Biological assays have dramatically improved in recent years due to the increasing use of living cells as "test tubes" for research studies. These cell-based assays have demanded that new technologies be developed for the life sciences in order to fully exploit the potential of designer drugs, stem cell engineering, and genetic medicine. The Allbritton Group is at the forefront of this technology development for biomedical and pharmaceutical research.
Micropallet Technology
In the area of cloning for cancer and stem cell studies, the Allbritton group demonstrated a novel and effective approach for the isolation of specific, single cells from a population of cells. Using principles borrowed from the electronics industry, microengineered arrays of extremely small structures (30 – 50 microns) termed micropallets are fabricated on the surface of a microscope slide. A laser is used to detach an individual micropallet and its attached cell from the slide whereupon it is collected. This strategy has been demonstrated for single-cell isolation with unprecedented survival and colony forming ability of single cells (>85%), thus dramatically improving the cloning process. This tool is now under development in an NIH-funded project with Mike Ramsey in the Department of Chemistry and colleagues in the Lineberger Cancer Center's Animal Models Facility to improve the process for creating genetically modified mice for medical research.
The Weeks laboratory is inventing high-throughput technologies for analyzing the structure of RNA. In one recent result, the lab has reported the architecture and secondary structure of an entire HIV genome. Ongoing work focuses on creating new RNA chemistries, on drug discovery, and on analyzing RNA structure-function relationships inside viruses and cells.
Francis Preston Venable Professor of Chemistry Joseph L. Templeton has been awarded the General Alumni Association's Faculty Service Award. The award was established in 1990 and honors faculty members who have performed outstanding service for the University or the association. Templeton was the faculty representative on the GAA board for 2009-10.
As Templeton — who served as chair of the chemistry department from 1990 to 1995 — has continued to teach a full course load, mentor graduate students, apply for grants and run his own research projects, he also has taken on several high-profile administrative assignments. Among other duties, he served as chair of the Faculty Council from 2006 to 2009, served as chair of the Summer Reading Program Book Selection Committee, on the Chancellor’s Advisory Committee, and the Faculty Executive Committee.
Brain norepinephrine and dopamine regulate a variety of critical behaviors such as stress, learning, memory, and drug addiction. In a study by the Wightman Group, published in the Journal of Neurochemistry, Jinwoo Park, Pavel Takmakov, and professor Wightman demonstrate differences in the regulation of in vivo neurotransmission for dopamine in the anterior nucleus accumbens (NAc) and norepinephrine in the ventral bed nucleus of the stria terminalis (vBNST) of the anesthetized rat.
Release of the two catecholamines was measured simultaneously using fast-scan cyclic voltammetry at two different carbon-fiber microelectrodes, each implanted in the brain region of interest. Simultaneous dopamine and norepinephrine release was evoked by electrical stimulation of a region where the ventral noradrenergic bundle, the pathway of noradrenergic neurons, courses through the ventral tegmental area/substantia nigra, the origin of dopaminergic cell bodies. The release and uptake of norepinephrine in the vBNST were both significantly slower than for dopamine in the NAc.
The dual-microelectrode fast-scan cyclic voltammetry technique along with anatomical and pharmacological evidence confirms that dopamine in the NAc and norepinephrine in the vBNST can be monitored selectively and simultaneously in the same animal. The high temporal and spatial resolution of the technique enables researchers to examine differences in the dynamics of extracellular norepinephrine and dopamine concurrently in two different limbic structures.
Histone lysine methylation is a critical marker for controlling gene expression. The position and extent of methylation controls the binding of effector proteins that determine whether the associated DNA is expressed or not. Dysregulation of histone protein methylation has been associated with a number of types of cancer, and development of inhibitors for the effector proteins is becoming an active area of research.
Mutation studies performed by scientists in the Waters Group, published in ChemBioChem, provide insight into the role of electrostatic interactions and hydrogen bonding in the differentiation of methylation states and have implications regarding the evolutionary pressure for selectivity in this protein–protein interaction. Moreover, the information from this study may help guide inhibitor development for this class of proteins.
Sample transport and electrokinetic injection bias are well characterized in capillary electrophoresis and simple microchips, but a thorough understanding of sample transport on devices combining electroosmosis, electrophoresis, and pressure-driven flow is lacking. In work published in Electrophoresis, researchers from the Allbritton Group evaluate the effects of electric fields from 0 to 300 V/cm, electrophoretic mobilities from 10-4 to 10-6 cm2/Vs, and pressure-driven fluid velocities from 50 to 250 µm/s on sample injection in a microfluidic chemical cytometry device. By studying a continuous sample stream, they found that increasing electric field strength and electrophoretic mobility result in improved injection and that COMSOL simulations accurately predict sample transport.
The effects of pressure-driven fluid velocity on injection are complex, and relative concentration values lie on a surface defined by pressure-driven flow rates. For high-mobility analytes, this surface is flat, and sample injection is robust despite fluctuations in flow rate. For lower mobility analytes, the surface becomes steeper, and injection depends strongly on pressure-driven flow. These results indicate generally that device design must account for analyte characteristics and specifically that this device is suited to high-mobility analytes. The team demonstrates that for a suitable pair of peptides fluctuations in injection volume are correlated; electrokinetic injection bias is minimized; and electrophoretic separation is achieved.
Mesoporous silica nanospheres (MSNs) are a promising material for magnetic resonance imaging (MRI) contrast enhancement, as they can carry high loadings of Gd(III) complexes. MSN-based MRI contrast agents can circumvent many of the limitations of small molecule contrast agents such as low contrast enhancement efficiency, potential toxicity, and the inability to specifically target disease tissues. Nanoparticle-based MRI contrast agents must be cleared in a timely fashion to avoid the long-term toxicity.
Researchers in the Lin Group, as published in the journal Small, report the incorporation of a cleavable Gd(III) chelate into the MSN material such that the chelate is rapidly cleared after injection. The material was further functionalized with poly(ethylene glycol) and a targeting ligand to impart biocompatibility and target specificity. The effectiveness of this material as a MRI contrast agent was effectively demonstrated in vivo with human colon and pancreatic adenocarcinoma cells; the chelate was successfully cleaved and cleared via the renal excretion pathway.
Published in the Journal of Physical Chemistry, C, Alessa Gambardella, Natalie Bjorge, and Katherine Alspaugh in the Murray Group, describe and compare the pH-dependent solution voltammetry (pH 1–13) of phosphate-stabilized, small (2 nm diameter) iridium oxide nanoparticles (IrIVOx NPs) with that of (flocculated) films of the same size nanoparticles on electrodes. The IrIVOx NPs show waves with one electron/one proton formal potential dependency for the IrV/IV redox transformation and (below pH 6) for the IrIV/III reaction. Above pH 6, the IrIV/III reaction becomes a one-electron/two-proton process, unlike the one-electron/one-proton reactivity of the nanoparticles in films.
The change is associated with surface oxide acid–base sites having pKA = 6 for solution phase nanoparticles that apparently are inactivated by the flocculation chemistry. Spectral isosbestic points are observed over the pH range of 5–8 for the dissolved nanoparticles. Controlled potential coulometry demonstrates that all of the Ir sites, throughout the nanoparticle, undergo the IrIV/III redox transformation. Whereas the IrIVOx NPs) are stabilized at different pH values by phosphate ligation, the associated equilibria are somewhat sluggish, as indicated by small spectral differences for equi-pH nanoparticle solutions mixed with phosphate by different procedures. The IrIVOx NPs) can also be capped with hydrophobic carboxylic acids, which allows extraction into nonpolar solvents such as CH2Cl2.