Tag Archives: RF2022-Week6

PI: Ru-Rong Ji, PhD; Department of Anesthesiology

Mentors: Aidan McGinnis, Yul Huh

Pain is good when it is short term but is a problem and serves no real purpose once it becomes long term or chronic. A form of chronic pain is neuropathy, or nerve damage, which has no cure nor effective treatment. To develop novel treatments for neuropathy, previous experiments involving a cytokine of interest showed strong pain relief in male mice but not in females. This is a big issue in medicine because studies have been done predominantly in male animals as scientists were not fully aware of sex differences within how the sensory system operates. Previously, experimental data showed that said cytokine’s receptor 1 is more expressed in male sensory neurons—also known as dorsal root ganglion (DRG) neurons—than in female DRG neurons. Currently, immunohistochemistry (IHC) has been used to identify and quantify the cytokine’s receptor 2 in male and female DRG and have showed no sexual dimorphism in expression level. The next step is to identify and quantify the cytokine’s receptor 3 in male and female DRG and potentially in spinal cords as well to identify the means by which the cytokine of interest alleviates neuropathic pain in males but not in females to then potentially create effective nerve pain treatments for both sexes in humans.

PI: Zhiqing Huang, MD PhD

Modern cancer research has been focused on how the tumor microenvironment (TME) affects cancer cell growth. This project’s purpose was to observe how the presence of the periostin (POSTN) protein as well as ascites in the TME affect the chemosensitivity of ovarian cancer cells, with the expectation that their presence would decrease chemosensitivity. This project was conducted by growing two lines of fibroblasts, one with and one without the POSTN gene; this produced medium that both contained and didn’t contain the POSTN protein. Ovarian cancer cells were then grown in this conditioned medium, along with ascites and an assay was performed to determine how growth in ascites and POSTN affected chemosensitivity after chemotherapy was administered to the tumor cells. Preliminary results from this project show that when cancer cells were grown in ascites, the presence of POSTN did decrease chemosensitivity after the administration of chemotherapy. These results support previously conducted research into the effects of POSTN on the chemosensitivity of cancer cell types. These results can be used to provide better therapies for ovarian cancer. Understanding how the TME affects tumor growth can lead to more targeted treatment of ovarian cancer based on what is present in a patient’s TME.

Mentors: Daichi Shonai, Scott Soderling, PhD, Cell Biology

Interrogation of endogenous proteins is pivotal to the study of cell types and disease etiology. CRISPR-Cas9-based approaches allow the insertion of DNA sequences into genes of interest (GOI), providing an opportunity to label endogenous proteins and thus advance proteomics. One such CRISPR-based method is homology-independent universal genome engineering (HiUGE), which utilizes adeno-associated virus (AAV) vectors to deliver both the payload lacking gene-specific sequences and the gene-specific gRNA that integrates the payload into the GOI. While universally compatible with genome targets accessible by CRISPR-Cas9, the question remains of whether a double- versus single-oriented payload or HiUGE donor might affect the target efficiency of this strategy. As such, we transfected NIH3T3-Cas9 cells with different HiUGE donors, conducted immunofluorescence (IF) staining to enable epitope tagging, and quantified tag expression to test the hypothesis that double-oriented HiUGE donors would demonstrate greater or even double the efficiency of their single-oriented counterparts. As we continue comparison between single-oriented HA and double-oriented HA-HA, KI D620N/WT VPS35 and KI WT/WT VPS35, and UltraID and Dual-UltraID, preliminary results show that although the difference across constructs varies, the dual-oriented donors not only successfully integrated despite larger size, but they also correspond to increased expression of tagged endogenous proteins.

Mentors: Jun Zeng, Dr. David Lawrence

The human gut microbiome performs several important services beneficial to host health. In hematopoietic stem cell transplant (HSCT) patients, who often undergo total parenteral nutrition (TPN), this microbiome can become disrupted. However, we do not understand exactlyhow microbiome function is altered. We investigated the consequence of TPN on the gut microbiome’s ability to digest fiber and produce short chain fatty acids (SCFAs) through a fermentation experiment, testing the hypothesis that the level of fiber fermentation is lower in TPN microbiota samples than in normal diet (ND) samples due to microbiome disruption. We introduced inulin to HSCT stool samples and left them to digest for a period of 24 hours. High-Performance Anion-Exchange and Gas Chromatography analyses were used to measure concentrations of inulin and SCFAs, respectively, in the resultant solutions. We found that when introduced to inulin, the TPN samples had a greater fold change in SCFA composition compared to that of ND patients and TBD EFFECT WAITING ON RESULTS on fiber digestion. This analysis suggests that fiber supplementation in TPN patients can aid in reestablishing a healthy gut during and post-treatment and adds to our understanding of dysbiosis in human patients.

Mentors: Naveen Natesh, Sajeesh Kumar, Ph.D., Pankaj Mogha, Ph.D., Shyni Varghese, Ph.D. (Department of Bi0medical Engineering)

Clonal heterogeneity is regularly observed in primary and secondary cancers, but the precise role of each of these cell populations in tumor evolution and clinical prognosis remains poorly understood. Particularly, the functional significance of tumor clones in cancer metastasis is a point of ongoing investigation. Current research suggests the presence of tumor clones with a heightened metastatic potential that are more capable of metastatic colonization. In mouse models of sarcoma, secondary lesions in the lung have been observed to arise from a single metastatic clone, despite cellular heterogeneity in primary tumors and their presence in circulation. This observation could be attributed to the differences in extravasation potential of different cell populations or changes in colonization. We address these questions using an array of in-vitro platforms. First, we examined the changes in extravasation potential using a vascular network model. Next, we examined the changes in adhesion and proliferation of different cell populations by using lung explants cultures and decellularized lung extracellular matrix (ECM)-based cultures. Furthermore, we are quantifying the strength of the cell attachment in the lung ECM using a shear flow device. Characterizing these cellular differences offers the potential to identify specific clonal populations as therapeutic targets at different stages of metastasis.

Bugs as bioindicators: what aquatic macroinvertebrates reveal about ecotoxicology

Rena Ouyang

Mentors: Behrens, Jonathan, Bernhardt, Emily, Ph.D. Department of Biology

Aquatic macroinvertebrates hold high importance in food webs, spending their larval and nymph stages in water before emerging out of the water as adults. Thus, these freshwater organisms can be indicative of water quality and stressors present within their ecosystems. Specifically, the effects of chemical pollution on macroinvertebrates in urban streams begs further research. We hypothesized that as the amount of water pollution decreases, these ecosystems can support higher density, size, and biodiversity. We focused on three sites in Durham, North Carolina over the course of one year: two sites along a creek heavily impacted by development and one reference site in a highly preserved forest. We expected to see variations in macroinvertebrates present with seasonal changes, but we still observed a general trend of biodiversity and size increasing with decreased contamination. The count of organisms yielded more complex results, as more polluted areas in certain months appeared to have higher density. However, when we apply a more holistic and in-depth approach, we consider that this increase in number does not necessarily constitute successful emergence. Therefore, this research suggests that higher water quality encourages more diversity and survival of aquatic macroinvertebrates, and therefore, other organisms in their ecosystems.

Mentors: YingYu Lin, Dr. Sharon Gerecht (Department of Biomedical Engineering)

A new protocol for cell differentiation of human-induced pluripotent stem cells (h-iPSCs) into endothelial cells (h-iECs) has been created to increase efficiency and reduce procedure duration for EC generations. The original protocol acquired twice the amount of time and relied on endogenous ETV2 rather than electroporation, reducing its efficiency. From h-iPSCs to mesodermal cells to h-iECs, this two-stage procedure is the basis of the original and new protocol. The difference lies within the second stage’s differentiation method: mRNA temporal modulation of ETV2 via electroporation. Induction of mRNA strand that encodes for transcription factor ETV2 is introduced through electroporation of mesodermal cells. This new approach should result in about 96% of h-iECs 48 hours after electroporation. Replication of the new protocol will confirm the accuracy of the procedure and provide further research on h-iECs development. With this new protocol, additional research on vascular systems, cell regeneration, and diseases including diabetic retinopathy can be achieved faster and more effectively.

Mentors: Anne Draelos, Ph.D., Liz O’Gorman, John Pearson, Ph.D (Department of Biostatistics and Bioinformatics)

The intersection of neuroscience and advanced computational software opens the door for increasingly complex and integrative experiments. Improv is a software platform that allows users to have flexibility in controlling experiments and provides live data analysis and visualizations. The original visualization of improv works sufficiently, however it lacks accessibility and effortless control from the user. I am developing an updated visualization that addresses these issues and integrates seamlessly with improv. Jupyter Notebook is the base for the visualization, which can easily be accessed through a web browser and gives users control over which plots and visuals are displayed. In order to create the various components of the visualization, data must be loaded in from improv. This process will be facilitated by ZMQ, a program that can communicate between Jupyter Notebook and improv’s data store and data acquirer. After efficiently loading the data, I am developing live updating plots of neural data and images. These plots are built through Python packages such as Matplotlib and jupyterplot, and they will be timed for their efficiency. Key features of the updated visualization include easy accessibility and broader control for experimentalists, thus providing insightful and more intuitive information of ongoing experiments for the user.

Mentors: Chengcheng Du, Shania Appadoo, Pelin Volkan, Ph.D. Department of Biology 

The social environment modulates animal behaviors in many aspects. Studies have shown that male Drosophila raised in social isolation perform differently from those presented in groups in the courtship behavior assay. However, the mechanism under this phenomenon is unclear. The olfactory system is integral to the normal courtship behavior of flies. In Drosophila, pheromones are detected by receptors expressed in sensory neurons. Two of these olfactory receptor neurons (ORNs), Or47b ORNs and Or67d ORNs,  have been found to be involved in regulating male fly courtship behaviors. Therefore, our hypothesis is that the difference between group-housed and single-housed male flies is due to the difference in detecting odors like pheromones in different environments. To test this, we studied if disturbing the detection of pheromones using genetic mutants of Or47b and Or67d will influence the courtship behaviors of male flies through a series of behavioral experiments this summer including isolating flies, group housing flies, and recording courtship behavior. Our findings suggest the influence of social experience on male flies’ courtship behavior partially relies on the normal detection of odors.