Metastasis is responsible for most deaths due to malignant melanoma. The clinical significance of micrometastases in the lymph is a hotly debated topic, but an improved understanding of the lymphatic spread of cancer remains important for improving cancer survival. Cellular magnetic resonance imaging (MRI) is a newly emerging field of imaging research that is expected to have a large impact on cancer research. In this study, we demonstrate the cellular MRI technology required to reliably image the lymphatic system in mice and to detect iron-labeled metastatic melanoma cells within the mouse lymph nodes. Melanoma cells were implanted directly into the inguinal lymph nodes in mice, and micro-MRI was performed using a customized 1.5-T clinical MRI system. We show cell detection of as few as 100 iron-labeled cells within the lymph node, with injections of larger cell numbers producing increasingly obvious regions of signal void. In addition, we show that cellular MRI allows monitoring of the fate of these cells over time as they develop into intranodal tumors. This technology will allow noninvasive investigations of cellular events in cancer metastasis within an entire animal and will facilitate progress in understanding the mechanisms of metastasis within the lymphatic system.

Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/Dgalactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 mM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N9-dimethyl-N-(iodoacetyl)-N9-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based sitesaturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.Keywords: GGBP; glucose/galactose-binding protein; low affinity; structure-based site-saturation mutagenesis; screening; focused library Abbreviations: GGBP, glucose/galactose-binding protein; IANBD, N,N9-dimethyl-N-(iodoacetyl)-N9-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine; wt, wild type; ORF, open reading frame; LOD, limit of detection; DPBS, Dulbecco's phosphate-buffered saline; PBS, phosphate-buffered saline.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.