Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qβ replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system.

Liposome display for in vitro selection and evolution of membrane proteins.

Liposome display is a novel method for in vitro selection and directed evolution of membrane proteins. In this approach, membrane proteins of interest are displayed on liposome membranes through translation from a single DNA molecule by using an encapsulated cell-free translation system. The liposomes are probed with a fluorescence indicator that senses membrane protein activity and selected using a fluorescence-activated cell sorting (FACS) instrument.

Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes.

Directed evolution of proteins is a technique used to modify protein functions through "Darwinian selection." In vitro compartmentalization (IVC) is an in vitro gene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment.

Construction of a gene screening system using giant unilamellar liposomes and a fluorescence-activated cell sorter.

We have constructed a gene screening system composed of an in vitro transcription-translation system encapsulated within giant unilamellar liposomes and a fluorescence-activated cell sorter (FACS), which allows high-throughput screening of genes encoding proteins of interest. A mock gene library of β-glucuronidase (GUS) was compartmentalized into liposomes at the single-molecule level, and liposomes exhibiting green fluorescence derived from hydrolysis of the fluorogenic substrate by the synthesized enzyme were sorted using FACS. More than 10-fold enrichment of GUS gene with higher catalytic activity was obtained when a single copy of the GUS gene was encapsulated in each liposome.

Nitric oxide release in human aortic endothelial cells mediated by delivery of amphiphilic polysiloxane nanoparticles to caveolae.

Microdomains such as lipid raft and caveolae are organized as functional compartments in plasma membrane of cells. In this study, we note the functional platform of caveolae with dual functions, internalization of external substances and cell signalings leading to nitric oxide release, and hypothesize that the switching of enzyme activity of endothelial nitric oxide synthase can be achieved by targeting caveolae with nanoparticles. We prepared polysiloxane nanoparticles and studied cellular uptake of the nanoparticles and its concomitant influence on the nitric oxide release in human aortic endothelial cells.

Substrata mechanical stiffness can regulate adhesion of viable bacteria.

The competing mechanisms that regulate adhesion of bacteria to surfaces and subsequent biofilm formation remain unclear, though nearly all studies have focused on the role of physical and chemical properties of the material surface. Given the large monetary and health costs of medical-device colonization and hospital-acquired infections due to bacteria, there is considerable interest in better understanding of material properties that can limit bacterial adhesion and viability. Here we employ weak polyelectrolyte multilayer (PEM) thin films comprised of poly(allylamine) hydrochloride (PAH) and poly(acrylic acid) (PAA), assembled over a range of conditions, to explore the physicochemical and mechanical characteristics of material surfaces controlling adhesion of Staphylococcus epidermidis bacteria and subsequent colony growth.

Amphiphilic poly(N-propargylamide) with galactose and lauryloyl groups: synthesis and properties.

An amphiphilic poly(N-propargylamide) with galactose and lauryloyl groups was synthesized by copolymerization of the corresponding N-propargylamide monomers using a Rh catalyst. The obtained copolymer formed a one-handed helical conformation and molecular aggregates in water. The observations by fluorescence microscopy in a cell culture experiment in the presence of dye-labeled copolymer indicated that the copolymer was incorporated into the cells.