Characterization of Thiol-Ene Crosslinked PEG Hydrogels.

The properties of synthetic hydrogels can be tuned to address the needs of many tissue-culture applications. This work characterizes the swelling and mechanical properties of thiol-ene crosslinked PEG hydrogels made with varying prepolymer formulations, demonstrating that hydrogels with a compressive modulus exceeding 600 kPa can be formed. The amount of peptide incorporated into the hydrogel is shown to be proportional to the amount of peptide in the prepolymer solution. Cell attachment and spreading on the surface of the peptide-functionalized hydrogels is demonstrated. Additionally, a method for bonding distinct layers of cured hydrogels is used to create a microfluidic channel.

Regulating specific growth factor signaling using immobilized branched ligands.

VEGF-binding peptide ligands are incorporated into hydrogel microspheres and reduce the amount of growth factor in solution. VEGF binding affinity is enhanced by creating ligands with a dimer structure. The spheres are able to knock down VEGF-mediated HUVEC growth and reduce calcium signaling. The binding interaction is reversible, allowing the spheres to be used as a VEGF delivery vehicle.

Specific VEGF sequestering and release using peptide-functionalized hydrogel microspheres.

Growth factor signaling plays an essential role in regulating processes such as tissue development, maintenance, and repair. Gene expression levels, diffusion, degradation, and sequestration by extracellular matrix components all play a role in regulating the concentration of growth factors within the cellular microenvironment. Herein, we describe the synthesis and characterization of hydrogel microspheres that mimic the ability of the native extracellular matrix to reversibly bind vascular endothelial growth factor (VEGF) out of solution. A peptide ligand derived from the VEGF receptor 2 (VEGFR2) was covalently incorporated into the hydrogel microspheres in order to achieve binding affinity and specificity. In addition to being able to both bind and release VEGF in a controllable manner, the microspheres were also shown to affect human umbilical vein endothelial cell (HUVEC) proliferation. The resulting microspheres may enable new strategies to specifically upregulate or downregulate growth factor signaling in the cellular microenvironment.

Identification of a functionally important loop in Salmonella typhimurium ArnT.

ArnT confers resistance to the antibiotic polymyxin in Salmonella typhimurium and Escherichia coli through the modification of lipid A, a major component of the outer surface of gram-negative bacteria. ArnT transfers a neutral aminoarabinose moiety onto the negative phosphate groups of lipid A, reducing the surface charge of the bacteria and preventing cationic peptides such as polymyxin from electrostatically recognizing and killing the bacteria. We previously reported the first expression, purification, and functional analysis of ArnT from S. typhimurium [Bretscher, L. E., Morrell, M. T., Funk, A. L., and Klug, C. S. (2006) Protein Expression Purif. 46, 33-39]. Our studies showed that ArnT is highly alpha-helical and described a new in vivo functional growth assay. Here, we use the cysteine-specific mPEG-mal to demonstrate that all eight of the native cysteines in S. typhimurium ArnT are in the reduced form and not involved in disulfide bonds and show that the cysteine-free protein is structurally and functionally intact as characterized by circular dichroism and the in vivo growth assay. Following this initial characterization, in vivo expression and function profiles were surveyed for 31 consecutive mutations within a putative ArnT loop. These studies identify for the first time 14 residues that are essential for function of the ArnT transferase and 3 additional residues that completely disrupt protein folding or insertion into the bacterial inner membrane.