Biological Sciences
Presenter(s): Lucy Chalekian
Advisor(s): Dr. Douglas Fudge, Gaurav Jain, Matthew Snyder, Andrew Lowe
Hagfishes are a diverse group of jawless marine fishes that are noteworthy for their ability to produce gill-clogging slime when threatened. The slime exudate ejected by the slime glands is made up of two main components: thread skeins and mucous vesicles. Although the biophysical mechanisms of exudate deployment in seawater are not understood, some details are known. Thread skeins must unravel from their coiled state and provide strength to the slime in the form of a network of silk-like threads. Deployment of mucous vesicles is known to involve the swelling of constituent glycoproteins their subsequent deformation into a vast mucous network that interpenetrates the slime thread network. Recent theoretical work suggests that thread skein unraveling would be greatly enhanced under conditions where the skein (or a loose piece of thread) is pinned to a solid surface. We hypothesize that the slime gland pore remains attached to ejected exudate, and acts as an anchor that allows mucus, and skeins embedded within it, to be loaded in tension, which facilitates unravelling and efficient slime formation. We have employed high-speed camera mounted on an Axio Zoom microscope to observe this process in detail. Our preliminary results suggest that hagfish slime exudate indeed requires an anchor for proper skein unraveling and slime formation.
Swelling Kinetics of Fresh Mucin Vesicles Project Proposal
Presenter(s): Anne Kenney
Advisor(s): Dr. Douglas Fudge
Hagfish produce a large quantity of defensive slime when attacked. This slime is a rapid forming dilute hydrogel comprised of two main components, thread filaments and membrane-bound mucin vesicles. Little is known about the behavior of the mucin vesicles immediately after secretion from the slime gland, as all studies on mucin vesicles have used stabilized vesicles. Slime is also formed rapidly after secretion, however the exact speed and kinetics of the gel formation of the slime has not been quantified. In this study I propose that the swelling kinetics of mucin vesicles in fresh exudate happens extremely fast forming the gel seen in slime deployment. This is purely a speculative proposal for future research. Knowing the swelling kinetics gives key insights about slime production and can help describe the process of skeins unraveling in a mucus gel when ejected. To observe the swelling kinetic, high speed video of fresh mucin vesicles being exposed to artificial sea water will be quantitatively analyzed. The change in area of the vesicle when exposed to the sea water will be measured and plotted to determine the rate of expansion. That rate of expansion will give a time frame for the gel formation when slime is ejected. These insights on the mucin vesicle swelling kinetics in fresh exudate will further contribute to research involving the mechanisms for hagfish slime formation.
Identification and Quantification of Secondary Metabolites in Pignut
Presenter(s): Jordan Farmer
Advisor(s): Dr. Hagop S. Atamian, Dr. Matthew Gartner, Dr. Peter Chang
Plants synthesize very diverse types of secondary metabolites throughout their life cycle. These secondary metabolites have specialized functions such as repelling pests and herbivores, attracting pollinators, and playing roles in different ecological functions. Altogether, secondary metabolites help the plant adapt to its specific environment and increase its chances of survival. Ancient records show that humans have been using plant secondary metabolites (commonly known as medicinal plant products) for treatment of diseases and illnesses. Nowadays, there is great interest in identifying functionally diverse secondary plant metabolites since they could aid in drug discovery. In addition, plant secondary metabolites are routinely used in food flavors, fragrances, insecticides, and dyes. The mint plant family (family: Lamiaceae) includes important plants such as basil, mentha, rosemary, sage, savory, oregano, thyme, lavender, and perilla. These plants possess a wide diversity of secondary metabolites which give them their distinctive smells and flavors. The objective of this project is to identify and quantify the secondary metabolites of an understudied plant species within the mint family called pignut (Hyptis suaveolens). The pignut is native to Mexico and South America and has been used in ancient times to treat diseases. The secondary metabolites were extracted from leaves of different wild pignut plants grown in our greenhouse using steam distillation method. The analysis of the extract was performed on a Gas Chromatograph Mass Spectrometer (GC-MS) instrument to identify the different compounds and analyze their concentrations. Our analysis showed variation in the quantities of some metabolites among the different wild plants. It would be interesting to further investigate the roles of those metabolites in plant adaptation as well their potential in medicine. The results generated in this project will provide valuable resources to future research aimed at utilizing the diversity of the pignut secondary metabolites for human well-being.
Screening for Salinity Tolerance and Weed Suppression Ability in Different Hairy Vetch Accessions
Presenter(s): Nina Rodricks, Kiana Saleminik, Alise Maripuu
Advisor(s): Dr. Hagop S. Atamian
Hairy vetch (Vicia villosa Roth) is a winter annual legume that is very frost tolerant and is grown as a cover crop during fall, winter, and early spring. Being a legume, hairy vetch fixes atmospheric nitrogen through its symbiotic association with soil rhizobium bacteria. Consequently, when planted during off season, hairy vetch enriches the soil quality and nutritional status resulting in higher yields in the following summer annuals such as corn, cotton and many vegetables. In addition, hairy vetch suppresses weed populations by producing abundant biomass and effectively competing for essential resources such as light, water and nutrients. Another mechanism of weed suppression is through production of chemicals that either inhibit weed seed germination or reduce weed growth through phytotoxicity. While hairy vetch is adapted to all soil textures, it is very sensitive to soil salinity. This limits its widespread adoption. In addition, its weed suppression ability is variable and depends on the plant genotype and the environmental conditions. The two objectives of this project is to 1) identify salt tolerant hairy vetch accessions by screening a panel of wild populations and 2) better understand the mechanisms of weed suppression. Seeds from nine wild hairy vetch accessions were grown between November and March in pots. Plant biomass, seed weight, and seed number data was collected. The plants were also subjected to salinity conditions and their performance evaluated. Finally, leaf and stem sap were extracted from the different accessions and tested for their ability to suppress the germination and growth of two weed species. Our results show differences between the nine hairy vetch accessions in terms of their salinity tolerance and weed suppression potential. These results will be used in future efforts to better understand the molecular mechanism of the salinity tolerance and identify the chemicals in hairy vetch that are responsible for suppressing weed growth and germination.
Genomic Correction of Pompe Disease Knock-in Mouse Myoblasts via CRISPR-Cas9 Homology-directed Repair
Presenter(s): Emilie Sandfeld
Advisor(s): Lindsay Waldrop
The goal of this study is to optimize CRISPR-Cas9 homology-directed repair (HDR) strategies to evaluate in vitro efficacy of genome correction in knock-in models of infantile-onset Pompe disease (PD). To accomplish this goal, we used a CRISPR-Cas9 knock-in system targeting the Gaa gene to introduce the known infantile-onset PD orthologues – Gaac.1826dupA (p.Y609*) or Gaac.1935C>A (p.D645E) - into C2C12 mouse myoblasts. We confirmed the molecular and biochemical analogy of our Gaac.1826dupA and Gaac.1935C>A knock-in myoblasts to PD by measuring GAA mRNA expression and enzymatic activity as well as glycogen accumulation. Next, for each Gaa knock-in line, we screened 6 CRISPR single-guide RNAs (sgRNA) and their respective single-stranded oligo DNA nucleotide donors (ssODNs) via nucleofection-mediated ribonucleoprotein (RNP) delivery. We then chose the top sgRNA candidates – with the highest levels of on-target nuclease activity and HDR template integration – to subclone into GFP-tagged CRISPR-Cas9 expression vectors (pX458). Lastly, we nucleofected CRISPR sgRNA-containing pX458 vectors with their respective ssODNs into knock-in myoblasts and used fluorescence-activated cell sorting to isolate GFP-positive cells. Overall genome correction efficacy was determined by TIDER (Tracking of Insertion, DEletions, and Recombination events) analysis. As determined by this study, the optimal genomic correction strategy will be used for future work to isolate, expand and characterize genome-corrected Gaa knock-in myoblasts prior to evaluating its therapeutic potential in an in vivo model system.