Development of collimator insert for linac based stereotactic irradiation.

The aim of this study is to develop collimator inserts of various sizes which are either not commercially available or are expensive to import. The dosimetry parameters such as tissue maximum ratio (TMR), off-axis ratio (OAR) and output factor of the developed collimator insert are compared with that of the commercial collimator insert (Radionics). In order to check the suitability of the collimator insert developed locally for clinical use and to standardize the method of development, a collimator insert of 15 mm identical to the one supplied by Radionics is developed with low-melting alloy (Cerrobend). Moreover for the clinical use of the developed collimator insert, certain acceptance tests are performed which include a collimator concentricity test, beam size check and radiation leakage test. The dose verification is carried out with a thermoluminescent dosimeter (7LiF rods) and an FBX chemical dosimeter in a human-head-shaped Perspex phantom filled with water. The variation between the calculated and measured dose is found to be within +2.4% for 7LiF rods and -2.0% for the FBX chemical dosimeter thus ensuring the suitability of the developed collimator insert for clinical use. This has encouraged us to standardize the method adapted to develop the collimator insert and to develop collimator inserts of different field sizes.

Multiple mechanisms for the inhibition of entry and uncoating of superinfecting Semliki Forest virus.

Recombinant Semliki Forest viruses (SFV) that express one or none of the viral structural proteins were used to infect cells and to analyze the fate of incoming superinfecting wild-type viruses. It was found that in addition to the previously described block in replication that superinfecting viruses encounter within 15 min of infection, other mechanisms of superinfection inhibition occurred at later times. Over a 6-hr infection period, inhibition was seen in binding of virus to the cell surface, in acid-activated penetration into the cytoplasm, and in uncoating of nucleocapsids. For each of these processes, the inhibitory mechanism was investigated. In summary, we found that infection evoked several independent mechanisms for blocking the entry and uncoating of superinfecting viruses. The results also offered new insights into the normal processes of penetration and uncoating of SFV.

High-resolution functional mapping of a cloned gene by genetic footprinting.

We describe an efficient method for introducing and analyzing a comprehensive set of mutations in a cloned gene to map its functional organization. The technique, genetic footprinting, uses a retroviral integrase to generate a comprehensive library of mutants, each of which bears a single insertion of a defined oligonucleotide at a random position in the gene of interest. This mutant library is selected for gene function en masse. DNA samples are isolated from the library both before and after selection, and the mutations represented in each sample are then analyzed. The analysis is designed so that a mutation at a particular location gives rise to an electrophoretic band of discrete mobility. For the whole library, this results in a ladder of bands, each band representing a specific mutation. Mutants in which the inserted sequence disrupts a feature that is required for the selected function, ipso facto, fail the selection. The corresponding bands are therefore absent from the ladder of bands obtained from the library after selection, giving rise to a footprint representing features of the gene that are essential for the selected function. Because the sequence of the inserted oligonucleotide is known, and its position can be inferred precisely from the electrophoretic mobility of the corresponding band, the precise location and sequence of mutations that disrupt gene function can be determined without isolating or sequencing individual mutants. This method should be generally applicable for saturation mutagenesis and high-resolution functional mapping of cloned DNA sequences.