CELLULAR AND MATERIALS-BASED STUDIES OF MARINE INVERTEBRATES TO ADVANCE BIOMINERALIZATION, ANTIFOULING AND NANOTECHNOLOGY FIELDS
|Principal Investigator||A. S. Mount|
|Relevance to Implications||Substantial|
|Class of Nanomaterial||Engineered Nanomaterials|
|Broad Research Categories||
|Anticipated Total Funding||n/a|
|Anticipated End Year||2011|
NON-TECHNICAL SUMMARY: We will develop technologies enabling sustainable use and management of aquatic and marine ecosystems. Toxic coatings are now used to prevent fouling on ship hulls and corrosion on aircraft. These antifouling chemicals on hulls, docks and offshore platforms threaten sea life and commercial catches of fish and shellfish. Disposal of aircraft anticorrosion coatings contaminates fresh water, thus affecting the environment, agriculture and drinking water supplies. For ships, both naval and maritime, we seek to replace the use of toxic copper antifouling paints with coatings that incorporate naturally occurring bio-molecules, which are safe and non-toxic. We are also investigating cellular based ceramic coatings for aircraft which may eliminate the use of highly toxic chromium coatings. Biomineralization remains one of the least understood areas of biology. The oyster has proven to be an ideal model to advance the basic science of calcification at a tissue, cellular, and molecular level. Our basic research about cellular mineralization mechanisms will be applied to the next generation of antifouling coatings for ships, and anticorrosion coatings for the aerospace industry. The evaluation of the safety nanotubes is crucial for the future success of nanotechnology. OBJECTIVES: 1.Validate our pioneering model (see Science 304:297-299) of cellular mediated calcification in bivalve mollusks. 2.Develop a safe and benign antifouling strategy that will disrupt larval settlement of marine surfaces through co-option of cellular signal transduction mechanisms. 3.Ascertain the safety of nanomaterials through toxicological study and cell biological investigation of lysolipid solubilized single walled nanotubes (LL-SWNTs). APPROACH: For cellular mediated calcification in bivalve mollusks: Collagen will be extracted and characterized from oyster tissues by a pepsinogen extraction technique followed by PAGE and western analysis. PCR primers prepared from MS peptide sequences will be used to obtain full length cDNA sequences using the RACE technique. Oyster microarrays will used to validate key signal transduction pathways. Hemocytes are collected before and after the induction of the biomineralization response. For SEM imaging hemocytes are incubated 20 minutes on glass and modified surfaces and fixed with 4% fresh paraformaldehyde solution and osmium tetraoxide then sputter coated. Alternatively, acetone washed glass cover slips are placed inside the shell forming region of the whole animal, in contact with the shell organ of the mantle tissue. The slips are collected at various time points and prepared for SEM analysis. Crystal diffraction studies are performed on polished specimens by EBSD. For antifouling studies of adult and larval marine invertebrates: Laser scanning confocal microscopy (LSM) and SEM imaging of blood cells from adult organisms (oysters and barnacles) which will be examined for evidence of focal adhesion formation (by vinculin), actin stress fibers (by phallodin) and the presence of NA receptors (by NA receptor antibodies). Apoptosis in control cells will be ascertained by several techniques, including detection of changes in DNA, a cytochrome C assay and detection of changes in plasma lipid membrane composition in living cells. Adhesive cellular assays will be tested on both control and engineered thin polymer films. Live whole organism larval assays (oysters and barnacles) will be performed using a Zeiss stereo Lumar fluorescence stereomicroscope by directly labeled fluorescent markers The larval cellular attachment process will also be examined in detail by SEM and LSM using fixed specimens and labeled gold antibodies and cellular markers. For cellular and whole organism response to nanomaterials (NM): Living Intact organisms (Daphnia and fat head minnow fry) will be examined by LSM followed by confirmatory studies using SEM and TEM. The organisms will be fixed for EM studies as described above. The onset of apoptosis, which is a direct measure of NM induced cytotoxicity in D. magna cells will be determined using a BrdU TUNEL assay and will be verified by the detection of phosphatidylserine in cellular membranes. Both living and fixed specimens of Daphnia will be tested.