Current Pilot Projects

Urara Hasegawa, Assistant Professor of Chemical Engineering, Kansas State University

Mentor: Stevin Gehrke, Fred Kurata Memorial Professor of Chemical Engineering, University of Kansas

Project Title: Nanoparticle Platform for Site-Specific Delivery of the Gasotransmitter Hydrogen Sulfide

Project Summary
Hydrogen sulfide (H2S) has emerged as an important signaling molecule in many diseases including cancer, cardiovascular diseases and neurodegenerative diseases. However, the mechanisms underlying the pathological functions of H2S has not been fully understood mainly due to the lack of a delivery technology that enables to supply a controlled amount of H2S to disease sites for desired period of time. The long-term goal of this research is to better understand the H2S biology involved in a variety of disease pathways including detailed molecular mechanisms as well as the effective H2S concentrations and exposure duration required for specific biological functions. The overall objective of this project is to develop a H2S delivery platform that enables disease-site specific H2S delivery at a controlled rate. The central hypothesis is that disease site-specific H2S delivery will be achieved by designing polymeric nanoparticles that release H2S in response to disease-specific enzymes. The rationale underlying the proposed research is that, the H2S delivery technology proposed in this project will be an innovative tool to investigate the pathological roles of H2S in disease-related cells. The hypothesis will be tested by pursuing two specific aims: 1) Development of enzyme-responsive H2S donor micelles; and 2) Development of enzyme-responsive H2S donor hydrogen nanoparticles. For the first aim, polymeric micelles, supramolecular assemblies of amiphiphilic block copolymers, will be formulated as a scaffold material having H2S donors which enables flexible material design to control release kinetics, stability of the nanostructure and interaction with enzymes. Under the second aim, hydrogel nanoparticles (nanogels) will be chosen as a scaffold material having H2S donors which enable sensitive response to increased levels of enzyme to release H2S at disease sites. Nanogels are composed of a crosslinked hydrated polymer network, which is expected to facilitate interaction with disease-specific enzymes. The proposed approach is innovative, because it integrates both a macromolecule-based H2S delivery system and a stimuli-responsive system that respond to disease-specific enzyme. This research will vertically advance the fundamental knowledge of pathological roles of H2S by clarifying the effective concentration and duration of H2S in diseased tissues. Ultimately, such knowledge has the potential to contribute to development of an innovative approach for the prevention and treatment of a variety of diseases.

Erik Holmstrom, Assistant Professor of Molecular Biosciences, University of Kansas

Mentor: Mark Richter, Professor of Molecular Biosciences, University of Kansas

Project Title: Inhibition of hepatitis C virus nucleocapsid-like particle assembly

Project Summary
Viruses are the most numerous biological entities on the planet and can often be extremely pathogenic and globally concerning. For example, the hepatitis C virus (HCV) is a widespread pathogen that infects more than 1 in 50 people worldwide and is responsible for a wide range of progressive hepatotropic diseases. Fortunately, nucleocapsid assembly is a fundamental biochemical pathway in the viral life cycle that can be exploited by anti-viral therapies. HCV nucleocapsid formation is driven by interactions between multiple copies of the multifunctional core protein (HCVcp) and the genomic RNA. Additionally, HCVcp is the most conserved of all 10 HCV proteins. For these reasons, the HCVcp represents a promising therapeutic target that has not yet been explored because many critical structural details associated with the assembly pathway remain unknown. Therefore, this proposal aims to provide a biophysical description of the HCV assembly pathway by studying the RNA-induced formation of nucleocapsid-like particles (NLPs). Using a wide range of single-molecule techniques, including a single-molecule FRET-based microfluidic mixing device, we will develop a structurally-motivated kinetic model for this process. Such a model will then be used to identify the mechanism of action associated with a variety of small molecule and peptide-based therapies reported to inhibit formation of multimeric assemblies of the HCVcp.


Past Pilot Projects

Mei He, Assistant Professor of Biological & Agricultural Engineering, Kansas State University (2016-2017)

Project Title: Microfluidic engineering of immunogenic exosomes for personalized cancer vaccine

Project Summary
Exosome, as a natural and safe therapeutic delivery system, is emerging in cancer immunotherapy, yet hard to harvest as a pure, immunogenic population. Consequently, co-purified exosome subtypes and extracellular microvesicles could confound our understanding on activation of immune pathways and effective cancer vaccination. This project specifically addresses technology challenges by introducing a lowcost microfluidic approach for large-scale production of clinical-grade immunogenic exosomes, and examining the roles of immunogenic exosomes in MHC-I antigen presentation pathway and antitumor responses. The study focuses on the immunogenicity of peptide-engineered exosomes in antitumor responses. The long term goal is to establish an enabling strategy for studying immunogenic roles of variable peptide-engineered exosomes in cancer immunotherapy and developing personalized cancer vaccines. Microfluidic high-throughput isolation of MHC-I specific exosomes will be uniquely streamlined with vesicular loading of antigenic peptides, and subsequent light-triggered release of intact, engineered exosomes in continuous-flow. The engineered immunogenic exosomes will be assessed to the degree of potency and activity in stimulating immune pathways that are critical for an effective antitumor response in vitro (e.g., CD8+ cytotoxic T cells and CD4+ T helper cells stimulating). Outcomes will increase understanding of fundamentals and roles of variable peptide-pulsed immunogenic exosomes in immune system, provide well defined models for in vivo study of immunogenic exosomes in tumor microenvironment, and gain knowledge of cell-free cancer vaccination system for designing personalized cancer vaccines.

J. Christian Ray, Assistant Professor of Molecular Biosciences, Center for Computational Biology, University of Kansas (2016-2017)

Project Title: An integrative platform for cell-resolution analysis of the acute-to-chronic transition in bacterial pathogens

Project Summary
This pilot project will develop a new method to understand how pathogens form chronic infections. Many experiments in model bacterial organisms, especially Escherichia coli, have provided tantalizing clues about how bacteria can resist the assaults of antibiotic treatment. In our emerging picture of bacterial robustness, it appears that colonies can transition into a slow-growing state that creates a stubborn infection. Recently, researchers have discovered that cellular lineage (that is, non-genetic inheritance of cellular contents) and the interaction of many different similar growth-regulating systems at once together create specific statistical patterns of heterogeneous robustness to antibiotic treatment. Ultimately, researchers believe that these factors are contributing to the acceleration of antibiotic resistance, among the most dangerous of emerging medical crises today. One of the worst offenders of antibiotic resistance is the most common cause of urinary tract infections, known as uropathogenic E. coli, or UPEC. The goal of our pilot project is to test the feasibility of a new, integrative method for understanding how heterogeneity in UPEC colonies contributes to its ability to form chronic infections. Our strategy is to create a microfluidic device that allows us to monitor, and capture, lineages of UPEC that can be subjected to global gene expression analysis with next-generation sequencing techniques (Aim 1). Because UPEC is closely related to the laboratory E. coli strain, which has had many valuable molecular tools and strains created for it, we will also use our transcriptomic analysis in conjunction with a comparative regulatory network approach to identify targets in UPEC that control its ability to maintain robust chronic infections (Aim 2).

Robert Unckless, Assistant Professor of Molecular Biosciences, University of Kansas (2016)

Project Title: Pathology, host defense and population of Drosophila innubial Nudivirus

Project Summary
DNA viruses are the infectious agents causing innumerable human diseases. Until now, scientists have lacked a natural model of DNA virus infection in Drosophila. The Drosophila innubila nudivirus (DiNV) and other nudiviruses are related to baculoviruses which primarily infect insects. They are large, both in size and gene content (>100 genes) and tend to be highly virulent. DiNV was detected in about 40% of wild-caught individuals and infected individuals showed significantly reduced lifespan and fecundity. Our immediate goal is to establish DiNV as a model system for the study of DNA virus infection in Drosophila. The long term goal is to understand pathology, host immune defense and host-virus co-evolution using the DiNV system. Our main objective is to gain enough knowledge about these aspects of the DiNV system to develop important, testable hypotheses for further research. Though the purpose of the grant is largely exploratory, we hypothesize that host defense against DiNV will be quite different from that of RNA virus infections and that we will uncover previously unknown pathways in the innate immune response. Our approach is to establish the virus in cell culture, then perform experimental infections to better understand pathology, virulence and host immune response. We will also study host-virus co-evolution in natural populations using the natural replication found in the Sky Island populations in the desert Southwest.

Justin Blumenstiel, Assistant Professor of Ecology & Evolutionary Biology, University of Kansas (2013-2014)

Project Title: Mechanisms of genome instability induced by transposable elements

Project Summary
Transposable elements (TEs) are selfish replicating elements that comprise about half of the human genome. Due to their proliferative and repetitive nature, they are a significant source of new mutation and chromosomal rearrangement. Studies over the past few years have provided growing evidence for TEs, through their mutator capacity, being an important contributor to cancer. Recent studies have also demonstrated that a genome defense mechanism mediated by small, silencing RNAs (piRNAs), limits potential TE damage by targeting TE transcripts for destruction. Under some circumstances, however, this mode of genome defense fails to control TE proliferation. In Drosophila, the mobilization of one single element family can result in global failure of TE control, chromosome damage and sterility. This syndrome of genome destabilization is known as hybrid dysgenesis. It is not clear how the mobilization of one element family can lead to cascading mobilization of other elements. A critical question is how this mobilization causes the machinery of genome defense by small RNAs to become compromised. The goal of this project is to test two specific models for how the piRNA machinery loses efficacy when TEs become mobilized. One model is that DNA damage itself is directly responsible for TE mobilization. A second model is that, as has been demonstrated with viruses, TEs encode suppressors of small RNA silencing. Thus, when one TE mobilizes, the piRNA machinery becomes directly antagonized, leading to the mobilization of other TEs. By testing these two models, we will provide significant insight into the mechanisms by which genome instability can be induced by TEs. In addition, by testing the TE encoded suppressor model, we may identify new potential mechanisms of oncogenesis.

Marco Bortolato, Assistant Professor of Pharmacology & Toxicology, University of Kansas (2013-2014)

Project Title: Transcriptomic analysis of disease pathways in animal models of Tourette syndrome

Project Summary
Tourette syndrome (TS) is a neurodevelopmental disorder with marked male predominance (M:F=4:1). The disease is characterized by multiple motor and vocal tics, which have a disrupting impact on social and occupational functioning. The current available therapies for TS have variable efficacy and induce significant side effects, highlighting the need for novel treatment and diagnostic biomarkers that can predict treatment response.The objective of this pilot NIH/COBRE grant is to study the molecular bases of the gender differences in TS and the mechanisms of action of finasteride in this disease. Finasteride is the inhibitor of 5J-reductase (5AR), the enzyme catalyzing the conversion of testosterone and other steroid precursors into their neuroactive metabolites. Recent evidence from our group suggests that FIN may be a highly efficacious therapy for TS with limited side effects. The proposed studies will be focused on transcriptomic changes in the animal model of TS with highest degree of homology, the D1CT-7 transgenic mice, which exhibit tic-like manifestations. The results of these studies will set the stage for future large-scale NIH-funded translational studies aimed at the development of new therapeutic strategies for TS with limited side effects.

Bradley Olson, Assistant Professor of Biology, Kansas State University (2013-2014)

Project Title: Multicellular evolution by reprograming cell cycle regulation

Project Summary
The long term objective of this project is to better understand the genetic basis of multicellular evolution. Despite multicellular evolution being a fundamental, and cancer relevant process, the genetic pathways required for multicellularity to evolve are poorly understood. This project will determine the genes important for multicellular evolution in a novel, metazoan relevant multicellular model system, the Volvocine algae, whose members include closely related unicellular and multicellular species. Importantly, the Volvocine algae regulate their cell cycle with homologs of the retinoblastoma (RB) tumor suppressor, where evolutionary changes in its function are linked to multicellular evolution. In Aim 1 of this proposal, cell cycle regulated gene expression will be determined by deep sequencing all messenger RNA (RNA-seq) in unicellular Chlamydomonas compared to multicellular Gonium. Second, RNA-seq will also be performed in Chlamydomonas and Gonium strains lacking RB (encoded by the MAT3 gene) to determine which genes have expression defects compared to wild-type and between the two species. This process will then be repeated in a pseudo-multicellular Chlamydomonas strain caused by the presence of the Gonium RB. In Aim 2, the promoter occupancy by the RB protein in Chlamydomonas and Gonium will be determined by chromatin immunoprecipitation, followed by deep sequencing (ChIP-seq) of RB bound genetic loci. These RB bound loci will be compared between Chlamydomonas and Gonium as well as to the expression data from Aim 1. In summary, this project will make significant advancements in our understanding of the genetic determinants of multicellularity, as well as determine the genome-wide architecture of the RB pathway in unicellular Chlamydomonas compared to multicellular Gonium.

Shenqiang Ren, Assistant Professor of Chemistry, University of Kansas (2013-2014)

Project Title: Single wall carbon nanotube platforms as near-infrared fluorescent sensors

Project Summary
The research objective of this proposal is to establish the single wall carbon nanotubes (SWCNTs) platform for adenosine 5’-triphosphate (ATP) and glucose sensing. Molecular recognition plays an important role in the design of therapeutics and sensing platforms. The research strategy is to explore functionalized single-chirality SWCNT platforms as new type molecular recognition motif, by using a binding pocket or interface to recognize the specific molecule. The project will be comprised of the following interrelated sub-programs: (1) Explore the design rules for building single-chirality SWCNT NIR fluorophore biosensor; (2) Understand the mechanism and relationship between structure and fluorescent emission changes of SWCNT by the target analyte binding, such as the wavelength and intensity changes due to charge-transfer, exciton quenching or solvatochromism. Biosensors based on the modulation of single-chirality SWCNT photoemission will demonstrate real time spatio-temporal detection.


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