2018 Research Projects

Research Project Descriptions

Investigating the Mouse Enteric Nervous System in Three Dimensions Using Optically Cleared Tissue

The enteric nervous system (ENS) is a complex autonomic network of neurons and glia that surrounds the gastrointestinal (GI) tract and has a profound impact on all digestive processes. Despite the importance of the ENS in motility and nutrient absorption and secretion, much remains unknown about its spatial organization and particular functions. The ETS Variant 1 (ETV1) transcription factor is expressed in ENS neurons all along the GI tract. Mice in which the Etv1 gene is knocked out are unable to properly form fecal pellets. However, the influence of ETV1 on neuronal circuit organization and function remains unknown. iDISCO is a recently developed solvent-based optical clearing technique that allows for 3D imaging of tissue. Here we test whether iDISCO is a workable clearing method for investigating the mouse ENS. Moving forward, we intend to use this 3D imaging approach to study the organization of the ENS in Etv1 mutant mice to better understand the impact of the loss of ETV1 on ENS circuitry.

YeastML: Machine Learning for QTL mapping in S. Cerevisiae

Quantitative Trait Loci (QTL) mapping is a valuable tool for determining the genetic causes of complex phenotypes by identifying specific loci that are correlated with phenotypic change. However, the loci identified by such studies often only explain a small fraction of the predicted heritability for the trait in question. The source of this “missing heritability” is an important question in quantitative genetics, and we are addressing this issue by using machine learning rather than the traditional linear regression model to identify QTL for colony size in S. Cerevisiae. We hypothesize that one of the sources of missing heritability is the fact that linear regression cannot take into account epistatic interactions between QTL. By using a nonlinear model such as a neural network, we hope to explain a larger portion of total heritability.

Efficient Cell Penetrating Chaperone Reagents to Regulate Mutant Huntingtin Protein Aggregation

Huntington’s Disease, a chronic neurodegenerative disease, is caused by mutant Huntingtin, a protein with abnormally long polyglutamine stretches that are prone to insoluble aggregation. Nuclear aggregation is the target for many developing therapeutic strategies, with molecular chaperones such as TRiC showing to modulate and mitigate toxic protein aggregation within cells. ApiCCT1, the apical domain protein subunit for TRiC, sufficiently interacts with mutant Huntingtin monomers to prevent protein aggregates while also penetrating the nuclear membrane where most toxic protein aggregation occurs. However, ApiCCT1 is very unstable, making it difficult to synthesize and purify efficiently. ApiCCT1 coupled with GFP allows for added stability for efficient protein delivery while also allowing for a fluorescent tag during experimentation. However, the negative surface charge and size of ApiCCT1-GFP hinders its exogenous delivery. Therefore, we will perform genetic and chemical modifications to allow for more efficient protein delivery while retaining the protein’s native functionality. Modified ApiCCT1-GFP will be used to characterize mutant Huntingtin aggregation in cells. The technology can also be applied to more biologically relevant cell cultures and demonstrate therapeutic targets for neurodegenerative disease research.

Reconstitution and Characterization of Orphan Assembly Line Polyketide Synthases

Many important polyketide natural products are produced through a process similar to fatty acid metabolism via a class of molecular assembly lines called polyketide synthases (PKSs). PKSs comprise a class of enzymes that acts on (methyl)malonyl substrates to produce structurally and pharmacologically diverse secondary metabolites that often have clinical or agricultural value. The availability of large sequencing databases has allowed for the detection of PKS clusters within genomes of many organisms, and in turn this process has accelerated discovery of bioactive natural products; however, the majority of PKS clusters are “orphan” PKS clusters which are currently uncharacterized in terms of their biological role and product. One mission of the Khosla Lab is to work toward characterizing these orphan PKS clusters. This particular project aims to characterize the product and understand the purpose of PKS clusters found within Azospirillum and Nocardia genuses through in vitro and in vivo reconstitution of the corresponding PKS modules, followed by chemical assays to determine resultant metabolites. Characterization of such orphan PKS clusters could lead to the discovery of novel antibiotics or agriculturally useful compounds, and it may also provide insight into an organism’s mode of pathogenicity.

Determining the Response of the Mu-Opioid Receptor to Ligands of Different Bias using Single-Molecule Förster Resonance Energy Transfer

Opioid receptors are members of a receptor super-family called G-protein coupled receptors (GPCRs), and are key targets for pain management drugs. GPCRs have seven transmembrane alpha helices, the sixth of which (TM-6) is known to move outward upon drug activation of the receptor.  While this motion creates a cavity for proteins in the cytoplasm, such as G proteins and arrestins, to bind to the receptor, the mechanism by which the receptor becomes biased for selecting one type of protein over another is poorly understood.  We synthesized fluorescently photostable dyes into probes that were inserted on TM-6 to measure changes in the position of TM-6 on a millisecond timescale using the technique of single-molecule Förster resonance energy transfer.  Future experiments seek to determine important conformational differences in the receptor that provide new insights into receptor bias. 

Phenotypic Cancer Response to Organ-Specific Extracellular Matrices

Metastasis, the process by which cancer spreads through the body, causes ninety percent of cancer-related deaths. The preference for cancer cells to metastasize to certain organs has been shown to depend on differences in the tissues’ microenvironments, but this relationship is not well characterized. To better understand the impact of the tumor microenvironment, especially the role of extracellular matrix (ECM) composition, on cell cluster malignancy, this project uses a decellularization and digestion protocol to isolate specific tissues’ ECMs and reconstitute them into hydrogels for 3D culture of organotropic breast cancer cell lines. The growth phenotype of these different cell lines in the tissue ECM hydrogels will be visually determined and then confirmed by staining for phenotype-characteristic biomarkers. Finally, the cellular microenvironment will be characterized by testing the physical properties and protein composition of the various tissue ECMs. Examining how differences in ECM composition affect the phenotype distribution of cells will provided a greater understanding of the factors that determine metastasis sites, which could in the future lead to the development of an assay that could identify probable metastasis sites for an individual patient’s cancer cells and allow physicians to develop more effective treatment plans.

Impact of Peripheral Cancer on the Brain

Among cancer patients, the neurological complications from chemotherapy, known as “chemobrain”, has been well studied. However, the link between cancer in the body and cognitive decline is not well understood. We examined the impact of melanoma on the brain by implanting B16-F10 cells in the flank of mice and quantifying inflammation and neurogenesis in the brains of mice with melanoma tumors. We used luciferase expressing B16-F10 cells to track the melanoma tumors and to ensure no metastatic growth on the brain. Moving forward, we will perform proteomic analysis on the blood plasma and tissues to look for secreted proteins from the tumor cells.