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2020
Friday, April 24th

Poster Number: 100

Characterization of Microbacterium liquefaciencs Bacteriophages Isolated from the Local Soil Environment on the Winthrop University Campus

Hunter Tristen Cannon, Winthrop University
Ayden Dybik, Winthrop University
Nicole R. Garcia, Winthrop University
Joel A. Haley, Winthrop University
Caleb M. Meyerand, Winthrop University
Dallas Nivens, Winthrop University
Lauren E. Patterson, Winthrop University
Alexis A. Ramirez, Winthrop University
Jordan N. Rucker, Winthrop University
Laela A. Walker, Winthrop University

Faculty Mentor: Kristi M. Westover, Ph.D., and Victoria J. Frost, Ph.D.

In the fourth year of Winthrop University’s involvement in the HHMI SEA—PHAGES program, two new bacteriophages were discovered and annotated using the host Microbacterium liquefaciens. Bacteriophages Mercedes and Leafus were isolated from soil on the Winthrop University campus in Rock Hill, South Carolina. DNA from each phage was isolated, and restriction enzyme digests were performed. Phage structures were also described using TEM microscopy. Once isolated, the DNA was sent to the University of Pittsburgh for genomic sequencing. DNA Master software and other programs, including Glimmer, GeneMark, Phamerator, the NCBI Blast Local Alignment Search Tool, the HHPred Bioinformatics Toolkit, and Starterator, were used to annotate and identify the functions of genes by comparing the sequences to other phages. The Mercedes genome length was found to be 40,230 base pairs (bp) long in a circularly permuted organization containing an estimated 59 genes. The Leafus genome length was found to be 42,000 bp long, also in a circularly permuted pattern containing an estimated 64 genes. While Leafus is a member of the EA1 subcluster, Mercedes has not been placed into a subcluster yet. The completed annotation should be able to shed light on its phylogenetic relationship with other EA cluster phages. The two most closely related phage sequences based on nucleotide similarity and gene structure are Neferthena, and EA5 subcluster phage and Chepli, an EA6 cluster phage. SEA—PHAGES research allowing for exploration of mycobacteriophage genomes will help researchers to ultimately develop gene libraries for biomedically relevant bacteriophages.

Poster Number: 102

Measuring Heat Related to the Disassembly and Reassembly of Ferritin using Isothermal Titration Calorimetry

Brandon Ellison, Winthrop University
Alexandra Perez, Winthrop University

Faculty Mentor: Nicholas Grossoehme, Ph.D., and F. Wayne Outten, Ph.D., University of South Carolina

Ferritin is an iron storage protein responsible for the accumulation of excess intracellular iron. Native ferritin self-aggregates into a nanocage structure containing a ferroxidase center that regulates the uptake and release of iron. In recent years, researchers have begun to explore using ferritin as a component in drug delivery. Ferritin is an attractive candidate because it is a native human protein that has the ability to encapsulate small molecules. Furthermore, it can be chemically or genetically modified to target very specific cells. One major limitation of drug delivery by ferritin lies in its inherent stability: harshly acidic conditions are needed to drive the disassembly of the nanocage. It was recently discovered that replacing the E-helix of human light chain ferritin with a GALA peptide repeat (hFtnL-GALA) would allow for the pH-induced disassembly to occur at a pH below 6, thus rendering ferritin a more attractive drug carrier under physiologically relevant conditions. This project aims to express and purify hFtnL-GALA with a subsequent thermodynamic characterization of the disassembly and reassembly of the nanocage. The chimeric protein is largely isolated in an insoluble form; consequently, the published protocol failed to produce enough protein for subsequent experiments. An alternate protocol was developed that leveraged urea to resuspend the insoluble fraction, followed by slow dilution to allow the protein to fold properly. Chromatographic analysis of the sample was consistent with an intact nanocage structure. Ongoing efforts are focused on developing a strategy that yields sufficient protein for further analysis.

Poster Number: 103

Mapping Brain-Wide Inputs to Two Distinct Thalamic Nuclei

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Juliana Quay, Winthrop University

Faculty Mentor: Ying Zhang, Ph.D.; Dheeraj Roy, Ph.D.; and Guoping Feng, Ph.D., Massachusetts Institute of Technology

The thalamus contains many nuclei, among which the anterior thalamic nuclei (ATN) and parafascicular thalamus (PF) have been found to be the most unique based on their single cell RNA-sequencing transcriptomic signature. Functionally, ATN has been implicated in learning, memory, and spatial navigation, whereas PF is thought to contribute to both fine and coarse motor actions. The outputs of these two structures have been fairly well characterized; however, much less is known about brain regions that send inputs to these nuclei. In order to identify the regions that serve as inputs to ATN versus PF in a brain-wide manner, we used the monosynaptic rabies virus tracing approach. We imaged representative coronal mouse brain sections and aligned each section to the standard mouse brain atlas, before quantifying the number of retrogradely labeled rabies virus-positive neurons in each brain region upstream of ATN and PF. These data allowed us to create a rank-ordered list of brain regions that serve as inputs to ATN and PF, which has not been reported in the literature. We found that the retrosplenial granular cortex (RSG) and cingulate cortex (Cg1) were the regions sending greatest input to ATN, while a sub-division of the superior colliculus (InG), the anterior pretectal nucleus (APTD), and a sub-division of the motor cortex (M2) sent the greatest input to PF. This work not only sets the stage for future input-specific circuit manipulations during mouse behavioral tasks, but it will also serve as a resource for the entire thalamic neuroscience field.

Poster Number: 104

Diet Analysis of Juvenile Dragonflies Using Group-Specific Polymerase Chain Reaction

Rachael Rowe
Whitney Player

Faculty Mentor: Cynthia Tant, Ph.D.

Aquatic food webs are complex, and understanding interactions in these food webs can give an indication of ecosystem health and stability, as well as movement of energy and nutrients through ecosystems. Previous studies have utilized both microscopic gut content analysis and stable isotopes to aid in constructing food webs in these ecosystems. However, gut content analysis is time-consuming, stable isotope analysis can be cost prohibitive, and both methods only identify general categories of food items. The application of newer, molecular-based approaches has the potential to provide previously unavailable taxonomic resolution in aquatic food webs (i.e., who is eating whom?). DNA-based methods have been used in other disciplines for diet analyses, but have not been widely applied in freshwater ecology. We collected juvenile dragonflies (Odonata, Anisoptera) from Winthrop Lake in Rock Hill, South Carolina, dissected gut contents, and extracted DNA from individuals in three genera. We amplified DNA via PCR, using group-specific primers targeting mitochondrial CO1 gene regions for midges (Diptera, Chironomidae) and mosquitoes (Diptera, Culicidae) to identify these potential prey in gut contents; we used gel electrophoresis as a presence/absence test for DNA from these prey groups. Occurrence of prey groups in gut contents varied by individual and by genus of dragonfly examined. With further refinement, these methods have the potential to provide previously unavailable detail on predator—prey interactions in these ecosystems.

Poster Number: 105

Optimization of RNA Isolation Methodology from Three-Dimensional Collagen Hydrogel Culture Systems

Nathaniel C. Kidd, Winthrop University
Chandler E. Burt, Winthrop University

Faculty Mentor: Matthew Stern, Ph.D.

Three-dimensional culture systems allow for more complex cellular interactions and organization than traditional two-dimensional culture, which better replicates an in vivo environment. Interestingly, cells placed on top of collagen hydrogels organize into a donut-like shape called a toroid, while cells mixed into the hydrogels do not organize into higher order structures. The goal of our project is to evaluate the signal transduction pathways and cellular mechanisms that mediate toroid formation. The specific goal of the work described here was to optimize RNA isolation methodology from cells cultured on or in collagen hydrogels – a procedure that is known to be technically challenging – and to use real-time RT-PCR to compare the expression of select genes during toroid formation. We tested and compared several different RNA isolation protocols and found that a method based on the use of cetyltrimethylammonium bromide (CTAB) prior to alcohol precipitation, which is more typically used in isolation of RNA from plants, proved to be the most consistently effective in our hands. We went on to conduct a twelve-hour time-course experiment where RNA was isolated from adipose derived stem cells cultured under toroid-forming conditions every two hours. This enables us to compare gene-expression profiles of cells during toroid formation via methods like real-time RT-PCR andRNA sequencing in the future. Such comparisons will provide valuable insight into the mechanisms of toroid formation and self-organization by stem cells in developmental and regenerative contexts.

Poster Number: 106

Isolating, Purifying, and Investigating Mycobacterial Lysogens

Allyssa L. Lewis, Winthrop University

Faculty Mentor: Victoria Frost, Ph.D.

Bacteria have shared an entangled evolutionary history with bacteriophages for the past three billion years. Some bacteriophages use a specific type of infectious pathway (lysogeny) that maintains their hosts’ viability, thus enabling a mechanism of coexistence. In certain phage genomes, annotation has revealed the presence of immunity-related genes, which suggests a mechanism of how some bacteriophages can protect their hosts and resist superinfection by other related bacteriophages. To investigate this further, two temperate (and previously annotated) mycobacteriophages (ExplosioNervosa and Rhynn) were isolated in an effort to create lysogens in their host cell, Mycobacteria smegmatis. Once created, the lysogens were purified and tested against their original infecting phage as well as an unrelated bacteriophage (Haimas) to see if they were able to resist superinfection. Tests showed that both Haimas and the original viruses were still able to infect the lysogens and cause them to lyse. The ability of bacteriophages to lyse their own lysogens raised the idea of spontaneous reversion: the prophages could have reverted to the lytic cycle due to a triggering condition in their environment. The ability of the host—phage relationship to respond to certain environmental signals warrants further investigation, as does manipulation of the genes linked with immunity and infection. Investigating the triggers and unraveling the mechanisms that fuel coevolution help further our understanding of the host—parasite equilibrium that exists today and highlights opportunities for future applications.

Poster Number: 107

Host Range Investigations of Novel Bacteriophages

Bethany M. Wise, Winthrop University

Faculty Mentor: Kristi Westover, Ph.D., and Victoria Frost, Ph.D.

Bacteriophages are viruses that infect bacterial cells, using them as hosts to express their genetic material and replicate. Some bacteriophages use a specific strain of bacterial host for this procedure, while others are less host-specific. This study aims to help understand this area of phage—bacteria interaction by investigating the ability of bacteriophages to replicate in alternative hosts. Bacteriophages originally isolated from Microbacterium foliorum were tested for their ability to infect and lyse Microbacterium testaceum, Microbacterium paraoxydans, Microbacterium liquefaciens, and Mycobacteria smegmatis. Spot titer assays demonstrated plaque formations on alternative hosts by two of the 16 phages (MonChoix and Sirkeiram). Three of the phages (Aries55, BravoCanis and Iann) were able to infect Mycobacteria smegmatis. In addition, two novel phages (Ixel and Nebulous) were isolated from the bacterial host M. liquefaciens. Using M. liquefaciens as the host, the infective abilities of Ixel and Nebulous were compared to those of MonChoix and Sirkeiram. The phenotypic measure of infectivity is termed Efficiency of Plating (EOP) and for both the M. foliorum isolated phages, the EOP was less (<1) when compared to the host phages. It is likely that the expression of particular genes in the genomes of phage and bacterial host is able to influence this phenomenon. A number of the phage genomes have been annotated. Genetic comparison and further testing phenotypically, will help elucidate whether specific, present genes function to enable phage to use a wide host range.

Poster Number: 108

Evaluating the Effects of Detergent Concentration on the Ultrastructure and Recellularization of Porcine Internal Thoracic Artery Scaffolds

Carlos E. Escoto-Diaz, Winthrop University
Jesse B. Kooistra, Winthrop University

Faculty Mentor: Matthew Stern, Ph.D.

Heart disease is the leading cause of death in developed countries, leading to an increase demand for coronary artery bypass graft surgeries. Currently, bypass surgery requires either that a vessel is harvested from the patient or that a donor graft is provided. An alternative approach is the use of tissue-engineered vascular grafts. The goal of this study is to evaluate the potential of scaffolds derived from porcine internal thoracic arteries (PITAs) for use in vascular tissue engineering. PITA scaffolds can be produced through the process of decellularization, which uses detergents to remove porcine (pig) cells while leaving behind the extracellular matrix of the tissue. We hypothesized that increasing detergent concentration during decellularization would affect the ultrastructure of PITA scaffolds and be associated with greater residual cytoxicity. We evaluated the ultrastructure of the scaffolds using scanning electron microscopy and observed that increasing detergent concentration was associated with greater scaffold porosity. We evaluated the residual cytotoxicity of the scaffolds using the alamarBlue viability assay and found that extensive rinsing is required to eliminate scaffold cytotoxicity. We verified the ability of endothelial cells to grow on/in the scaffolds using a combination of the alamarBlue viability assay and fluorescent microscopy. Taken together, our results show that PITA scaffolds with different ultrastructural features can be prepared and repopulated with endothelial cells as long as the scaffolds are properly rinsed. Establishing an effective procedure for recellularization of PITA scaffolds will ultimately aid in the development of a clinically relevant alternative to our current options for vascular grafts.

Poster Number: 109

Investigation of an Indirect Defense Mechanism of Chapmannia floridana in Florida Scrub

Mackenzie Jenkins

Faculty Mentor: Jennifer Schafer, Ph.D.

Glandular trichomes (i.e., sticky hairs) that entrap carrion act as indirect defenses in some plant species. The sticky hairs attract predators that consume the carrion. Predators then deter herbivores from harming the plant, leading to an increase in the survival and/or reproduction of the plant. Flowering stems of Chapmannia floridana (Florida Alicia), a perennial plant endemic to Florida, are covered in sticky hairs, which may act as an indirect defense. For 84 flowering C. floridana individuals across six habitats in the Florida scrub ecosystem, we counted the number of fruits, flowers, and buds present and documented any damage to reproductive structures. In addition, we counted the number of carrion, herbivores, and predators on each flowering stem. There was a positive relationship between the length of the trichome-covered portion of the stem and the number of carrion trapped. Only 19% of flower buds, 15% of flowers, and 10% of fruits we counted were damaged. We found predatory spiders on 6% of flowering stems and herbivores such as grasshoppers and caterpillars on 37% of flowering stems. For scrubby flatwoods and firelane habitats, we found no association between habitat and damage. There was no association between carrion presence and damage; however, the probability of damage to reproductive structures decreased as the number of trapped carrion increased. Overall, our results suggest that herbivory of C. floridana reproductive structures is relatively low and that glandular trichomes on C. floridana may be acting as an indirect defense.

Poster Number: 110

Investigating the Responsiveness of Embryonic Chick Retinal Ganglion Cells to Semaphorin-3A

Shane Ira C. Lacanin, Winthrop University
Fatoumata Nancy Cisse, Winthrop University
Allison T. Reed, Winthrop University

Faculty Mentor: Eric Birgbauer, Ph.D.

During embryonic development, neural connections are made when axons grow over long distances to find their synaptic targets, guided by terminal structures called growth cones. Growth cones either continue to grow or collapse, retracting and growing in a different direction, in response to attractive or repulsive cues. One such axon guidance cue is Semaphorin-3A (Sema-3A). Luo et al. (1993) showed that Sema-3A causes growth cone collapse from embryonic chick dorsal root ganglion cells (DRGs) but not from chick retinal ganglion cells (RGCs). However, the experiment was repeated in Dr. Birgbauer’s lab, and we found that Sema-3A can cause growth cone collapse of chick RGCs. There are several possible hypotheses on why the results of those two experiments differed. There could be a dose-dependent difference or an age-dependent difference. There could also be a time-dependent difference, since the previous research assay was for 60 minutes while we used a 15-minute assay. We have tested these hypotheses and found a dose-dependent growth cone collapse in chick RGCs. Also, the time response of Sema-3A was investigated, revealing that RGCs exhibited a peak of collapse after 15 minutes, and that they started regenerating at 20 minutes. We also examined the responsiveness of different embryonic ages to Sema-3A, and it was found that as the chick embryo develops, the chick RGCs maintain a significant level of responsiveness (p < 0.05). We have also investigated the expression of Sema-3A receptors, Neuropilin-1 and Neuropilin-2, as well as their co-receptors in the chick retina by RT-PCR. We found that all of these are expressed except for Class B plexins and Neuropilin-1. In conclusion, we have found strong evidence that Sema-3A induces growth cone collapse in embryonic chick RGCs. We found that the retinal growth cone collapse is dose-dependent and developmentally relevant. Also, embryonic chick RGC growth cones rapidly desensitize to Sema-3A, explaining the difference from Luo et al.

Poster Number: 111

Optimization of Culture Conditions for the Simultaneous Recellularization of Porcine Internal Thoracic Artery Scaffolds with Multiple Cell Types

Holdyn C. Ferguson, Winthrop University

Faculty Mentor: Matthew Stern, Ph.D.

The increasing prevalence of ischemic diseases has generated a growing need for heart bypass surgeries. The goal of our research is to tissue engineer vascular grafts from scaffolds derived from decellularized porcine internal thoracic arteries that can be recellularized with patient-specific cells and restore function more effectively than current methods. The ability to recellularize scaffolds with multiple cell types, including endothelial cells (ECs), smooth muscle cells (SMCs), and adipose-derived mesenchymal stem cells (ADSCs) is important for imparting function to an engineered vessel. However, an important question that arises is what type of cell culture media should be used to allow the different cell types to grow together. We hypothesized that mixtures of two media types that support growth of two of the cell types of interest could be identified. To test our hypothesis, we grew ECs in different combinations of 1) EC and SMC medium and 2) EC and ADSC medium. We also grew ADSCs in different combinations of EC and ADSC medium. We monitored the viability of the cells and assessed the expression of CD31 by ECs cultured in different media combinations. In all cases, the growth of the cell type of interest was no different in a 50:50 combination of its medium and the other medium than growth in 100% of its own medium. In addition, CD31 expression was maintained by ECs under all experimental conditions. These results suggest that 50:50 mixtures of culture medium will support the growth of two cell types within our vascular scaffolds.