Todd Sulchek

Cell Stiffness Is a Biomarker of the Metastatic Potential of Ovarian Cancer Cells

The metastatic potential of cells is an important parameter in the design of optimal strategies for the personalized treatment of cancer. Using atomic force microscopy (AFM), we show, consistent with previous studies conducted in other types of epithelial cancer, that ovarian cancer cells are generally softer and display lower intrinsic variability in cell stiffness than non-malignant ovarian epithelial cells. A detailed examination of highly invasive ovarian cancer cells (HEY A8) relative to their less invasive parental cells (HEY), demonstrates that deformability is also an accurate biomarker of metastatic potential. Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. Our results indicate that cell stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells.

Maleimide Cross‐Linked Bioactive PEG Hydrogel Exhibits Improved Reaction Kinetics and Cross‐Linking for Cell Encapsulation and In Situ Delivery

Engineered polyethylene glycol-maleimide matrices for regenerative medicine exhibit improved reaction efficiency and wider range of Young’s moduli by utilizing maleimide cross-linking chemistry. This hydrogel chemistry is advantageous for cell delivery due to the mild reaction that occurs rapidly enough for in situ delivery, while easily lending itself to “plug-and-play” design variations such as incorporation of enzyme-cleavable cross-links and cell-adhesion peptides.

High-speed tapping mode imaging with active Q control for atomic force microscopy

The speed of tapping mode imaging with the atomic force microscope (AFM) has been increased by over an order of magnitude. The enhanced operation is achieved by (1) increasing the instrument’s mechanical bandwidth and (2) actively controlling the cantilever’s dynamics. The instrument’s mechanical bandwidth is increased by an order of magnitude by replacing the piezotube z-axis actuator with an integrated zinc oxide (ZnO) piezoelectric cantilever. The cantilever’s dynamics are optimized for high-speed operation by actively damping the quality factor (Q) of the cantilever. Active damping allows the amplitude of the oscillating cantilever to respond to topography changes more quickly. With these two advancements, 80μm×80 μm high-speed tapping mode images have been obtained with a scan frequency of 15 Hz. This corresponds to a tip velocity of 2.4 mm/s.

Explosives: A microsensor for trinitrotoluene vapour

Sensing devices designed to detect explosive vapours are bulky, expensive and in need of technological improvement — dogs remain the most effective detectors in the fight against terrorism and in the removal of land-mines. Here we demonstrate the deflagration of trinitrotoluene (TNT) in a small localized explosion on an uncoated piezoresistive microcantilever. This explosive-vapour sensor, which has a detection capability that is comparable to that of a dog, should enable extremely sensitive, miniature detection devices to be used on a large scale.

Insertion of Membrane Proteins into Discoidal Membranes Using a Cell-Free Protein Expression Approach

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.

Characterization and optimization of scan speed for tapping-mode atomic force microscopy

Increasing the imaging speed of tapping mode atomic force microscopy (AFM) has important practical and scientific applications. The scan speed of tapping-mode AFMs is limited by the speed of the feedback loop that maintains a constant tapping amplitude. This article seeks to illuminate these limits to scanning speed. The limits to the feedback loop are: (1) slow transient response of probe; (2) instability limitations of high-quality factor (Q) systems; (3) feedback actuator bandwidth; (4) error signal saturation; and the (5) rms-to-dc converter. The article will also suggest solutions to mitigate these limitations. These limitations can be addressed through integrating a faster feedback actuator as well as active control of the dynamics of the cantilever.

High-speed atomic force microscopy in liquid

High-speed constant force imaging with the atomic force microscope (AFM) has been achieved in liquid. By using a standard optical lever AFM, and a cantilever with an integrated zinc oxide (ZnO) piezoelectric actuator, an imaging bandwidth of 38 kHz has been achieved; nearly 100 times faster than conventional AFMs. For typical samples, this bandwidth corresponds to tip velocities in excess of 3 mm/s. High-speed AFM imaging in liquid will (1) permit chemical and biological AFM observations to occur at speeds previously inaccessible, and (2) significantly decrease measurement times in standard AFM liquid operation.

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Mercury vapor detection with a self-sensing, resonating piezoelectric cantilever

A microcantilever with an integrated piezoelectric film is demonstrated as a mercury vapor detector. The cantilever is self-sensing and self-actuating, and therefore does not need alignment of an external, optical detection system. This gives the new sensor system an advantage in array applications. Mercury vapor, when adsorbed onto gold on the cantilever, causes the stiffness, and therefore the natural frequency, of the cantilever to increase as a result of mercury gold amalgamation. This shift is detected using the piezoelectric portion of the cantilever in conjunction with a bridge circuit and amplifier. A mercury concentration of 93 ppb in nitrogen is detected.

Curcumin nanodisks: formulation and characterization

UNLABELLED Nanodisks (NDs) are nanoscale, disk-shaped phospholipid bilayers whose edge is stabilized by apolipoproteins. In the present study, NDs were formulated with the bioactive polyphenol curcumin at a 6:1 phospholipid-to-curcumin molar ratio. Atomic force microscopy revealed that curcumin-NDs are particles with diameters <50 nm and the thickness of a phospholipid bilayer. When formulated in NDs, curcumin is water soluble and gives rise to a characteristic absorbance spectrum with a peak centered at 420 nm. Fluorescence spectroscopy of curcumin-NDs provided evidence of self-quenching. Incubation of curcumin-NDs with empty NDs relieved the self-quenching, indicating redistribution of curcumin between curcumin-loaded and empty NDs. In HepG2 cells, curcumin-NDs mediated enhanced cell growth inhibition as compared with free curcumin. In a cell culture model of mantle cell lymphoma, curcumin-NDs were a more potent inducer of apoptosis than free curcumin. The nanoscale size of the complexes, combined with their ability to solubilize curcumin, indicates NDs may have in vivo therapeutic applications. FROM THE CLINICAL EDITOR Nanodisks (NDs), disk-shaped phospholipid bilayers stabilized by apolipoproteins, are shown entrap curcumin and improve its delivery to HepG2 and mantle cell lymphoma cells in culture. These novel nanocomplexes demonstrate interesting therapeutic application potentials.