April 10, 2015

Characterization of CP43 and PIF1 Genes in Medicago truncatula

Nathan Henning '15

Medicago truncatula is a model organism used to study the symbiotic relationship between plants and the nitrogen fixing soil bacteria Rhizobia. By studying the genes that are involved in this symbiosis the goal is to transplant the symbiotic relationship into agriculture staples (e.g. corn, wheat, etc) that do not utilize similar symbiotic relationships with nitrogen fixers. This would help to reduce our reliance on nitrogen based fertilizers. To find plant genes that might be involved in regulating this relationship, a large-scale Genome Wide Association Study (GWAS) was used. The purpose of this study was to look at two of the genes found by this GWAS. One of these genes codes for a PIF1-like DNA helicase, while the other codes for a CP43 Chlorophyll Apoprotein. In order to study and characterize these two genes, hairpin RNA constructs (hpRNA) were inserted into Medicago truncatula to reduce expression of the two candidate genes. HpRNA constructs were successfully inserted into Medicago truncatula and reduced expression of the two candidate genes by 80-85%. We found that there were no significant physical phenotype changes for height of the plants or number of nodules on the plant roots in comparison to unmodified plants for either hpRNA construct. However, preliminary results indicate that reduction of CP43 gene expression with the hpRNA construct alters the strain of Rhizobia that nodulates the Medicago roots. This indicates that the CP43 gene may play some role in the strain specificity of the symbiotic relationship between Medicago truncatula and Rhizobia. Further research is being done using a CRISPR construct that will eliminate rather than reduce gene expression.

The Significance of CUL3 Knockout on Human Colorectal Cancer Cells

Zoe Lautz '15

Cancer, the second leading cause of death in the US, is caused by mutations in select genes that alter cellular function leading to uncontrolled proliferation. Understanding the specific genes that drive cancer can lead to the generation of novel cancer therapies. To identify novel genes that drive colorectal cancer (CRC) in mice, Starr et. al. employed a transposon-based insertional mutagenesis system. One of the genes identified, APC, is mutated in 70-80% of human CRCs. In addition to genes that are known to cause CRC, 77 novel genes were also identified. One of those genes, CUL3, was analyzed for its role in a human CRC cell line in this study. CUL3 gene knockout was performed using the CRISPR/Cas9 system, which targets mutations to specific genes, thereby knocking out that gene’s function. Three different sites in the CUL3 gene were targeted for mutation and resulted in the creation of 41 separate cell lines with potential CUL3 knockout. Of those 41 cell lines, 25 exhibited qualitatively abnormal phenotypes 10 days after transfection. These phenotypes include slowed growth (25 of 25 cell lines), increased cell size (16 of 25 cell lines), and variation of cell adherence to culture flask surface (11 of 25 cell lines). Knockout was confirmed in 6 cell lines by using PCR in the region of the gene targeted for mutation and sequencing the PCR product. Each cell line was quantitatively evaluated for metabolic activity (or cell growth rate) using an MTS assay. If CUL3 knockout is shown to reduce overall cell growth and increase susceptibility to chemotherapy, this would support the development of new therapies for CRCs that target CUL3 function.

nab-Paclitaxel plus Gemcitabine as Treatment for Metastatic Pancreatic Cancer

Gina Groshek '15

Pancreatic cancer will affect 1 in 67 people in their lifetime, resulting in nearly 49,000 new cases and 40,560 deaths annually. Because early pancreatic cancer does not display symptoms, it often goes undiagnosed until it has metastasized, receiving a Stage IV diagnosis. The five year survival rate for Stage IV exocrine pancreatic cancer is only 1%. The goal of treatment is often geared towards stopping the progression of cancer and treating the symptoms to allow a few more months of survival for the patient. Combination treatment including gemcitabine have been unsuccessful in significantly increasing life expectancy after diagnosis with acceptable adverse effects. A phase 1/2 trial showed positive results from nab-paclitaxel plus gemcitabine to treat and prolong life in pancreatic patients. This phase 3 clinical study tests the efficacy and safety of this combination therapy, as well as the overall survival, progression-free survival, and response rate. Patients from 11 countries and 151 testing facilities were randomly assigned to receive 1000mg/m2 of body-surface area gemcitabine once a week for 7-8 weeks, then on days 1, 8, and 15 every four weeks, or to receive 125mg/m2 of body-surface area nab-paclitaxel followed by 1000mg/m2 of body-surface area gemcitabine on days 1, 8, and 15 every four weeks, receiving treatment until disease progression. Tumors were assessed every 8 weeks using spiral computed tomography or magnetic resonance imaging. The results of this clinical trial indicate that nab-paclitaxel plus gemcitabine increased overall survival from 6.7 months to 8.5 months (p<0.001). Survival rates at 1- and 2-years were also significantly increased with the combination treatment (p<0.001 and p=0.02, respectively). Progression-free survival increased and response rates tripled in patients receiving the combination therapy. Patients experienced some increased adverse events, though these were manageable. Based on the efficacy and acceptable safety of this combination therapy as determined in this study, FDA approval should be sought to provide an opportunity for metastatic pancreatic adenocarcinoma patients to increase overall and progression-free survival.