Bicyclic Peptides as a New Modality for Imaging and Targeting of Proteins Overexpressed by Tumors.

Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5%ID/g to 12%ID/g at 1 hour post injection (p.i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1-MMP expression levels. Fatty acid modification of the bicyclic peptide extended its circulating half-life, resulting in increased tumor signals (36%ID/g at 6 hours p.i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2%ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy.Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of high-contrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.

A 25-year replication of Katz et al.'s (1988) metaphor norms.

Research in metaphor processing has made extensive use of the normed metaphor database created by Katz, Paivio, Marschark, & Clark (Metaphor and Symbolic Activity, 3, 191-214, 1988). Because of the plasticity of figurative language, we conducted a renorming of selected metaphors from the database on a new student population. Correlations between Katz et al.'s and the present data showed that the pattern of responses has remained highly consistent across time and populations. The consistency of the normative ratings allows us to be confident in future research that will use the Katz et al. collection.

Using eye movements to evaluate the cognitive processes involved in text comprehension.

The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Measuring eye movements during reading is one of the most precise methods for measuring moment-by-moment (online) processing demands during text comprehension. Cognitive processing demands are reflected by several aspects of eye movement behavior, such as fixation duration, number of fixations, and number of regressions (returning to prior parts of a text). Important properties of eye tracking equipment that researchers need to consider are described, including how frequently the eye position is measured (sampling rate), accuracy of determining eye position, how much head movement is allowed, and ease of use. Also described are properties of stimuli that influence eye movements that need to be controlled in studies of text comprehension, such as the position, frequency, and length of target words. Procedural recommendations related to preparing the participant, setting up and calibrating the equipment, and running a study are given. Representative results are presented to illustrate how data can be evaluated. Although the methodology is described in terms of reading comprehension, much of the information presented can be applied to any study in which participants read verbal stimuli.

Development of a fluorescence intensity assay for the mitotic serine/threonine protein kinase Aurora-A.

The Aurora kinases are a group of serine/threonine protein kinases that regulate key steps during mitosis, and deregulation of these proteins (e.g., by gene amplification or overexpression) has been linked to a wide variety of tumor types. Thus, Aurora-A and Aurora-B have been intensely studied as targets for anticancer therapy and are now clinically validated targets. Here we report on the development of a novel fluorescence intensity binding assay for Aurora-A kinase inhibitors using a fluorescently labeled probe compound that shows intramolecular quenching when unbound but exhibits a dramatic increase in fluorescence when bound to Aurora-A.

Local factors regulating growth and function of human thyroid cells in vitro and in vivo.

In vitro culture of thyroid follicles is often employed to study thyroid cell biology and the control of thyroid follicular cell growth. For acceptance as a valid model, cultures should maintain differentiated function, which can be measured as the organification of [Formula: see text] and/or the de novo synthesis of thyroid hormones. In this article, the properties and merits of the various in vitro cultures of thyroid follicular cells and the potential effects of thyroid-specific, secreted products (thyroid hormones, thyroglobulin) and autocrine factors (proteases, growth factors and inhibitors) on thyroid growth and function, are explored. The regulation of the secretion of autocrine/paracrine factors by thyroid follicular cells is reviewed and methods by which cells may defend themselves from the effects of bioactive growth factors are discussed with particular reference to FGF signalling. The role and regulation of plasminogen activator activity, which may be central to the release and/or activation of growth factors and their receptors, and the secretion of angiogenic factors are described.

The ATP-operated clamp of human DNA topoisomerase IIalpha: hyperstimulation of ATPase by "piggy-back" binding.

We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction.