We synthesized a recombinant version of Mimivirus (MV) DNA topoisomerase 1B protein possessing an unusual 5'OH ligation activity (MV-ligase). This permits a novel type of probe for the detection of apoptotic cell clearance. In this project w will develop such new fluorescent probes for in situ research. The probes will visualize only those active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells) which engulf and digest apoptotic cell DNA. At the subcellular level, the probes will be the first specific markers of activated phagolysosomes - cellular organelles performing the actual phagocytic degradation of apoptotic cell corpses. Although the phagocytic phase is critical for efficient execution of apoptosis, there are currently no in situ probes specific for phagolysosomes digesting apoptotic cells. The project will close this technological gap and will provide a useful molecular tool. It will create an advantageous product with wide applicability in studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders. The Specific Aims of the proposal are: 1) to develop the MV-ligase-based probes that will selectively label phagolysosomes of the cells that engulfed apoptotic nuclei and to test the probes using in vitro models. 2) To develop the first in situ assay for targeted detection of apoptotic cell clearance in tissue sections and to test and optimize its sensitivity, specificity and applicability in several apoptotic models.
Public Health Relevance Statement: Public Health Relevance: The proposed project will result in the development of a new nano-scale technology for the needs of medical diagnostics and pathology. The technology will advance molecular imaging and will provide new enabling tools for molecular pathology and cell biology. It will allow precise evaluation of the effects of therapy in diseases such as various cancers, as well as stroke and Alzheimer's disease.
Project Terms: Acanthamoeba; Alzheimer's Disease; Apoptosis; Apoptotic; Area; Attention; base; Biochemical; Biological Assay; cancer therapy; Cell Nucleus; Cells; Cellular biology; Characteristics; Cytoplasm; Deoxyribonucleases; design; Detection; Development; Diagnostic; Disease; DNA; DNA Topoisomerases; DNA Viruses; Enzymes; Evaluation; Fluorescent Probes; Formaldehyde; Freezing; Generations; Goals; Imagery; Immune System Diseases; In Situ; in vitro Model; in vivo Model; Infection; Inflammation; innovation; Label; Ligase; Ligation; Malignant Neoplasms; Measures; Medical; Methods; Modeling; Molecular; molecular imaging; molecular pathology; nanoscale; Noise; novel; Organelles; Outcome; Paraffin Embedding; Parasites; Pathology; Phagocytes; Phagolysosome; Phase; Preparation; Property; Proteins; public health relevance; Recombinants; Research; research study; Resolution; Sampling; Sensitivity and Specificity; Signal Transduction; Site; Source; Specificity; Spottings; Staining method; Stains; stroke; Structure; Surface; Suspension substance; Suspensions; Technology; Testing; Tissues; tool; Topoisomerase; Virus; Work
