|Friday, April 20th|
Hallie V. Smith, Winthrop University
Faculty Mentor: Victoria Frost, Ph.D., and Kristi Westover, Ph.D.
Bacteriophages are viruses that inject their genomes into specific bacterial hosts to replicate. As members of the Howard Hughes Medical Institute’s SEA-PHAGES (Science Education Alliance – Phage Hunters Advancing Genomics and Evolutionary Science) program, freshman at Winthrop University have isolated 21 phages from soil using the host bacterial strain Mycobacteria smegmatis. This semester, the host bacterial strain Microbacterium foliorum was piloted to increase the collection and knowledge of diversity within actinobacteriophage in Rock Hill. The novel phage Scamander was isolated directly from local soil. The subsequent purification and amplification protocols entailed multiple plaque assays, which made use of techniques such as picking a plaque from plates and various dilutions of phage lysate to obtain a pure phage sample with a high titer. Transmission electron microscopy showed that Scamander has a long, flexible tail, which is characteristic of the siphoviridae morphotype. Phage DNA was extracted and the genome was sequenced by the Pittsburgh Bacteriophage Institute. Annotation of the genome is in progress and the following programs are being used to determine specific gene data: protein blasts from PhagesDB, HHPred, and NCBI; gene start-site calling programs such as Starterator, GeneMaster, and Glimmer; and genome comparison maps on Phamerator. When complete, the annotated genome will reveal functions of certain genes, while also serving as a comparison for other existing and, as yet, undiscovered Microbacterium phages. The increasing database of bacteriophages and their characteristics helps to expand the understanding of the genomic diversity of viruses, particularly those that infect bacterial hosts in the phylum Actinobacteria.
Jennifer N. Schroen, Winthrop University
Faculty Mentor: Matthew Stern, Ph.D.
Cellular physiology is regulated by both biochemical and mechanical stimuli received from the environment. Traditional cell culture experiments typically focus on manipulation of the biochemical stimuli present in cell culture medium, while largely ignoring the role of mechanotransduction in the cellular processes being studied. A growing body of literature demonstrates that systematic manipulation of the physical/mechanical environment of cultured cells can be effectively used to drive a desired outcome – such as stem cell differentiation into a particular lineage. We are interested in the use of adipose-derived mesenchymal stem cells (ADSCs) as a plentiful and easily obtained source of patient-matched multipotent stem cells for tissue engineering applications, including skeletal muscle tissue engineering. While ADSCs are capable of robust in vitro differentiation into several lineages, their ability to undergo (skeletal) myogenic differentiation is relatively limited. We hypothesized that the culture of ADSCs on flexible silicone membranes combined with the application of uniaxial stretch would increase the ability of ADSCs to differentiate down the myogenic lineage. Here, we describe the development and testing of a culture system that allows us to tune the material properties of the silicone membranes used as substrates for cell culture and apply precise regimens of uniaxial stretch to cells cultured on the membranes. Our results show that both culture on silicone membranes and exposure to uniaxial stretch alter the properties of ADSCs under standard growth conditions. Future work will seek to identify a combination of biochemical and mechanical stimuli that improves the efficiency of myogenic differentiation of ADSCs within this system.
Hannah Roark, Winthrop University
Sleep is a critical component of the body’s diurnal rhythm and for preparation and recovery from athletic competition. Therefore, it is a necessity for athletes to sleep for the minimum recommended amount (7-9 hours) each night. If there are disturbances with the timing, and/or quality of sleep, the psychological and physiological recovery processes are inevitably compromised. Poor sleep can cause diminished athletic performance, increased fatigue, and impaired cognition. In addition to exceptional physiological demands, collegiate athletes face many extra-athletic demands that can lead to insufficient sleep (e.g., studying, emergencies, etc.). Long-distance travel is common among most collegiate athletic competitions. Thus, travel fatigue can cause sleep disturbances, which can lead to a worsened mood, a reduced quality of sleep on the road, and decreased overall motivation levels. Travel between time zones can also cause jet lag and inadequate sleep quality and quantity. Sports teams will often schedule multiple matches per week, which does not allow the athletes adequate time for metabolic recovery. This can lead to overtraining and musculoskeletal injuries. Previous research indicates athletes benefit from prescriptions for additional sleep beyond their normal intake. Athletes who get adequate sleep demonstrate increased accuracy, enhanced mood, and, for example, faster sprint times in their sports. Taken together, there are many factors that can potentially impair sleep quality in collegiate athletes. Thus, the purpose of this literature review is to outline the importance of sleep in athletes, factors that can cause sleep loss, and the effects of reduced sleep on athletic performance.
Hannah Hopfensperger, Winthrop University
Multilineage-differentiating stress enduring (Muse) cells are a unique subpopulation of mesenchymal stem cells (MSC) that are pluripotent and have the ability to self-renew for multiple generations. Muse cells also have an advanced ability to receive damage signals, survive in stress-filled environments, and exhibit low tumorigenic activity. Due to their expression of the cell surface antigen SSEA-3, Muse cells in a heterogeneous population of MSCs can be separated from non-Muse cells. We hypothesized that human adipose derived stem mesenchymal cells (ADSCs) can be sorted into Muse and non-Muse populations on the basis of SSEA-3 expression using a magnetic cell sorting strategy. To test our hypothesis, cells were magenetically sorted, and the expression of genes associated with enhanced developmental potency was compared between Muse and non-Muse populations of ADSCs using real-time PCR. Our working hypothesis is that Muse ADSCs will exhibit significantly greater levels of expression of genes associated with enhanced developmental potency than non-Muse ADSCs. In addition to gene expression studies, both Muse and non-Muse cells were plated on poly-HEMA plates to assess their ability to form M-clusters – a known characteristic of Muse cells. Future work will include sorting human ADSCs into Muse and non-Muse populations using fluorescence activated cell sorting (FACS), as well as testing the developmental potential of Muse cells in three-dimensional culture systems.
Hunter G. Sellers, Winthrop University
Faculty Mentor: Nicholas Grossoehme. Ph.D.
Iron is essential to an overwhelming majority of life on Earth; however, in aerobic conditions, it can take on multiple oxidation states and create harmful oxidative species that must be regulated to maintain the health of the cell. Many bacteria use one of the common metal regulatory proteins (e.g., FUR) to maintain safe levels of iron in the cell, but genome analysis of S. pneumonia indicates that it lacks any of the standard sensors. Interestingly, the presence of extracellular iron triggers an intracellular uptake response; this process involves three proteins: StkP (membranous kinase), RitR (transcription factor), and PhpP (phosphatase). It is likely that the intracellular iron sensor is linked to this uptake system; in fact, we hypothesize that the intracellular sensor is built directly into this system. Noting that PhpP is a magnesium-dependent enzyme, we hypothesize that perhaps PhpP is activated by intracellular iron in S. pneumonia, thus providing the intracellular iron sensor that it needs. Using a combination of UV-visible and fluorescence spectroscopy, we tested this hypothesis. Using para-nitrophenylphosphate assays (PNPP, a surrogate for phosphorylated RitR) along with manganese as an aerobic condition-friendly surrogate, we demonstrated that PhpP is activated by manganese. Using fluorescence competition experiments with the metal-binding fluorophore Mag-Fura-2, we quantified the affinity of PhpP for manganese (Kd = 2.16 mM) and magnesium (Kd = 185.1 mM). Together, these results support the hypothesis. Future work will focus on testing ferrous iron activation of PhpP.