Kuo-Sheng Ma

c-Jun triggers apoptosis in human vascular endothelial cells.

In endothelial cells (ECs), the transcription factor c-Jun is induced by a variety of stimuli that perturb EC function. To extend our understanding of the role of c-Jun in EC physiology, we have directed overexpression of c-Jun in human umbilical vein ECs by using a tetracycline-regulated adenoviral expression system. In this study, we report a novel observation using this system. Specific expression of c-Jun is a sufficient trigger for ECs to undergo apoptosis, as demonstrated by a set of combined assays including an ELISA specific for histone-associated DNA fragmentation, DNA laddering, and TdT-mediated dUTP nick end labeling (TUNEL). Tetracycline can effectively shut off c-Jun overexpression and prevent EC apoptosis. Cleavage of poly(ADP-ribose) polymerase was also detected in ECs overexpressing c-Jun. Moreover, inhibitors of cysteine proteases blocked the apoptosis, suggesting a caspase-associated mechanism involved in proapoptotic effects of c-Jun. To gain further insight into the role of c-Jun as a pathophysiological regulator of EC death, TAM67, a dominant-negative mutant of c-Jun, was overexpressed in human umbilical vein ECs to abrogate endogenous c-Jun/activator protein-1 activation. H(2)O(2)-triggered apoptosis was largely attenuated in ECs overexpressing TAM67. Together, these results suggest that c-Jun, as a proapoptotic molecule, may play a role in mediating the cell death program in vascular endothelium.

Lipoprotein Promotes Caveolin-1 and Ras Translocation to Caveolae Role of Cholesterol in Endothelial Signaling

To explore the role of LDL in caveolin-Ras regulation in human endothelial cells (ECs), we incubated confluent human umbilical vein endothelial cells (HUVECs) with LDL. This resulted in a high steady-state caveolin-1 (Cav-1) expression at both the mRNA and protein levels. LDL exposure appeared not to regulate the abundance of Cav-1. Immunofluorescence staining showed that Cav-1 protein migrated from the cytoplasm to the cell membrane after LDL exposure. Cav-1 protein and cholesterol partitioned mainly into the caveola fractions, and LDL increased both Cav-1 and cholesterol in these fractions. Ras protein in caveola fractions was also increased by LDL. Increased Ras was detected in Cav-1 immunoprecipitated samples, and conversely, increased Cav-1 was found in Ras-immunoprecipitated samples. We also demonstrated LDL-increased Ras activity in HUVECs by measuring the GTP/GTP+GDP ratio of Ras with [32P]orthophosphate labeling in the cells. Finally, we determined the binding of [3H]-labeled free cholesterol and recombinant H-Ras to Cav-1 fusion proteins in vitro. Both cholesterol and Ras bound to full-length GST–Cav-1, scaffolding domain (61–101), and C-terminal (135–178) Cav-1 fusion peptides. Addition of cholesterol enhanced Ras binding to the full-length and scaffolding domain of Cav-1 but not to the C-terminal Cav-1. These findings strongly suggest a role for Cav-1 in cholesterol trafficking and cholesterol-mediated intracellular signaling, which may mediate EC activation by LDL.

In situ Synthesis of DNA Microarray on Functionalized Cyclic Olefin Copolymer Substrate

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Versatile surface functionalization of cyclic olefin copolymer (COC) with sputtered SiO2 thin film for potential BioMEMS applications

Cyclic olefin copolymer (COC) is a new class of thermoplastic polymers used for a variety of applications ranging from bio-sensing to optics. However, lack of functional groups and intense surface hydrophobicity hamper further development and application of this material. Here, we describe fabrication and characterization of SiO2–COC hybrid material (oxCOC), which provides a desirable substrate for microfluidic devices and subsequent surface modifications. The deposited SiO2 thin film on COC surface was characterized by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The new SiO2–COC hybrid was found to have similar high optical transmission properties as that of pristine COC. Profilometric and AFM analysis revealed no dramatic change in morphology or surface roughness of functionalized COC. The SiO2–COC hybrid appeared to be stable in most of the solvents evaluated and could be further modified by other compounds, such as 3-aminopropyltriethoxy silane (APTES). The new SiO2–COC hybrid material and the robust fabrication method are expected to enable a variety of BioMEMS applications.

Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells.

Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-kappaB (NF-kappaB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-kappaB-dependent expression was not affected. The AP-1/Ets (-891 to -908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression.

Development of integrated protection for a miniaturized drug delivery system

A bi-layer structure comprising a thin gold layer and a polypyrrole (PPy) film is developed as a valve. To create such structures, the thin metal layer is used as a working electrode, and the polypyrrole film is electrochemically deposited on the metal electrode. A layer of Cr deposited under a stationary part of the bi-layer flap adheres strongly to the gold layer and anchors the hinge of the flap, while the layer of polyimide deposited under a movable part of the flap adheres weakly to the gold layer of the flap. This bi-layer flap structure functions as an actuator valve for the opening and closing of an aperture. The actuation mechanism of the valve is based on a volume change in the PPy layer as cations move in and out of the polymer film during reduction and oxidation reactions. A bias of 1.2 V is used to actuate the flap. A method is proposed to increase the drug release systems functionality by placing the drug release flap within a protective enclosure that also serves as a drug reservoir. This integrated protection ensures reliable operation of the drug release flap unencumbered by surrounding tissues when used in vivo. A prototype system using a PDMS drug reservoir has been successfully tested in PBS buffer solution. The proposed integrated protection system holds promise for implantable biomedical devices.

Fabrication of plastic biochips

A versatile surface functionalization procedure based on rf magnetron sputtering of silica was performed on poly(methylmethacrylate), polycarbonate, polypropylene, and cyclic olefin copolymers (Topas 6015). The hybrid thermoplastic surfaces were characterized by x-ray photoelectron spectrometer analysis and contact angle measurements. The authors then used these hybrid materials to perform a sandwich assay targeting an HIV-1 antibody using fluorescent detection and biotinylated peptides immobilized using the bioaffinity of biotin-neutravidin. They found a limit of detection similar to arrays on glass surfaces and believed that this plastic biochip platform may be used for the development of disposable immunosensing and diagnostic applications.

Detection of DNA Hybridization by Adjacent Impedance Probing

In this paper we present the novel adjacent impedance probing (AIP) technique for DNA hybridization detection. In our design, the DNA hybridization site was employed only for the biorecognition event (this site does not necessarily need an underlying conductor surface). A bare adjacent electrode was used for detection of an impedance change. An enzymatic reporter produced the deposition of an insulator on the adjacent electrode. The adjacent impedance probing (AIP) technique is employed to alleviate the low-signal or high-noise problems caused by ssDNA capture probes adsorbed (nonspecifically through non-Au-S binding) on the electrode surface. Data showed that hybridization resulted in a twofold increase in impedance.

Low-cost and portable labware for computing curriculum using scalable mobile sensory platform

Mobile embedded system is an excellent candidate to provides depth, breadth, and rigorousness for meeting the emerging workforce and education needs in science, technology, and engineering. However, the high requirements of investment in resources and instructors make the mobile embedded system education impractical for universities and colleges that lack the resources and build-ups. This work-in-progress paper presents a novel low-cost and portable labware for hands-on labs and projects using Android smartphones and scalable sensory platform. It is easy-to-adopt, promotes students with authentic and creative learning, and supports wide dissemination.

Optimized in situ DNA synthesis on patterned glass

This paper describes studies of patterned arrays on glass surfaces and their use as spatially separated reactors for in situ synthesis of DNA using an inkjet synthesizer. Photolithographic methods were employed to fabricate arrays composed of homogenous circular features containing a hydroxyl-terminated silane coupled to the surface of the glass via a siloxane bond. Features are embedded within a background matrix composed of a fluorosilane attached to the glass. Due to the differential wettability of the two silanes, whereby the hydroxyl-terminated silane and fluorosilane are hydrophilic and hydrophobic respectively because of their head groups, the patterned circular features are able to constrain liquid within a defined site. The silanization result was analyzed using X-ray photoelectron spectroscopy (XPS) to optimize silanization time and solvent. Synthesis was then performed using a custom-built inkjet system using phosphoramidite chemistry. Base-by-base analysis using fluorescent labeling showed consistent coupling efficiency on synthesis of a 50-mer homopolymer.