Yue Chen - US grants

[unreadable] DESCRIPTION (provided by applicant): Unique characteristics of the C. perfringens enterotoxin (cpe) promoter and the biology of the C. perfringens type A has made it possible to deliver a large amount of foreign protein to the terminal ileum (a major location of the mucosal immune inductive sites in gastro-intestinal tract) and therefore, could be used as a general oral delivery vector of antigens. However, there are two potential problems with this delivery vector. First, C. perfringens type A produces two extracellular toxins, alpha toxin (pic) and theta toxin (pfoA) which could cause gas gangrene and pose a potential danger when used as a vaccine vector. Secondly, the C. perfringens expressed foreign antigen from a plasmid has potential problems related to instability and transferring a plasmid-encoded antibiotic resistant gene in the environment. Our hypothesis is that a pic /pfoA' mutant of cpe-negative C. perfringens, with the capacity to produce a high level of foreign proteins from its chromosome without antibiotic selection, will be an ideal vector to deliver the antigen to the immune inductive PPs in the GALT. The overall objective of this proposal is to construct a safe recombinant C. perfringens type A that is incapable of producing toxins and stably expresses high levels of foreign protein gene without involvement of antibiotic resistant gene. This will be accomplished by a) knocking out the chromosomal pfoA gene of a cpe negative ATCC 3624 C. perfringens type A using a mobile group II target-tron method; b) inserting a foreign gene cassette into the chromosomal pic gene of the pfoA' mutant by the target-tron methods to create a pIc'/pfoA' mutant and measure expression of the foreign protein from the chromosomal DNA in vitro and in vivo. Successful completion of this study will create a safe, low-cost and efficient oral delivery vector for vaccine and therapeutic agents. [unreadable] [unreadable]

Project Summary Mounting evidence have demonstrated proline hydroxylation (Hyp) as a fundamental posttranslational modification that are highly responsive to the changes in cellular metabolic environment. During cancer development, rapid proliferation of cancer cells in solid tumors suffers from limited oxygen supply. The hypoxia microenvironment prevents its hydroxyproline-dependent degradation of HIF? proteins and activates hypoxia- response cellular pathways that promote cancer cell survival in hypoxia. In addition to oxygen, the regulatory enzyme prolyl hydroxylases are also sensitive to the concentration of iron and key mitochondria metabolites including succinate, fumarate and alpha-ketoglutarate, making the pathway a critical metabolic sensor in cells. Extensive studies have demonstrated that proline hydroxylation of substrate proteins regulates protein-protein interactions or substrate protein degradation. Despite of its important roles in cell physiology and success in targeted analysis of individual substrates, system-wide characterization and functional quantification of the pathway have been hindered by the lack effective tools and strategies for global site-specific identification of proline hydroxylation targets. Our overall hypothesis and long-term goal is that systematic characterization of ?proline hydroxylome? through the development of functional proteomics approaches will lead to mechanistic understanding of the novel Hyp-mediated metabolic regulations in development and diseases. To achieve this goal, we have developed and applied an immunoprecipitation-assisted strategy for global identification of proline hydroxylation targets. With this strategy, we will tackle the challenge of systematic discovery and quantification of proline hydroxylation proteome. We will develop new quantitative proteomics workflows and apply the strategies for the identification and validation of endogenous prolyl hydroxylase targets. Using temporal dynamics analysis, we will also reveal the target proteins that are subject to Hyp-dependent protein degradation. Integrated data analysis will reveal the regulatory enzyme of the novel Hyp substrates and therefore enable confident validation as well as functional characterization. In addition to the target-specific degradation, our preliminary proteomics analysis showed that proline hydroxylation may regulate global protein homeostasis through the regulation of proteasome activities. We will develop endogenous model systems and novel quantitative mass spectrometry technology to determine the functional significance of proline hydroxylation on proteasome subunits and how such regulation affect global protein homeostasis. Overall, we anticipate that the development and application of functional proteomics technology for system-wide analysis of proline hydroxylation targets will reveal novel metabolic-sensing pathways and potentially lead to paradigm- shifting concepts in the fields of cancer, metabolic diseases and aging.

ABSTRACT The objective of this proposal is to create and validate a minimally-invasive Magnetic Resonance (MR) - compatible concentric tube robot for Intracerebral Hemorrhage (ICH) evacuation. About 1 in 50 people suffer from ICH in their lifetime. ICH occurs when blood leaked from a ruptured vessel accumulates and forms a blood clot (hematoma) in the cerebrum. The 30-day mortality for ICH is about 40% with half of all deaths occurring in the acute phase, especially in the first 48 hours. Blood spilled outside of the intracranial vessels is toxic to surrounding neurons, causing inflammation and perihematomal edema seen as early as 12-24 hours after hemorrhage. These deleterious effects motivate emergent treatment to save at-risk brain tissue. We propose three specific aims as follows. Specific Aim 1: Create the Robot. We will optimize the current robot prototype design to construct a new, compact, and precise MRI-compatible robot to deploy the aspiration cannula within the MRI scanner. We will develop the robot path planning algorithm and embedded electronics to enable accurate robot control. Specific Aim 2: Enable Image Guidance. We will track the aspiration cannula in MRI space by integrating wireless MRI microcoils on the cannula. Real-time MRI will allow the surgeon to monitor the cannula position and hematoma-brain boundary during the procedure. We will create a navigation workstation to control the robot, enable treatment monitoring including diffusion tensor imaging, and provide a virtual boundary to enhance safety. Specific Aim 3: Experimental Validation. We will first characterize robot targeting accuracy and its path- following ability in the benchtop environment. We will then perform phantom studies in a 3T MRI scanner to evaluate system accuracy and optimize the navigation workstation. We will conclude with four sheep studies to evaluate the system with realistic tissue properties, perfusion, and other relevant biological effects. We will collect robot accuracy data, quantify hematoma evacuation percentage, and determine damage to healthy brain tissue.