and saline (39.67s vs 39.14s). In contrast, met-diestrus female rats displayed a higher SI time when given saline rather than CNO (98.13s vs 75.89s). However, there was a trend for male rats given CNO to spend more time interacting than male rats given saline (62.76 s vs 52.60 s). Treatment order did not affect the SI time. Overall, these data suggest that BLA-BNST inhibition may increase SI in males, but not females.
Optimization of in vitro CD8 T cell differentiation protocols for efficient adoptive cell immunotherapy in cancer
Noah Vanderhyde, Alexander Schutte, Azad Darbandi, Tianhao Xu and Gustavo J Martinez, Center for Cancer Cell Biology, Immunology, and Infection, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago IL.
CD8+ T cells are pivotal in combating intracellular pathogens and for tumor immune surveillance. Upon recognizing their cognate antigen presented by antigen-presenting cells (APCs), naïve CD8+ T cells are activated, rapidly proliferate and differentiate into a heterogeneous pool of effector cells that display cytotoxic activity. Conventionally, effector cells are further categorized into two main subsets: short-lived effector T cells (SLECs) as KLRG1hi CD127lo and memory precursor effector cells (MPECs) as KLRG1lo CD127hi. The current protocols for differentiating effector or memory CD8 T cells in vitro are not optimal. Thus, the present study aimed at comparing different conditions to better differentiate effector and memory cells. We utilized IL-2, IL-15 and IL-7 cytokines, which are required for the activation and/or maintenance of T cells. Our results show that culture of activated CD8+ T cells with IL-7 and IL-15 in the absence of IL-2 better recapitulates the expression of memory-associated markers. We also began to optimize protocols for immunotherapy by adoptively transferring activated antigen-specific CD8 T cells into tumor-bearing mice. The ultimate goal is to further manipulate these cells to enhance the anti-tumor activity. Using new modified cells for adoptive transfer may prove as a new therapy for cancer clearance
Use of Colchicine to Highlight the Soma of Neurons in the Bed Nucleolus of the Stria Terminalis and the Central Amygdala Staining for VIP, CRF and Enk
K. M. Wallis, A. Martin, R. Chudoba, P. Lis, J. Dabrowska, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
Colchicine is a chemical compound which causes depolymerization of microtubules. This leads to the inability for the cell to transport neuropeptides such as Vasoactive Intestinal Peptide (VIP), Corticotrophin Releasing Factor (CRF), and Enkephalin (Enk). Using Immunohistochemistry (IHC), it is possible to image these molecules under a fluorescence microscope or a confocal. Under normal conditions, VIP, CRF and Enk would be transported to the terminals via microtubules, however without this function the molecules would remain in the soma of the neuron and should make it easier to image after an IHC as the concentration in the soma would make them well defined.
A longitudinal analysis of the benefits of music therapy in patients with dementia
Madeline Webb and Dr. John Calamari, Psychology Department, Rosalind Franklin University of Medicine and Science; North Chicago, IL 60064
Dementia refers to a group of cognitive disorders characterized by loss in memory, comprehension, language, learning and/or judgement abilities. Although a large portion of the population of those over the age of 80 have dementia (25%), the underlying causes are not known (Shiltz et. al). It is extremely important to catch early warning signs of dementia to be able to better treat the symptoms. Many studies have been done to try to help find treatments for dementia. One form of intervention that has been shown to reduce dementia symptoms is music therapy (Sole et. al). The current pilot study aimed to investigate and better understand the longitudinal effects of a music therapy intervention on people experiencing significant cognitive impairment. One scale developed to assess the effects of music therapy in dementia patients is the Music in Dementia Assessment Scales (MiDAS scale; McDermott et. al). This MiDAS scale, is a behavioral observation rating scale with observers asked to rate behavior across five distinct categories: Interest, Response, Initiation, Involvement, and Enjoyment. The MiDAS scale, was used in to assess the 28 study participants response to the Musical Memories program conducted at Lake Forest Place, a memory care facility for people with sever cognitive impairment. During this study, music therapists and other staff assessed participants using the MiDAS scale while the Lake Forest Symphony played music to them as outlined in the music therapy program. Participants were assessed ten times over a ten-week period. Preliminary results indicate that participants experienced a significant, positive increase in mood and engagement with their environment during music therapy throughout the study. Results from the current study provide preliminary evidence for the benefits of music therapy for older adults suffering from dementia.