Structure and transfer of the vanA cluster in vanA-positive, vancomycin-susceptible Enterococcus faecium, and its revertant mutant.

Of 18 vanA-positive vancomycin-susceptible Enterococcus faecium isolates, vanRS in the vanA cluster was detected in all isolates, while vanHAX was detected in only 2 isolates. Following exposure to glycopeptides, 22.2% of vancomycin-susceptible E. faecium (VSE) converted into vancomycin-resistant E. faecium. The vanA cluster of the revertant mutant was transferred to the VSE isolates.

Characterization of two newly identified genes, vgaD and vatH, [corrected] conferring resistance to streptogramin A in Enterococcus faecium.

We characterized two new streptogramin A resistance genes from quinupristin-dalfopristin-resistant Enterococcus faecium JS79, which was selected from 79 E. faecium isolates lacking known genes encoding streptogramin A acetyltransferase. A 5,650-bp fragment of HindIII-digested plasmid DNA from E. faecium JS79 was cloned and sequenced. The fragment contained two open reading frames carrying resistance genes related to streptogramin A, namely, genes for an acetyltransferase and an ATP efflux pump. The first open reading frame comprised 648 bp encoding 216 amino acids with a predicted left-handed parallel ß-helix domain structure; this new gene was designated vatH. [corrected] The second open reading frame consisted of 1,575 bp encoding 525 amino acids with two predicted ATPase binding cassette transporters comprised of Walker A, Walker B, and LSSG motifs; this gene was designated vgaD. vgaD is located 65 bp upstream from vatH, [corrected] was detected together with vatH [corrected] in 12 of 179 quinupristin-dalfopristin-resistant E. faecium isolates, and was located on the same plasmid. Also, the 5.6-kb HindIII-digested fragment which was observed in JS79 was detected in nine vgaD- and vatH-containing [corrected] E. faecium isolates by Southern hybridization. Therefore, it was expected that these two genes were strongly correlated with each other and that they may be composed of a transposon. Importantly, vgaD is the first identified ABC transporter conferring resistance to streptogramin A in E. faecium. Pulsed-field gel electrophoresis patterns and sequence types of vgaD- and vatH-containing [corrected] E. faecium isolates differed for isolates from humans and nonhumans.

Simple fabrication of a smart microarray of polystyrene microbeads for immunoassay.

We describe a simple method to fabricate an array of polystyrene microbeads (PS microbeads) conjugated with an elastin-like polypeptide (ELP) on a glass surface using a removable polymer template (RPT). A thin layer of adhesive was spun-cast on glass and cured by UV radiation. Micropatterns of an RPT were then transferred onto the surface by microcontact printing. The adhesion of PS microbeads on the surface depended on the adhesion performance of the adhesive layer, which could be adjusted by irradiation time. An array of PS microbeads conjugated with ELP was used for a smart immunoassay of prostate-specific antigen (PSA), a cancer marker. By controlling the phase transition of ELP molecules, PSA molecules were selectively adhered or released from the bead surface. The selective and reversible binding of PSA molecules on the bead surface was characterized with fluorescence microscopy.

Polystyrene microbeads modified with an elastin-like biopolymer for stimuli-responsive immunodetection.

An efficient and controlled method for immunodetection, using polystyrene (PS) microbeads conjugated with an elastin-like polypeptide (ELP), was investigated. ELP is a temperature-sensitive polymer that exhibits a hydrophilic-hydrophobic phase transition at the lower critical solution temperature. To introduce amine groups, ELP was modified with lysine for conjugation with PS microbeads functionalized with carboxylic groups. Prostate-specific antigen (PSA), a cancer marker, was detected from a biomolecular mixture using ELP-conjugated PS microbeads and ELP-conjugated antiPSA. External stimuli were used to reversibly separate the PSA-antiPSA complex. The stimuli-responsiveness of the ELP provided a designable generic biosurface for diagnostic detection.

"Smart" biopolymer for a reversible stimuli-responsive platform in cell-based biochips.

The rapid response of a smart material surface to external stimuli is critical for application to cell-based biochips. The sharp and controllable phase transition of elastin-like polypeptide (ELP) enabled reversible cell adhesion on the surface by changing the temperature or salt concentration in the system. First, ELP micropatterns were prepared on a glass surface modified into aldehyde. The lysine-containing ELP (ELP-K) was genetically synthesized from E. coli for conjugation with the aldehyde on the glass surface. The phase transition of ELP was monitored in PBS and cell culture media using UV-visible spectroscopy, and a significant difference in transition temperature (Tt) was observed between the two solution systems. The micropatterning of ELP on the glass surface was performed by microcontact printing a removable polymeric template on the aldehyde-glass followed by incubation in ELP-K aqueous solution. The ELP micropatterns were imaged with atomic force microscopy and showed a monolayer thickness of approximately 4 nm. Imaging from time-of-flight secondary ion mass spectroscopy confirmed that the ELP molecules were successfully immobilized on the highly resolved micropatterns. Cell attachment and detachment could be reversibly controlled on the ELP surfaces by external stimuli. The hydrophobic phase above Tt resulted in the adhesion of fibroblasts, while the detachment of cells was induced by lowering the incubation temperature below Tt. The smart properties of ELP were reliable and reproducible, demonstrating potential applications in cell-based microdevices.

A cell-repellent sulfonated PEG comb-like polymer for highly resolved cell micropatterns.

This paper investigates the chemical modification of a cell-repellent poly(ethylene glycol) (PEG)-based polymer to enhance its hydrophilicity with sulfonate groups, and its application in the fabrication of a cell microarray. First, a polymer comprised of a methyl methacrylate (MMA) backbone with PEG side-chains (PMMA-b-PEG) was synthesized from three monomers by radical polymerization and purified. Despite the hydrophilic side-groups in the amphiphilic polymer, the backbone structure's hydrophobicity allows for local adsorption of biomolecules in incubation media with or without serum. To enhance the hydrophilicity of the polymer, we tethered sulfonate groups to the hydroxyl groups on the PEG side chains (PMMA-b-PEG-SO3). The sulfate groups' physical and mechanical movement competitively repels biomolecules approaching the PMMA-b-PEG surface. Polymers modified with sulfonate were characterized by contact angle measurement, FT-IR, NMR, AFM and GPC. PMMA-b-PEG and PMMA-b-PEG-SO3 were successfully micropatterned on polystyrene and glass surfaces, and cell attachment was performed in either serum-free or serum-containing media, resulting in highly resolved cell micropatterns.

Polymeric optical microscreen for high-resolution surface plasmon resonance microarray imaging.

In this study, we demonstrate a simple method to fabricate surface plasmon resonance (SPR) imaging microarrays using polymer micropatterns. The use of a micrometer-scale polymeric optical screen (microPOS) passivates the region deposited with polymer by completely removing SPR signals or by saturating the SPR signal far beyond the detection range of SPR imaging. Two schemes were suggested to create a surface microPOS by either micropatterning a thick insulating layer before deposition of a metal layer (complete removal of SPR) or after deposition of a metal layer (saturation of SPR signal). The two schemes were successfully applied for the imaging of biological adsorption with a high imaging resolution of approximately 100 microm/pattern and 10 microm separation. The validity of the system was verified with a biotin-streptavidin system as a model for the systematic binding of biomolecules. Further, binding of prostate-specific antigen (PSA) onto the anti-PSA SPR microarray was demonstrated as a useful method for detecting a cancer marker.