Minimizing delivery time and monitor units in static IMRT by leaf-sequencing.

Intensity-modulated radiation therapy (IMRT) requires the determination of the appropriate multileaf collimator settings to deliver an intensity map. The purpose of this work was to attempt to reduce the number of segments required for IMRT delivery and the number of monitor units required to deliver an intensity map. An intensity map may be written as a matrix. Leaf sequencing was formulated as a problem of decomposing the matrix into a series of sub-matrices. Sets of random intensity matrices were created and the segmentations produced by applying different algorithms were compared. The number of segments, important if verification and record (VR) overhead is significant, and beam on times were examined. It is shown that reducing the value of the matrix entries by the maximum amount at each stage results in the smallest number of steps. Reducing the 2-norm (sum of the squares) of the matrix entries by the maximum amount at each step results in the smallest beam on time. Three new algorithms are introduced, two of which produce results that are superior to those generated by the algorithms of other researchers. The resulting methods can be expanded upon to include tongue and groove effects and leaf inter-digitization. With square random matrices of the order 15, the reduction in beam time and segmentation is up to 30-40%. Compared to previous algorithms, those presented here have demonstrated a reduction in the beam on time required to deliver an intensity map by 30-40%. Similarly, the number of segments needed to deliver an intensity map is also reduced.

Binding of ETS family members is important for the function of the c-sis/platelet-derived growth factor-B TATA neighboring sequence in 12-O-tetradecanoylphorbol-13-acetate-treated K562 cells.

The c-sis/platelet-derived growth factor (PDGF)-B TATA neighboring sequence (TNS) is a promoter element that is required for the full induction of this gene in K562 erythroleukemia cells undergoing 12-O-tetradecanoylphorbol-13-acetate-mediated megakaryoblastic differentiation. Nuclear factors from K562 cells can bind to the c-sis/PDGF-B TNS, generating four complexes in electrophoretic mobility shift assays. One of these complexes was previously shown to contain Sp family members. In this work, we provide evidence implicating two of the remaining complexes as belonging to the ETS family of transcription factors. This includes the identification of a novel constitutive TNS-binding complex containing the ETS family member ELK-1. The binding of both ETS-like complexes was disrupted by mutations in a central CCGGAA core within the TNS and, for one of the complexes, could be promoted by bringing the sequences flanking the core closer to a consensus ETS binding site. The molecular weights of these TNS-binding factors were estimated by UV cross-linking analysis and found to be consistent with those of several ETS family transcription factors, including ELK-1. A consensus ELK-1 binding site could compete for the binding of both putative ETS-like factors, and the novel complex could be disrupted by the addition of an antibody raised against ELK-1. Transient transfection analysis using mutant TNS promoter-reporter constructs demonstrated a strong correlation between the binding of the ETS-like factors and the transcriptional activity of the TNS. In contrast, mutations that prevented the binding of Sp family transcription factors had no effect on promoter activity. Thus, ETS family members, such as ELK-1, are not only capable of binding to the TNS but seem to be necessary for the function of this element in differentiating K562 cells.

Functional analysis of the SIS proximal element and its activating factors: regulated transcription of the c-SIS/PDGF-B gene in human erythroleukemia cells.

The SIS proximal element (SPE) is essential for the basal transcription of the c-sis/PDGF-B gene as well as the lineage-specific, activated transcription of this gene seen in megakaryocytes. In gel mobility shift analyses, the SPE element forms three gel-shift complexes; the t(op) and b(ottom) complexes were detected in nuclear extracts from both untreated and phorbol 12-myristate 13-acetate ('tetradecanoylphorbol acetate', TPA) treated K562 cells, whereas the m(iddle) complex was detected only in nuclear extracts from TPA-treated K562 cells. Site-directed mutagenesis of the SPE revealed a CCACCC motif that was essential for promoter activity as well as the formation of all three SPE gel-shift complexes. Nested-deletion analyses showed that the SPE was required for TPA-inducibility of c-sis/PDGF-B transcription. Antibody supershift analyses demonstrated that the t gel-shift complex contained both Sp1 and Sp3, and that the b complex contained only Sp3. In vitro transcription assays demonstrated that both Sp1 and Sp3 could support c-sis/PDGF-B transcription independent of each other in untreated K562 cells. However, overexpression of Sp1/Sp3 failed to significantly increase the c-sis/PDGF-B transcription in K562 cells.

Transcriptional regulation of the SIS/PDGF-B gene in human osteosarcoma cells by the Sp family of transcription factors.

Expression of PDGF-B, the gene encoding the platelet-derived growth factor B chain, has been implicated as a participant in an autocrine growth loop in the human osteosarcoma cell line U2-OS. In previous work, we identified a primary site in the PDGF-B promoter, the SIS proximal element (SPE), which is critical for transcription of the PDGF-B gene in U2-OS cells. We also identified Sp1 as one of the SPE-binding proteins in U2-OS nuclear extracts. In the present work, we have identified another SPE-binding protein to be Sp3. Gel mobility shift assays showed that both Sp1 and Sp3 require the CACCC motif within the SPE for binding. In vitro transcription assays showed that Sp1 or/and Sp3 is necessary for transcription of the PDGF-B gene. Cotransfection experiments functionally demonstrated that Sp1 and Sp3 can independently or additively activate the PDGF-B promoter through the SPE as well as a synthetic promoter. However, the CACCC motif within the SPE is not the only site within the minimal PDGF-B promoter through which Sp1/Sp3 acts; additional nested deletion analyses showed that multiple cis-acting elements within the minimal promoter are required for full level transcription of the PDGF-B gene in U2-OS cells.

Suppression of single and double nonsense mutations introduced into the diphtheria toxin A-chain gene: a potential binary system for toxin gene therapy.

We have previously shown that ablation of specific cells can be achieved through the transcriptionally regulated expression of the diphtheria toxin A-chain (DT-A) gene in both cell culture and transgenic mice. Such targeted toxin gene expression provides a novel approach to cancer and acquired immunodeficiency syndrome (AIDS) therapy. The use of mutants of DT-A with attenuated toxicity may allow targeting of cells for which only moderately selective gene regulatory elements are available. Alternatively, conditional mutants might be used to target cells in which conditions can be established for suppression of the mutation. We have investigated the effects of mutating selected serine codons to amber (TAG) nonsense codons in the DT-A coding sequence. In transient transfection of HeLa cells, DT-A activity was markedly reduced by the introduction of a single amber codon and was virtually eliminated by two amber mutations. Cotransfection of a serine inserting suppressor tRNA expression plasmid substantially restored DT-A expression from both single and double amber mutants. Expression of the same suppressor tRNA also suppressed a previously described amber mutation at the tyrosine codon 28 in DT-A. Thus, nonsense suppression can be used to control the expression of DT-A in mammalian cells, potentially allowing binary control over the targeting of tissues for selective ablation.

SIS/PDGF-B promoter isolation and characterization of regulatory elements necessary for basal expression of the SIS/PDGF-B gene in U2-OS osteosarcoma cells.

Platelet-derived growth factor BB, encoded by the SIS/PDGF-B gene, is a potent mitogen for cells of mesenchymal origin, and the SIS/PDGF-B gene is expressed in a large percentage of human mesenchymal tumor cells establishing a growth-promoting, autocrine growth circuit. A 4-kb fragment, containing the SIS/PDGF-B promoter, was isolated from a human genomic library, and a series of 5'-nested deletions and linker-scanning mutants were used to identify positive regulatory elements that are necessary for the constitutive expression of this gene in human U2-OS osteosarcoma cells. A 250-bp fragment, lying immediately 5' to the SIS/PDGF-B mRNA initiation site (+1), retained full promoter activity, and positive regulatory elements at -228 to -219, -97 to -88 (SIS distal element) and -58 to -39 (SIS proximal element, SPE) were identified. Insertion of the 20-bp SPE into a heterologous, minimal promoter resulted in >5-fold transcriptional activation which was ablated by mutations to the SPE. High resolution mutagenesis within the 20-bp SPE, indicated the necessity of a CACCC motif for activity. Gel shift analysis of SPE-binding proteins in U2-OS nuclear extracts identified Sp1 and two additional binding factors that could be competed away from SPE binding by adding excess consensus Sp1 or CACC oligonucleotides. The individual and aggregate roles of the SPE and two weaker positive regulatory elements in regulating SIS/PDGF-B transcription in these tumor cells is considered.

Antimicrobial activity of environmental surface disinfectants in the absence and presence of bioburden.

Thirty-nine products representing six categories of disinfectants (alcohols, chlorines, dilute glutaraldehydes, iodophors, phenolics, and quaternary ammonium compounds) were first tested in the absence of bioburden, using four test methods with five test organisms. Products that performed best were retested with the same methods and organisms in the presence of both serum and whole blood, using 3- and 10-minute contact times. Only products containing high ethyl alcohol had consistently high antimicrobial activity regardless of the test method, test organism, or contact time used both in the absence and presence of bioburden. Although these specific formulations demonstrated ability to penetrate and inactivate high concentrations of microorganisms within heavy bioburden, optimum disinfection of environmental surfaces is highly formulation dependent. Other products tested showed deficiencies that contraindicate their use as environmental surface disinfectants in clinical dental settings.

A suspension method to determine reuse life of chemical disinfectants during clinical use.

In-use testing of disinfectants is necessary to ensure efficacy over time. The current official procedure for testing disinfectants, the Association of Official Analytical Chemists (AOAC) use-dilution method, cannot be adapted to repeated sampling techniques of use-life testing. It is therefore necessary to use an alternative method when evaluating the activity of a disinfectant under actual use. The Clinical Research Associates (CRA) suspension method was developed to fill this need. It consists of adding 0.5 ml of a standard culture to 5.0 ml of test disinfectant and sampling the mixture after 10 min for surviving bacteria. When this test was compared with the AOAC use-dilution method under a simulated use situation, the two methods were generally equivalent in identifying disinfectant inactivation. In addition, the CRA method was less time consuming, easier to perform, and less variable than the AOAC method. Use of the CRA method in a clinical study demonstrated the need for reuse claims to be based on clinical use studies rather than on laboratory testing only.