Measurement of the Electron Magnetic Moment.

The electron magnetic moment, -µ/µ_{B}=g/2=1.001 159 652 180 59 (13) [0.13 ppt], is determined 2.2 times more accurately than the value that stood for fourteen years. The most precisely determined property of an elementary particle tests the most precise prediction of the standard model (SM) to 1 part in 10^{12}. The test would improve an order of magnitude if the uncertainty from discrepant measurements of the fine structure constant α is eliminated since the SM prediction is a function of α. The new measurement and SM theory together predict α^{-1}=137.035 999 166 (15) [0.11 ppb] with an uncertainty 10 times smaller than the current disagreement between measured α values.

Switchable damping for a one-particle oscillator.

The possibility to switch the damping rate for a one-electron oscillator is demonstrated for an electron that oscillates along the magnetic field axis in a Penning trap. Strong axial damping can be switched on to allow this oscillation to be used for quantum nondemolition detection of the cyclotron and spin quantum state of the electron. Weak axial damping can be switched on to circumvent the backaction of the detection motion that has limited past measurements. The newly developed switch will reduce the linewidth of the cyclotron transition of one-electron by two orders of magnitude.

On the incorrect use and interpretation of the model for colloidal, spherical crystal growth.

HYPOTHESIS: The standard model for diffusion and surface kinetics driven growth of a single spherical particle in solution is applied incorrectly throughout the literature. This leads to inaccurate values for parameter values, such as the diffusion and surface kinetics coefficients. The model cannot even distinguish between diffusion or surface kinetics driven growth. FINDINGS: It is shown that crystal growth occurs in two distinct stages. The standard model only holds during the late time. Fitting to experimental data, including the early time, leads to incorrect values for the coefficients. It is shown that diffusion and surface kinetics are interchangeable in the model and so indistinguishable. The growth is controlled by a single non-dimensional group. Previous studies, where more independent parameters are calculated have redundancy. The Gibbs-Thomson relation plays an important role but, in the cases studied here, this is only noticeable during the first growth stage where the model does not hold. For the first time an explicit relation for the variation of the radius with time is given. Excellent agreement with experimental data on CdSe growth is shown.

Modelling the cardiovascular system for assessing the blood pressure curve.

A four compartment model of the cardiovascular system is developed. To allow for easy interpretation and to minimise the number of parameters, an effort was made to keep the model as simple as possible. Using a standard method (Matlab function fminsearch) to calculate the parameter values led to unacceptable run times or non-convergence. Consequently we developed an algorithm which first finds the most important model parameters and uses these as a basis for a four stage process which accurately determines all parameter values. This process is then applied to data from three ICU patients. Good agreement between the model and measured arterial pressure is demonstrated in all cases.

Dysregulation of interleukin 5 expression in familial eosinophilia.

BACKGROUND: Familial eosinophilia (FE) is a rare autosomal dominant inherited disorder characterized by the presence of lifelong peripheral eosinophilia (>1500/µL). Mapped to chromosome 5q31-q33, the genetic cause of FE is unknown, and prior studies have failed to demonstrate a primary abnormality in the eosinophil lineage. OBJECTIVE: The aim of this study was to identify the cells driving the eosinophilia in FE. METHODS: Microarray analysis and real-time PCR were used to examine transcriptional differences in peripheral blood mononuclear cells (PBMC), and in purified cell subsets from affected and unaffected family members belonging to a single large kindred. Cytokine levels in serum and PBMC culture supernatants were assessed by suspension array multiplexed immunoassays. RESULTS: Whereas IL-5 mRNA expression was significantly increased in freshly isolated PBMC from affected family members, this was not accompanied by increased mRNA expression of other Th2 cytokines (IL-4 or IL-13). Serum levels of IL-5 and IL-5 receptor α, but not IgE, were similarly increased in affected family members. Of note, IL-5 mRNA expression was significantly increased in purified CD3+ CD4+, CD14+, CD19+, and ILC2 cells from affected family members, as were IL-5 protein levels in supernatants from both stimulated PBMC and ILC2 cultures. CONCLUSIONS: These data are consistent with the hypothesis that the eosinophilia in FE is secondary to dysregulation of IL-5 production in PBMC (and their component subsets).

Reactive oxygen species mediate inflammatory cytokine release and EGFR-dependent mucin secretion in airway epithelial cells exposed to Pseudomonas pyocyanin.

Despite the long-appreciated in vivo role of the redox-active virulence factor pyocyanin in Pseudomonas airway infections and the importance of airway epithelial cells in combating bacterial pathogens, little is known about pyocyanin's effect on airway epithelial cells. We find that exposure of bronchiolar epithelial cells to pyocyanin results in MUC2/MUC5AC induction and mucin secretion through release of inflammatory cytokines and growth factors (interleukin (IL)-1ß, IL-6, heparin-bound epidermal growth factor, tissue growth factor-α, tumor necrosis factor-α) that activate the epidermal growth factor receptor pathway. These changes are mediated by reactive oxygen species produced by pyocyanin. Microarray analysis identified 286 pyocyanin-induced genes in airway epithelial cells, including many inflammatory mediators elevated in cystic fibrosis (granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte CSF, chemokine (C-X-C motif) ligand 1 (CXCL1), serum amyloid, IL-23) and several novel pyocyanin-responsive genes of potential importance in the infection process (IL-24, CXCL2, CXCL3, CCL20, CXCR4). This comprehensive study uncovers numerous details of pyocyanin's proinflammatory action and establishes airway epithelial cells as key responders to this microbial toxin.

Spin coating of non-Newtonian fluids with a moving front.

We investigate axisymmetric spin coating of power law and Ellis fluids. The flow is driven by centrifugal force, gravity and surface tension. For power law and Ellis models a single equation for the fluid film height is obtained. For a Newtonian fluid the flux only involves linear derivative terms which allows the flux to be easily split for a numerical scheme. For power law and Ellis models the derivatives appear as nonlinear terms. To overcome this we develop an alternative numerical scheme to solve for the film height. Neglecting surface tension and gravity the power law model shows a central spike which is reduced by the introduction of surface tension and gravity. In certain cases the shear thinning power law model predicts slower spreading than the Newtonian model. The Ellis fluid shows no central spike, even for zero surface tension and the film always spreads further than the Newtonian fluid.

Application of non-Newtonian models to thin film flow.

The paper describes an investigation into the use of lubrication models on thin film flow. Power law, Ellis, and Carreau models are compared for free surface flow and flow within a channel. It is shown that the Ellis law (or a slight modification) can give very similar viscosity curves to Carreau. The three models are then compared for thin film flow with a constant height free surface. For low shear rates the power law model can give very inaccurate predictions. Having shown Carreau and Ellis may produce similar results we then study flow in a channel for Ellis and power law fluids. Again the power law can give inaccurate results due to the high viscosity around the turning point for the velocity. Finally, we briefly describe the modification to include surface tension in the free surface flow model.

Thymidylate synthase as a molecular target for drug discovery using the National Cancer Institute's Anticancer Drug Screen.

Thymidylate synthase (TS) is a critical cellular target for cancer chemotherapeutics, particularly the fluoropyrimidine and antifolate classes of antineoplastic agents. One of the primary mechanisms of clinical insensitivity to these agents is through the overexpression of the target enzyme, TS. Thus, there is a need for the development of agents which selectively target TS-overexpressing malignant cells. To this end, we conducted a search for agents which potentially selectively target TS-overexpressing cells using two separate algorithms for identifying such compounds in the NCI Drug Repository by comparing cytotoxicity profiles of 30000 compounds with the TS expression levels measured by Western blot analysis in 53 cell lines. Using the traditional COMPARE analysis we were unable to identify compounds which maintain a selective ability to kill high TS-expressing cells in a subsequent four cell line validation assay. A new algorithm, termed COMPARE Effect Clusters analysis, enabled the identification of a particular drug cluster which contained compounds that maintained a selective ability to kill TS-overexpressing cell lines in the validation assay. While the identified compounds were selectively cytotoxic to TS-overexpressing cells, we found that they were not specifically targeting TS as a mechanism of action. Apparently, the overexpression of TS was providing a marker for sensitivity. This identified class of compounds which appears to be selectively cytotoxic against cells which overexpress TS may be useful for the development of therapeutics for those whose cancers overexpress TS de novo.

Screening for and identification of novel agents directed at renal cell carcinoma.

We were interested in identifying novel agents for renal cell carcinoma (RCC) by screening for activities that model renal tumor biology. Searching for relative renal cell sensitivity and leukemia insensitivity among cytotoxicity profiles in the NCI Drug Screen database, we identified 16 potential agents with renal selectivity. We evaluated the agents in 10 RCC cell lines (of primary and metastatic origin) isolated from 5 patients. The 50% inhibitory concentrations (IC50) in these cell lines ranged from 0.019 +/- 0.013 to 11.4 +/- 0.55 microM and were comparable with values obtained with renal cell lines in the NCI Drug Screen panel. Because RCC are slowly growing tumors, we evaluated the compounds on rapidly (27% S phase) or slowly (6% S phase) growing cells. In contrast to doxorubicin, where cytotoxicity was restricted to rapidly proliferating cells, three compounds (NSC 280074, 281613, and 281817) were more cytotoxic in slowly proliferating cells. NSC 72151 and 268965 were equitoxic for both populations. NSC 94889, 638850, and 630938 were more cytotoxic in rapidly growing cells. In in vitro time exposure studies, four compounds, NSC 268965, 280074, 281613, and 281817, were maximally cytotoxic with as little as 3 h exposure time. From an analysis comparing the p53 genotype of the 60 cell lines of the National Cancer Institute (NCI) Drug Screen with the cytotoxicity profiles for the 16 putative renal compounds, 13 compounds were classified as likely to be indifferent to p53 status. We also developed a panel specificity detection method for the NCI Drug Screen database to evaluate the prevalence of renal sensitive compounds. Of the 16 studied compounds, 14 were among those identified as renal sensitive by the statistical analysis. Lastly, we found reduced tumor growth in mice with established renal human tumor xenografts after treatment with two of the renal active compounds. These studies describe compounds with potential renal activity that are candidates for preclinical development for renal cell carcinoma.

Wild-type p53 marginally induces endogenous MDR-1 mRNA without causing a measurable drug resistance in human cancer cells.

The notion that wt p53 downregulates MDR-1 links p53 mutations to multidrug resistant phenotype. Alternatively, it has been envisioned that wt p53 protects cells against DNA damaging drugs by inducing MDR-1. Opposing conclusions on the relationship between MDR-1 and p53 have been predominantly based on the effects of p53 on MDR-1 promoter-constructs. We found that introduction of wt p53 slightly induced MDR-1 mRNA in three cell lines having endogenous mt p53. Wt p53-mediated induction of endogenous MDR-1 may represent a rudiment of cellular protection against toxic compounds earlier in evolution. Marked induction of p21WAF1/CIP1 (p21) mRNA was observed in all cell lines; and lower levels of wt p53 were required to induce p21 than MDR-1. Pgp was undetectable and wt p53 did not increase resistance to an MDR-1 substrate, suggesting the changes in MDR-1 mRNA may be functionally insignificant. Unlike endogenous MDR-1, the expression of an MDR-1 promoter (-434/+147 fragment) - luciferase construct was unchanged or even inhibited by wt p53 that may be secondary to wt p53-mediated cytotoxicity. Thus, partial promoter constructs may not accurately represent endogenous MDR-1.

An informatics approach identifying markers of chemosensitivity in human cancer cell lines.

We have used a sensitive and reproducible method of measuring mRNA expression to compare basal levels of 10 transcripts in the 60 cell lines of the National Cancer Institute's in vitro anticancer drug screen (NCI-ACDS) under conditions of exponential growth. The strongest correlation among these target genes was between levels of CIP1/WAF1 and BAX. Levels of the three major growth arrest and DNA damage-inducible gene transcripts, (GADD34, GADD45, and GADD153), which are coordinately regulated in response to many stresses, were also correlated across the 60 cell lines. Although the stress induction of several of the transcripts studied here has been shown to be dependent on wild-type p53 status, basal levels of only CIP1/WAF1 and BAX were found to correlate with p53 status. As expected, basal expression of O6 alkyl guanine alkyl-transferase correlated well with resistance to O6-alkylating agents (r = -0.44) but not with resistance to alkylators with different mechanisms of action (r = -0.04). When basal expression levels of the 10 genes across the NCI-ACDS panel were compared with sensitivities to a panel of 122 standard chemotherapy agents, the most striking relationship was a strong negative correlation (r = -0.3) between basal BCL-X levels and sensitivity to drugs in all of the mechanistic classes except one class of antimetabolites. Sensitivities to a maximally diverse sample of 1200 from 70,000 compounds tested in the NCI-ACDS of agents were also negatively correlated with BCL-X levels. A novel application of factor analysis revealed that the newly discovered associations were independent of previously demonstrated sensitivity factors such as p53 mutation status and native population doubling time. A similar pattern of correlation was seen for Bcl-X(L) protein levels. Conversely, BAX and BCL2, two other genes associated with regulation of apoptosis, showed no overall correlation with drug sensitivities. This suggests that BCL-X may play a unique role in general resistance to cytotoxic agents, with the cell lines demonstrating relative resistance to 70,000 cytotoxic agents in the NCI-ACDS being characterized by high BCL-X expression.

Systematic variation in gene expression patterns in human cancer cell lines.

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90.

BACKGROUND: To our knowledge, 17-allylamino,17-demethoxygeldanamycin (17AAG) is the first inhibitor of heat shock protein 90 (Hsp90) to enter a phase I clinical trial in cancer. Inhibition of Hsp90, a chaperone protein (a protein that helps other proteins avoid misfolding pathways that produce inactive or aggregated states), leads to depletion of important oncogenic proteins, including Raf-1 and mutant p53 (also known as TP53). Given its ansamycin benzoquinone structure, we questioned whether the antitumor activity of 17AAG was affected by expression of the NQO1 gene, which encodes the quinone-metabolizing enzyme DT-diaphorase. METHODS: The antitumor activity of 17AAG and other Hsp90 inhibitors was determined by use of a sulforhodamine B-based cell growth inhibition assay in culture and by the arrest of xenograft tumor growth in nude mice. DT-diaphorase activity was determined by use of a spectrophotometric assay, and protein expression was determined by means of western immunoblotting. RESULTS: In two independent in vitro human tumor cell panels, we observed a positive relationship between DT-diaphorase expression level and growth inhibition by 17AAG. Stable, high-level expression of the active NQO1 gene transfected into the DT-diaphorase-deficient (by NQO1 mutation) BE human colon carcinoma cell line resulted in a 32-fold increase in 17AAG growth-inhibition activity. Increased sensitivity to 17AAG in the transfected cell line was also confirmed in xenografts. The extent of depletion of Raf-1 and mutant p53 protein confirmed that the Hsp90 inhibition mechanism was maintained in cells with high and low levels of DT-diaphorase. 17AAG was shown to be a substrate for purified human DT-diaphorase. CONCLUSION: These results suggest that the antitumor activity and possibly the toxicologic properties of 17AAG in humans may be influenced by the expression of DT-diaphorase. Careful monitoring for NQO1 polymorphism and the level of tumor DT-diaphorase activity is therefore recommended in clinical trials with 17AAG.

The complexity of radiation stress responses: analysis by informatics and functional genomics approaches.

Molecular responses to genotoxic stress are complex and are mediated by a variety of regulatory pathways. One key element in cellular response is the stress gene transcription factor p53, which can regulate nearly 100 genes that have already been identified. Although p53 plays a central role in the cellular response to DNA-damaging agents such as ionizing radiation (IR), other pathways can also have important roles. One example is the transcriptional responses associated with IR-induced apoptosis, where induction of some genes is limited to p53 wild-type (wt) cells that also have the ability to undergo rapid apoptosis after irradiation. In contrast, other genes are triggered after IR in lines undergoing rapid apoptosis regardless of p53 status. From this and other examples, it is apparent that the pattern of stress gene expression is cell type specific in both primary and transformed lines. The premise will be developed that such differences in stress gene responsiveness can be employed as molecular markers using a combination of informatics and functional genomics approaches. An example is given using the panel of lines of the NCI anticancer drug screen where both the p53 status and sensitivity to a large collection of cytotoxic agents have been determined. The utility of cDNA microarray hybridization to measure IR-stress gene responses has recently been demonstrated and a large number of additional IR-stress genes have been identified. The responses of some of these genes to IR and other DNA-damaging agents varied widely in cell lines from different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses; this also highlights the need for informatics approaches to discover and prioritize hypotheses regarding the importance of particular cellular factors. The aim of this review is to demonstrate the utility of combining an informatics approach with functional genomics in the study of stress responses.

Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin.

This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.

Cytotoxic activities of Mannich bases of chalcones and related compounds.

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

Mining the NCI anticancer drug discovery databases: genetic function approximation for the QSAR study of anticancer ellipticine analogues.

The U.S. National Cancer Institute (NCI) conducts a drug discovery program in which approximately 10,000 compounds are screened every year in vitro against a panel of 60 human cancer cell lines from different organs of origin. Since 1990, approximately 63,000 compounds have been tested, and their patterns of activity profiled. Recently, we analyzed the antitumor activity patterns of 112 ellipticine analogues using a hierarchical clustering algorithm. Dramatic coherence between molecular structures and activity patterns was observed qualitatively from the cluster tree. In the present study, we further investigate the quantitative structure-activity relationships (QSAR) of these compounds, in particular with respect to the influence of p53-status and the CNS cell selectivity of the activity patterns. Independent variables (i.e., chemical structural descriptors of the ellipticine analogues) were calculated from the Cerius2 molecular modeling package. Important structural descriptors, including partial atomic charges on the ellipticine ring-forming atoms, were identified by the recently developed genetic function approximation (GFA) method. For our data set, the GFA method gave better correlation and cross-validation results (R2 and CVR2 were usually approximately 0.3 higher) than did classical stepwise linear regression. A procedure for improving the performance of GFA is proposed, and the relative advantages and disadvantages of using GFA for QSAR studies are discussed.