NCUR CBS Abstracts

Hosam Alkhatib
“Improving disadvantaged middle-school students’ academic habits and interests in STEM through engaging STEM-related experiences in a university setting.”
Hosam Alkhatib, Erik Skoe, Paul Siliciano

In today’s educational system, socioeconomically disadvantaged middle-school students commonly have poor academic habits and low interest in pursuing STEM-related educations and careers. Research shows that poor academic habits often result from a lack of familial educational background and students’ unpursued and unidentified academic interests. Further, research shows that students exhibit greater interest toward classroom topics when exposed to them in hands-on, real-world experiences. This study aims to expose middle-school students to STEM-related topics in an active, real-world setting to promote their interests in STEM and improve their academic habits. In our study, this real-world setting takes the form of a university campus. We have developed an ongoing program in which groups of eighth-grade students are brought to a myriad of interdisciplinary scientific domains within the University of Minnesota. Students are selected preferentially if they do not have family members who have attended college or are defined by their teachers as not performing to their fullest potential. The event on campus will consist of engaging and immersive tours of research and health professional settings. These experiences are selected based off of studies indicating what content is mentally and visually stimulating to adolescents, and will include biology, chemistry, engineering, agricultural sciences, and healthcare. Data acquisition will consist of administering pre-post surveys to the students to quantitatively and qualitatively assess the extent to which the students’ academic habits and relative interests in STEM are affected by involvement in our program. We expect that after experiencing STEM in the university setting and interacting personally with professors, socioeconomically disadvantaged students will have increased interest in STEM, ultimately leading to improved academic habits. After concluding this study, we hope to collaborate with the University of Minnesota to implement this program as an indefinite Twin Cities outreach program.

“Investigating fatty acid export following lipid droplet degradation in skeletal muscle.”
Hosam Alkhatib, Douglas Mashek, Mostafa Ali

Fat accumulates in the form of lipid droplets (LDs) in numerous tissues including muscle. Excessive intramyocellular LD accumulation contributes to the development of insulin resistance and other metabolic abnormalities. The primary fat type stored in LDs is triacylglycerol, which can serve as fuel in muscle metabolism. Degraded LDs release fatty acids (FAs) into the cytoplasm that can be metabolized through mitochondrial -oxidation. LD degradation in skeletal muscle has not been extensively studied, and it was conventionally thought that FAs released from LD degradation are directly transferred from LDs to mitochondria. The purpose of this study is to elucidate the cellular mechanism primarily responsible for LD turnover in skeletal muscle. In this study, we used a radioactive tracer to track FA trafficking in skeletal muscle under varying conditions. C2C12 myotubes were lipid loaded with 500 µM oleate containing a radioactive tracer ([14C]oleate), which is incorporated into the cells during a 16-hour “pulse-period.” The media from the pulse-period was then replaced with new media that differed in nutrient content or contained various inhibitors of lipid catabolic pathways for a six-hour “chase-period.” Following the chase period, the media and cells were harvested for lipid extraction analysis using thin-layer chromatography. The radioactivity for each lipid fraction was quantified, making it possible to determine the location and relative amounts of each lipid fraction following the cellular treatment. Our findings show that myocellular FAs are exported from the cell, a process termed “fatty acid efflux.” Further, we found that these FAs are effluxed prior to being oxidized in the mitochondria. Finally, we show that lipophagy, the recently discovered mechanism responsible for LD turnover in liver cells, is not responsible for LD turnover and FA efflux in skeletal muscle. However, the mechanism responsible for fatty acid efflux is unknown and is the focus of our ongoing work.


Ella Borgenheimer
“Tracking cell lineages of differentiated progenitor cells of the embryonic mouse thalamus.”
Ella Borgenheimer, Yasushi Nakagawa

The thalamus is a vital structure of the brain responsible for several crucial functions of the nervous system including motor control, learning, and sensory processing. Progenitor cells are crucial to specification of functional circuits within the brain, though there is currently little knowledge on the division and migratory processes of a single progenitor cell and how it individually contributes to the nervous system. This project aims to explore the mechanisms behind the fate of thalamic progenitor cells as they divide and become postmiotic. Using the transcription factor Olig3, which is specifically expressed in the thalamus and cortex, thalamic progenitor cell differentiation can be further explored. This specificity can be used to express the DNA recombinase CreER and mediate nucleus translocation using the genetic cassette MADM (mosaic analysis of dual markers). By utilizing the spatially and temporally specific activation of Cre-mediated interchromosomal recombination of the MADM alleles, we are able to express EGFP and TdTomato fluorescent proteins in progenies of a recombined stem cell and track the fates of all the progenies derived from EGFP- or TdTomato-expressing cells. Fluorescently tagged thalamic progenitor cells can be analyzed and tracked for individual cell lineage patterns that result in postmiotic differentiation. By tracking a single lineage of thalamic progenitor cells, the results showed that principle sensory nuclei of the thalamus have a high tendency to share asymmetrically dividing progenitor cells, and that asymmetric divisions produce laterally located nuclei first and then more medially located nuclei later.


Christina Landis
“Determining the effect of A3B overexpression on the mutational landscape of ovarian and fallopian tube epithelial cells in vivo.”
Christina Landis, Tim Starr

Apobec (A3B) is a human oncogene which codes for a DNA cytosine deaminase. The presence of this enzyme can lead to base changes from cytosine to uracil, mutating the DNA. This gene has been linked to breast cancer in humans through previous studies. Due to the prevalent connection between breast and ovarian cancer, this experiment aims to determine if a link between A3B and ovarian cancer in humans exists. This experiment will be performed using a mouse model. The experimental mice have had a cre-adenovirus injected into their ovaries and have been allowed to live out to varying time points. The cre complex recognizes two lox sites that flank a stop codon upstream from the A3B gene. After recognizing the lox sites, cre will excise anything between them, including the stop codon which will then allow for expression of the A3B gene. After reaching their specific time point, different organs have been collected and DNA has been extracted from the ovaries. This DNA will then be used in differential DNA denaturation (3D) PCR to determine the level of cytosine to uracil base changes. If the samples are able to denature at lower temperatures, the experimental data would suggest a higher composition of adenine and thymine base pairs indicating the expression of A3B. The expected results of this experiment will indicate the mice which were injected with the cre-adenovirus will have high expression of A3B in the ovaries and not the other organs.


Samantha Lee
“Rapid generation of multiplexed and conditional CRISPR transgenic mouse models.”
Samantha C. Lee, Walker S. Lahr, Beau R. Webber, Branden S. Moriarity

Cancer is a genetically heterogeneous disease in which altered expression of genes result in a disruption in normal somatic cell growth. Researchers have developed numerous mouse models of cancer that rapidly and accurately models the disease. This has largely relied on the generation of transgenic mice expressing the Cre recombinase system, which can be used to delete or activate specific genes in a particular cell/tissue type in the mouse. This is very laborious and time consuming to generate the best Cre-based models of cancer developing, most requiring the breeding of 3-4 different transgenic models into a single mouse. This barrier has largely precluded their use for pre-clinical drug testing. To address this barrier, we plan to utilize the CRISPR/Cas9 nuclease system to delete or activate specific genes that will produce cancer in the same exact fashion as the Cre-based models. Critically, we implemented multiplexed CRISPR gRNAs that can target 10 or more genes at one time, with a single transgenic mouse. We also targeted CRISPR activity in a conditional manner, allowing us to target only the cells or tissues we desire. Using this method, we will be able to generate experimental mice from a single breeding regardless of the number of genes to be deleted or activated, removing the need for laborious and costly breeding schemes to generate mouse models of cancer based on the Cre system. We engineered this CRISPR system in mouse embryonic stem (ES) cells that will be used to generate our multiplexed conditional CRISPR (MCC) transgenic animals. Subsequent MCC mice will be bred and aged for tumor development to validate our system.


Ajinkya Limkar
"Development of vaccines for treatment of opioid abuse using carrier proteins suitable for pharmaceutical manufacturing and clinical use.”
Ajinkya Limkar, Valeria Gradinati, Federico Baruffaldi, Andrew Lees, Marco Pravetoni

In the United States, 2.6 million people are dependent on heroin and prescription opioid analgesics such as oxycodone, causing over 54,000 opioid-related overdose deaths in 2016. Treatment of opioid abuse and prevention of overdose results in $78.5 million in healthcare costs. Although safe and effective medications are available, only 1 out of 5 opioid users benefit from pharmacotherapy. To address this unmet medical need, therapeutic vaccines for opioid use are being developed. Vaccines such as these are effective against opioid abuse as they lead to a strong humoral immune response that targets the drug directly. The components of the humoral response have been shown to inhibit opioid passage to the brain, thus decreasing the rewarding effects of the drug, which lead to a decrease in opioid use over time. To ensure vaccine translation, it is critical that vaccines are built using individual components suitable for pharmaceutical manufacturing and compliant with the Food and Drug Administration (FDA) guidelines are used. In this study, two oxycodone conjugate vaccines containing carrier proteins generated in the Escherichia coli expression system were synthesized. These immunogens consisted of oxycodone-based haptens (OXY) conjugated to the E. coli-expressed (Eco) diphtheria cross-reactive material (CRM197) and nontoxic tetanus toxin fragment (rTTHc). It was found that these new immunogens induced expansion of hapten-specific B cell lymphocytes, generated effective oxycodone-specific serum IgG antibodies, reduced oxycodone distribution to the brain, and reduced oxycodone-induced toxicity in mice. Furthermore, the efficacy of bioconjugates containing EcoCRM197 and EcoTTHc was equivalent to oxycodone vaccines containing the well-established carrier proteins keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) and native CRM197. Thus, it was concluded that opioid vaccines containing EcoCRMTM and rTTHc are candidate immunogens for future vaccine development.


Lauren Madden
“Orientation selective spinal cord stimulation.”
Lauren Madden, Julia Slopsema, Lauri Lehto, Cuellar Ramos, Igor A. Lavrov, Shalom Michaeli, Matthew Johnson

Introduction and Background: Clinical evidence has found that electrical stimulation of the spinal cord can have therapeutic effects for pain modulation and restoration of motor function after spinal cord injury. Epidural stimulation is thought to stimulate a large region of the spinal cord surrounding the active electrodes. Improvements can be made to provide more spatially selective activation tailored to the spinal cord anatomy. Previous research has shown that controlling the primary direction of the electric field can illicit orientation selective activation in the brain. Here we applied this concept of orientation selectivity to the spinal cord using a computational model of spinal cord stimulation. Methods: Here we built a Finite Element Model (FEM) of lumbar (L1) and sacral segments of a rat spinal cord traced from anatomical slices and four electrodes arranged in a diamond shape. The FEM was interpolated along six anatomically placed axons. Stimulation was applied by rotating the primary direction of the electric field around the four contacts using square pulses with amplitudes determined by phase offset sinusoids. The activation thresholds of the axons were then found using NEURON simulations. Results: Depending on the location within the spinal cord, each axon trajectory had the lowest activation threshold for a single electric field direction. In contrast, the highest threshold was found in the antiparallel electric field direction, with threshold gains ranging from 4.21 – 16.92 (maximum/minimum thresholds). Conclusions: This study indicates that the orientation of the electric field generated by a four-contact electrode array can provide for spatial specificity when targeting axons with a high degree of directionality, which will next be tested in an in-vivo rat model.


Clairice Pearce
“Analyzing the potentiality of Zika virus infection in the human medulloblastoma.”
Clairice M. Pearce, Maple L. Shiao, Walter C. Low

Zika Virus (ZIKV) has recently raised global alarm due to its association with microcephaly in infants of infected mothers. Current evidence suggests that this condition is a consequence of neural stem cell death as mediated by the virus. Since it is known that neural stem and brain tumor cells possess similar properties, we explored the feasibility of developing ZIKV as an oncolytic virus against the human medulloblastoma. This brain cancer is the most prevalent amongst children and has been proven difficult to treat through chemotherapy, radiation, and surgical procedures. Thus, we sought to characterize ZIKV infectivity and the presence of receptors known to facilitate infection in the medulloblastoma cell line DAOY. More specifically, AXL, TIM-1, DC-SIGN, and TYRO3 receptors were found to be present in DAOY cells by immunohistochemistry. Cells were then incubated with ZIKV for 3 days, and RNA was isolated and used for qPCR. Changes in gene expression provided further evidence for cell infection by AXL, MERTK, and TYRO3 receptor involvement. Interestingly, AXL and MERTK expression decreased after 3 days infection as compared to control cells, while TYRO3 expression was significantly upregulated. ZIKV envelope, NS2, and NS5 proteins also demonstrated a fold change of 10,000 or greater, indicating high viral infectivity. Together, these results provide further support for the potency and potential of ZIKV as an oncolytic virus in a medulloblastoma-targeted treatment.


Livia Songster
“Role of MyTH4-FERM myosin in filopodia initiation within Dictyostelium discoideum.”
Livia D. Songster, Margaret A. Titus

Filopodia are dynamic cellular structures consisting of bundled actin filaments that allow cells to detect chemical cues and interact with their environment. Filopodia formation is used in neuronal growth and metastasis of some cancers. MyTH4-FERM (MF) myosin proteins are essential for filopodia assembly across phylogenetically-distant species, but the mechanism of filopodia initiation is not well understood. The social amoeba Dictyostelium discoideum is being used as a model system to determine the role of an MF-myosin, DdMyo7, in filopodia initiation. Wild-type D. discoideum extend multiple filopodia, whereas myo7-null cells extend none. Full-length GFP-tagged DdMyo7 localizes to filopodia tips of wild-type cells and rescues filopodia formation in myo7-null cells. To determine if DdMyo7 requires a lever arm and dimerization domain for filopodia formation, two GFP-tagged DdMyo7 mutants were made, one with a dimerization domain deletion (?DD) and the other with a lever arm and dimerization domain deletion (?3IQ-DD). Both mutants rescue filopodia in myo7-null cells, so the potential role of the MF2 domain in dimerization of the deletion mutants was tested. These mutants were expressed in wild-type cells and protein expression was confirmed by western blot. Live-cell confocal microscopy was used to image filopodial localization of ?DD-MF2 and ?3IQ-DD-MF2 in wild-type cells. ?DD-MF2 can localize to filopodia tips, but ?3IQ-DD-MF2 cannot, suggesting that this mutant cannot dimerize. Next, I will test the ability of these mutants to rescue filopodia formation in myo7-null cells. I predict that in ?3IQ-DD-MF2 will not rescue any filopodia and ?DD-MF2 will rescue a small number of filopodia, since this mutant was able to localize to filopodia tips in wild-type cells. The results of this project will provide insight into features of MF myosins involved in dimerization and indicate regions of DdMyo7 that bind to other actin-interaction proteins.


Sophia Vrba
“Development of a Pichinde virus-based vaccine vector for the treatment of tuberculosis.”  
Sophia Vrba, Wanjun Zhu, Vikram Verma, Anna Tischler, Yuying Liang

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, is one of the top ten causes of death worldwide. Although it can be treated through a combination of antibiotics, multi-drug resistant and extensively drug resistant tuberculosis strains continue to emerge and spread. The current vaccine for TB, the BCG vaccine, is only effective against the most severe forms of childhood disseminated TB and has limited effectiveness in adults and adolescents. Therefore, there needs to be another effective prophylactic and therapeutic method. To address this need, a trisegmented Pichinde Virus-Based Vaccine Vector (rP18tri) was developed for the prevention and/or treatment of TB. Control of TB infection is mediated primarily by cellular immune responses, and the rP18tri-based viral vector can strongly induce both humoral and cellular immunity. The virus vector is composed of three single strands of RNA. The L RNA segment contains the RNA polymerase and the Z protein; S1 contains the nucleoprotein in the negative sense, and the TB antigen, Ag85, in the positive sense; S2 contains the envelope glycoprotein precursor in the positive sense, and the TB antigens EsxA and EsxH in the negative sense. The vaccine vector plasmids were created through traditional cloning techniques and the live viral vaccine was generated after plasmid transfection. Expression of the three TB antigens, Ag85, EsxA, and EsxH, in the viral vaccine-infected cells was detected by western blot analysis with TB antiserum. The rP18tri-induced TB-specific T cell immune response in mice will be characterized by tetramer analysis to detect TB-specific CD4 and CD8 T cells through direct flow cytometry (FACs) on mouse blood samples. It is expected that the rP18tri vector will induce strong TB-specific cellular immunity. Future experiments will include assessing the vaccine against an infectious strain of TB, and the immunized mice should display protection against the TB.