Nuclear Testing

Abnormal morphology of the nucleus is considered a hallmark of neoplastic transformation, but nuclear deformation may also indicate the influence of mechanical stimuli. Understanding this latter phenomenon should help guide cell and tissue engineering strategies, so it is not surprising that a recent article in Biophysical Journal investigates a better way to measure the shape of cell nuclei for such applications. Traditionally, nuclear dimensions have been gathered using laborious analysis of fluorescent microscopy images. Not only is this process inefficient, but, if cell fixation is required, it provides only a snapshot of nuclear morphology at a given moment. Chalut et al., now describe the adaptation of angle-resolved low coherence interferometry (a/LCI) for nuclear morphology measurements in non-neoplastic cells. This method is based on measuring light scattering by cells and organelles, and then using data processing to filter out undesired contributions. These experimental signals are then compared to theoretical scattering calculations based on the calculated properties of homogenous spheres. Although real-life nuclei deviate from this theoretical ideal, the authors show that measurements of even the most aspherical nuclei differed by no more than 3% from values obtained using the traditional technique. Chalut et al. showed the applicability of the a/LCI approach to measuring nuclear deformation in primary porcine chondrocytes that were exposed to different osmotic environments, as well as to measurements of nuclei in murine macrophages held in two different orientations by a micropatterned grating. As the measurements are about five times faster than traditional image processing, use of a/LCI for assessing nuclear perturbations promises to be an informative way of assessing changes in physiology and gene expression that occur as a cell responds to its physical world.

Chalut et al. Label-free, high-throughput measurements of dynamic changes in cell nuclei using angle-resolved low coherence interferometry. Biophysical Journal [Epub ahead of print, March 7, 2008].

RhoSS-y Hue

A new rationally designed compound for intracellular thiol detection called RhoSS is described by Pires and Chmielewski, in a recent paper in Organic Letters. The probe contains two disulfide bonds that can be reduced by cellular thiols; this reduction reveals reactive sulfhydryl groups that are freed to attack carbamate bonds within the compound, collapsing it to produce rhodamine 110. Detection of cellular thiols (of which glutathione is the most common) is of interest because redox homeostasis is disrupted in a number of disease states, including cancer, cardiovascular disease, and neurological conditions. To explore the utility of their new thiol indicator, Pires and Chmielewski, compared RhoSS side-by-side with RhoCC, a compound identical except for the presence of carbon rather than sulfur bonds. In vitro experiments confirmed that RhoSS, but not RhoCC, fluoresced when incubated with dithiothreitol (DTT) or reduced glutathione. Tests in HeLa and MCF-7 cells showed that RhoSS was cell permeable and became fluorescent within cells in a time-dependent manner, behavior that was less pronounced when cells were pretreated with N-ethylmaleimide, a thiol-blocking agent. Detection of RhoSS-produced rhodamine 110 was possible via both confocal microscopy and flow cytometry, and no decrease in viability was seen in cells treated with RhoSS at concentrations up to 80 µM. This work extends the suite of caged rhodamine 110 derivatives, which have been previously used for detection of enzymatic activities such as proteases and esterases, and provides a new tool for investigating redox homeostasis in living cells.

Pires, M.M. and J. Chmielewski. 2008. Fluorescence imaging of cellular glutathione using a latent rhodamine. Organic Letters 10:837-840.

Preferential Treatment

Nothing makes us feel more pampered than to have our preferences recognized and catered to accordingly. And while a day at the lab is never going to be confused with a stint in a spa, States and colleagues have perhaps brought the two a bit closer than before. In Bioinformatics, they describe a web interface to PubMed that monitors user behavior in order to rank articles according to the deduced preferences of the searcher. The tool, MiSearch, is designed to help tame the overwhelming volume of scientific literature, in which relevant articles are just as easily buried as retrieved. While the Related Articles link on PubMed does return results that are similar to the original query, it can't know exactly which elements of the parent article are of most interest and which are dispensable. One way to overcome this is to ask users to rate results as to appropriateness, but few have the patience to provide interactive feedback. Instead, States et al., record the user's search history and the articles he or she selects or views, using these preferences to rank the results returned, with those calculated as most likely to be viewed being placed at the top. This ranking process allows the user to be less restrictive when inputting search terms, lessening the chances of missing a relevant result, while at the same time not having to search through pages of inappropriate articles. Establishing a profile does not require a login and can be based on IP; however, for users who share a computer or who have discrete research interests, username-based searches can also be performed. Researchers looking for a little more personal attention can access MiSearch at misearch.ncibi.org.

States et al. MiSearch adaptive PubMed search tool. Bioinformatics [Epub ahead of print, March 11, 2008].

A Real Drag

Frictional resistance usually gets a bad rap, but an article appearing in a recent Langmuir suggests that it's anything but a brake on progress. Gorti et al., a team based at Purdue, describe an immunoassay strategy that relies on changes to drag coefficients during Brownian motion in order to detect the presence of analyte in a sample. The work was inspired by a desire to develop faster, smaller-scale alternatives to microarray- or mass spectrometry-based detection systems. The assay involves 5-nL reaction vessels, submicron fluorescent microparticles that are functionalized with antibodies, and an imaging system outfitted with particle-tracking software. If the target antigen is present in the microreactor, it can be expected to interact with the antibodies, exerting drag on the random motions of the miniature beads to which they are conjugated. As a test case, Gorti et al., developed an assay to detect the filamentous bacteriophage M13 using an antibody directed against one of its capsid proteins. The diffusion coefficient of antibody-coated microparticles was calculated as a function of phage concentration; as a control, unconjugated beads that fluoresced in a different color were compared in parallel. The differences in mobility trends of the two microparticle types clearly showed the damping effect of the 930-nm-long viruses on the 690-nm-diameter microparticles. The authors calculate that their method has a sensitivity of 500 viruses/nL, but concede that detection of smaller analytes would be extremely challenging with their setup. However, the authors point out that microparticles with diameters as small as 40 nm can be imaged successfully, and their calculations suggest that analytes of 1 nm diameter (the approximate size of the 45 kDa protein ovalbumin, for example) could be detected under those circumstances. There's a lot more to be shown, but this intriguing 30-minute assay may ultimately succeed in dragging immunodetection into the fast lane.

Gorti et al. 2008. Immunoassays in nanoliter volume reactors using fluorescent particle diffusometry. Langmuir 24:2947-2952.