Focused Ion Beam Fabrication

Contains reports on thirteen research projects and a list of publications.Defense Advanced Research Projects Agency/U.S. Army Research Office Contract DAAL03-88-K-0108National Science Foundation Grant ECS 89-21728MIT Lincoln Laboratory Innovative Research ProgramSEMATECH Contract 90-MC-503Micrion Contract M08774U.S. Army Research Office Contract DAAL03-87-K-0126IBM Corporatio

Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression

Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h). In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes

Oxidizing capacity of periodate activated with iron-based bimetallic nanoparticles

Nanosized zerovalent iron (nFe0) loaded with a secondary metal such as Ni or Cu on its surface was demonstrated to effectively activate periodate (IO4 -) and degrade selected organic compounds at neutral pH. The degradation was accompanied by a stoichiometric conversion of IO4 - to iodate (IO3 -). nFe 0 without bimetallic loading led to similar IO4 - reduction but no organic degradation, suggesting the production of reactive iodine intermediate only when IO4 - is activated by bimetallic nFe0 (e.g., nFe0-Ni and nFe0-Cu). The organic degradation kinetics in the nFe0-Ni(or Cu)/IO 4 - system was substrate dependent: 4-chlorophenol, phenol, and bisphenol A were effectively degraded, whereas little or no degradation was observed with benzoic acid, carbamazepine, and 2,4,6-trichlorophenol. The substrate specificity, further confirmed by little kinetic inhibition with background organic matter, implies the selective nature of oxidant in the nFe0-Ni(or Cu)/IO4 - system. The comparison with the photoactivated IO4 - system, in which iodyl radical (IO3 •) is a predominant oxidant in the presence of methanol, suggests IO3 • also as primary oxidant in the nFe0-Ni(or Cu)/IO4 - system.close0

Photosensitized Oxidation of Emerging Organic Pollutants by Tetrakis C-60 Aminofullerene-Derivatized Silica under Visible Light Irradiation

We recently reported that C-60 aminofullerenes Sunlight immobilized on silica support (aminoC(60)/silica) efficiently produce singlet oxygen (O-1(2)) and inactivate virus and bacteria under visible light irradiation.(1) We herein evaluate this new photocatalyst for oxidative degradation of 11 emerging organic contaminants, including pharmaceuticals such as acetaminophen, carbamazepine, cimetidine, propranolol, ranitidine, sulfisoxazole, and trimethoprim, and endocrine disruptors such as bisphenol A and pentachlorophenol. Tetrakis aminoC(60)/silica degraded pharmaceuticals under visible light irradiation faster than common semiconductor photocatalysts such as platinized WO3 and carbon-doped TiO2. Furthermore, aminoC(60)/silica exhibited high target-specificity without significant interference by natural organic matter. AminoC(60)/silica was more efficient than unsupported (water-suspended) C-60 aminofullerene. This was attributed to kinetically enhanced O-1(2) production after immobilization, which reduces agglomeration of the photocatalyst, and to adsorption of pharmaceuticals onto the silica support, which increases exposure to O-1(2) near photocatalytic sites. Removal efficiency increased with pH for contaminants with a phenolic moiety, such as bisphenol A and acetaminophen, because the electron-rich phenolates that form at alkaline pH are more vulnerable to singlet oxygenation.close1

Sox10 Controls Migration of B16F10 Melanoma Cells through Multiple Regulatory Target Genes

It is believed that the inherent differentiation program of melanocytes during embryogenesis predisposes melanoma cells to high frequency of metastasis. Sox10, a transcription factor expressed in neural crest stem cells and a subset of progeny lineages, plays a key role in the development of melanocytes. We show that B16F10 melanoma cells transfected with siRNAs specific for Sox10 display reduced migratory activity which in turn indicated that a subset of transcriptional regulatory target genes of Sox10 is likely to be involved in migration and metastasis of melanoma cells. We carried out a microarray-based gene expression profiling using a Sox10-specific siRNA to identify relevant regulatory targets and found that multiple genes including melanocortin-1 receptor (Mc1r) partake in the regulation of migration. We provide evidences that the effect of Sox10 on migration is mediated in large part by Mitf, a transcription factor downstream to Sox10. Among the mouse melanoma cell lines examined, however, only B16F10 showed robust down-regulation of Sox10 and inhibition of cell migration indicating that further dissection of dosage effects and/or cell line-specific regulatory networks is necessary. The involvement of Mc1r in migration was studied in detail in vivo using a murine metastasis model. Specifically, B16F10 melanoma cells treated with a specific siRNA showed reduced tendency in metastasizing to and colonizing the lung after being injected in the tail vein. These data reveal a cadre of novel regulators and mediators involved in migration and metastasis of melanoma cells that represents potential targets of therapeutic intervention