5 Apr 2019
Looking for Maize Genes Involved in Cold Response: Producing Knockouts for Arabidopsis Homologs of Maize Candidate Genes Using a CRISPR/Cas9 Approach
Katie Hillmann '19
Most of today’s maize is cultivated outside its original climate zone, where yields are constrained by the changes in climate. Maize is especially vulnerable to high temperatures and drought stress, both of which negatively affect corn yields. An important strategy to combat this is early sowing, which avoids the effects of summer droughts and high temperatures in many places around the globe. However, maize is a cold sensitive species (Sanghera et al., 2011), making improvement to cold stress crucial for its adaption. The relatively new system CRISPR (Clustered regularly-interspaced short palindromic repeats)/Cas9 offers the potential to study cold-stress related genes through targeted mutagenesis. Using an enzyme called Cas9 and guide RNA, scientists can target a specific region in the genome and make a double stranded break so that any DNA can then be added or removed through DNA repair mechanisms (Jiang, Yang, & Weeks, 2014). Our research project aimed at investigating cold response in maize through development of an application that can be used to analyze the function of plant genes. We designed and implemented CRISPR/Cas9 technology on a model organism Arabidopsis thaliana (rockcrest) to knock out eleven plant genes (from the website database arabidopsis.org) that would produce easily distinguishable phenotypic traits once mutated, or were homologs to potential stress candidates in maize. Using published CRISPR/Cas9 protocols (Čermák et al., 2017) we selected appropriate gRNA regions to create approximately 200 bp out of frame deletions in coding parts of the genes and constructed transformation vectors, using golden gate cloning technology. The vectors at each of the cloning steps were analyzed by restriction digests, colony PCR, and sequencing, demonstrating the success of vector assembly. The T0 plants were transformed with T-DNA transformation vectors for select genes and T1 seeds were harvested and screened for transformants. Results showed the success of using CRISPR/Cas9 to create transgenic plants. Further investigation of mutant response to cold stress conditions is necessary to investigate the involvement of maize candidate genes in controlling cold tolerance in maize.
A Possible Radiosensitizer Enhances Response to Radiation Therapy in Solid Tumors by Inhibiting Mitochondrial Metabolism
Julia Frost '19
Hypoxia, a common feature of solid tumors occurs because the supply of oxygen is insufficient to meet metabolic needs of the tumor. Tumor hypoxia causes radiation therapy to be less effective in the treatment of cancer. A paper done by Benej et al.(2018) discusses the importance of effective radiation treatment by increasing tumor oxygenation before radiation therapy. Previous studies have looked at decreasing tumor hypoxia by trying to increase oxygen delivery to the tumors, however this study looks at decreasing the tumor demand for oxygen through the inhibition of mitochondrial respiration. This study proposed the drug papaverine as a candidate for a metabolic radiosensitizer of hypoxic tumors. They picked this drug because it decreased the tumors demand for oxygen by reversibly inhibiting mitochondrial complex 1. This strategy for enhancing radiation therapy will not sensitize normal surrounding tissue making this an effective strategy for killing tumor cells without further damage to healthy tissue. Through this research they were also able to find derivatives of papaverine that have the potential to become radiosensitizers in clinical settings with fewer side effects. Within this study they found that papaverine and its derivatives successfully improves hypoxia in model tumors and enhances response to radiation therapy.
The Role of Serum Cortisol in Assessing Equine Survival
Danielle Meister ‘19
The hypothalamic-pituitary-adrenal axis (HPA) is the neuroendocrine component of the stress response. In this process, the hypothalamus releases the corticotropin-releasing factor (CRF), and when the CRF is bound, it causes the adrenocorticotropic hormone (ACTH) to be released. The ACTH binds to the adrenal cortex receptors, which then produces the release of cortisol. Critical illness-related corticosteroid (CIRCI) occurs when there is a deficiency in the cortisol level. This deficiency can be found in people and animals that have sepsis. Sepsis is a life-threatening condition that is characterized by the imbalance of chemicals released into the bloodstream in response to an infection. This imbalance can cause incidences of shock, multiple organ dysfunction syndrome (MODS), and death. The ability to diagnose CIRCI in these septic patients would allow the intervention of physiological doses of corticosteroids. Blood was taken from young foals (51 septic symptoms and 11 healthy) to get a baseline of the cortisol present. A 10ug and 100ug dose of cosyntropin were administered 90 minutes apart, while cortisol levels were checked at 30, 120, and 180 min points. The blood levels were then analyzed through chemiluminescent and ultrafiltration assays. The assays were used to quantitatively determine the basal total cortisol concentration, the free cortisol fraction, and the total cortisol concentration. After statistical analysis, it was found that foals have unique plasma cortisol binding dynamics and a significantly higher percent of free cortisol and total cortisol concentration in the 5-7 days-old range. There was no significant difference in total cortisol concentration between adult horses and foals 12 hours to 7 days of age. These results do not support the hypothesis of free cortisol concentration being a better indicator than total cortisol concentration in sepsis patients.