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The purple sea urchin, Strongylocentrotus purpuratus , possesses a sophisticated innate immune system that functions without adaptive capabilities and responds to pathogens effectively by expressing the highly diverse SpTransformer gene family (formerly the Sp185/333 gene family). The swift gene expression response and the sequence diversity of SpTransformer cDNAs suggest that the encoded proteins have immune functions. Individual sea urchins can express up to 260 distinct SpTransformer proteins, and their diversity suggests that different versions may have different functions. Although the deduced proteins are diverse, they share an overall structure of a hydrophobic leader, a glycine-rich N-terminal region, a histidine-rich region, and a C-terminal region. Circular dichroism analysis of a recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1) demonstrates that it is intrinsically disordered and transforms to α helical in the presence of buffer additives and binding targets. Although native SpTrf proteins are associated with the membranes of perinuclear vesicles in the phagocyte class of coelomocytes and are present on the surface of small phagocytes, they have no predicted transmembrane region or conserved site for glycophosphatidylinositol linkage. To determine whether native SpTrf proteins associate with phagocyte membranes through interactions with lipids, when rSpTrf-E1 is incubated with lipid-embedded nylon strips, it binds to phosphatidic acid (PA) through both the glycine-rich region and the histidine-rich region. Synthetic liposomes composed of PA and phosphatidylcholine show binding between rSpTrf-E1 and PA by fluorescence resonance energy transfer, which is associated with leakage of luminal contents suggesting changes in lipid organization and perhaps liposome lysis. Interactions with liposomes also change membrane curvature leading to liposome budding, fusion, and invagination, which is associated with PA clustering induced by rSpTrf-E1 binding. Longer incubations result in the extraction of PA from the liposomes, which form disorganized clusters. CD shows that when rSpTrf-E1 binds to PA, it changes its secondary structure from disordered to α helical. These results provide evidence for how SpTransformer proteins may associate with molecules that have exposed phosphates including PA on cell membranes and how the characteristic of protein multimerization may drive changes in the organization of membrane lipids.

Naja atra cathelicidin (NA-CATH) is a 34-amino acid highly cationic peptide identified in Chinese cobras to possess potent toxicity against gram-negative and gram-positive bacteria and low toxicity against host cells. Here, we report the NMR solution structure of the full-length NA-CATH peptide and its interaction with liposomes. The structure shows a well-defined α-helix between residues Phe3 to Lys23, on which one surface is lined by the side-chains of one arginine and 11 lysine residues, while the other side is populated by hydrophobic residues. The last eleven amino acids, which are predominately aromatic and hydrophobic in nature, have no defined structure. NMR data reveal that these residues do not interact with the hydrophobic residues of the helix, indicating that the C-terminal residues have random conformations. Fluorescence requenching experiments, in which liposomes serve as a mimic of the bacterial membranes, result in fluorophore leakage that is consistent with a membrane thinning or transient pore formation mechanism. NMR titration studies of the peptide-liposome interaction reveal that the peptide is in fast exchange with the liposome, consistent with the fluorescent studies. These data indicate that full length NA-CATH possesses a helical segment and unstructured C-terminal tail that disrupts the bilayer to induce leakage and lysing., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Poly(dimethylsiloxane), PDMS, a versatile elastomer, is the polymer of choice for microfluidic systems. It is inexpensive, relatively easy to pattern, and permeable to oxygen. Unmodified PDMS is highly hydrophobic. It is typically exposed to an oxygen plasma to reduce this hydrophobicity. Unfortunately, the PDMS surface soon returns to its original hydrophobic state. We present two alternative plasma treatments that yield long-term modification of the wetting properties of a PDMS surface. An oxygen plasma pretreatment followed by exposure to a SiCl4 plasma and an oxygen-CCl4 mixture plasma both cause a permanent reduction in the hydrophobicity of the PDMS surface. We investigate the properties of the plasma-treated surfaces with X-ray photoelectron spectroscopy (XPS) and contact angle measurements. We propose that the plasma treated PDMS surface is a dynamic mosaic of high- and low-contact-angle functionalities. The SiCl4 and CCl4 plasmas attach polar groups that block coverage of the surface by low-molecular-weight groups that exist in PDMS. We describe an application that benefits from these new plasma treatments, the use of a PDMS stencil to form dense arrays of DNA on a surface.

We have investigated the effects of cross-linking perturbations on lipid phase-domain coalescence. Our model system explores cross-linking in the fluid-disordered phase of two-phase vesicles. Here, we quantify the vesicle population shift from the expected predominance of two-domain, two-phase configuration to a multidomain vesicle majority. We have found that the increase in multidomain vesicles is a distinct outcome from the cross-linking of biotinylated lipids and avidin. Analysis of our cross-linking data suggests that avidin forms clusters on the surface of the fluid-disordered domains, resulting in a large immobile fraction and restricted diffusion. In cellular membranes, receptor concentrations are similar to our experimental model, and we expect similar cluster formations, leading to nonideal mixing and lateral heterogeneity. We have induced and quantified a global response by cross-linking only a small percentage of lipids in our system, similar to receptor-ligand interactions on the cell membrane. Common activities, such as ligand-receptor coupling, contribute to lateral heterogeneity and membrane protein clustering, adding to cell membrane complexity. Fundamental studies into subtle shifts such as cross-linking events, which induce global cellular response, are pertinent to understanding membrane activities and effects of external stimuli.

We have examined the deformation of vesicles on surface topography. From known vesicle shape dynamics, we expect temperature to play a major role; however, when cooling vesicles in the presence of a low-permeability solute, the mechanism of deflation is not clear. We investigate giant unilamellar vesicles on topography to quantify their cooling over a range of temperatures. The volume constraints point to a transient defect pore model as being responsible for vesicle deflation over the temperature range studied.

The familiar biconcave shape of the red-blood cell (RBC) deforms as the cell travels through capillaries. Its dimpled configurations are unique cell shapes and display malleability to form echinocytes, discocytes and stomatocytes, in response to external perturbations. Sheetz and Singer introduced intercalating species to the exterior lipid leaflet of the membrane to promote cup-shaped stomatocytes, and observed that additives to the interior had the opposite effect. Shape transformations appear to be controlled via the RBC bilayer and the asymmetric surface areas of the two leaflets [Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4457]. Our system promotes area-difference between the lipid bilayer leaflets from a fully symmetrical system and has mimicked the RBC discoid. In our analysis, we explore the system energetic and geometric confinements, which points to transient pores as enablers for the vesicles to deflate and thereby to assume lower profiles.

We describe a new patterning technique that employs microcontact printing to replace preformed labile self-assembled monolayers (SAMs) selectively; we call this "microdisplacement printing". We demonstrate that this technique results in ordered molecular regions of both the patterning ("displacing") molecule as well as the remnant labile film, here 1-adamantanethiolate. The existence of the 1-adamantanethiolate SAM before patterning hinders lateral surface diffusion of the patterning molecules, and therefore permits the use of molecules that are otherwise too mobile to pattern by other methods.

DNA computing on surfaces is where complex combinatorial mixtures of DNA molecules are immobilized on a substrate and subsets are tagged and enzymatically modified (DESTROY) in repeated cycles of the DNA computation. A restriction enzyme has been chosen for the surface DESTROY operation. For the READOUT operation, both cycle sequencing and PCR amplification followed by addressed array hybridization were studied to determine the DNA sequences after the computations.

A multi-base encoding strategy is used in a one word approach to surface-based DNA computation. In this designed DNA model system, a set of 16 oligonucleotides, each a 16mer, is used with the format 5'-FFFFvvvvvvvvFFFF-3' in which 4-8 bits of data are stored in eight central variable ('v') base locations, and the remaining fixed ('F') base locations are used as a word label. The detailed implementations are reported here. In order to achieve perfect discrimination between each oligonucleotide, the efficiency and specificity of hybridization discrimination of the set of 16 oligonucleotides were examined by carrying out the hybridization of each individual fluorescently tagged complement to an array of 16 addressed immobilized oligonucleotides. A series of preliminary hybridization experiments are presented and further studies about hybridization, enzymatic destruction, read out and demonstrations of a SAT problem are forthcoming.