Influence of Ru on structure and corrosion behavior of passive film on Ti-6Al-4V alloy in oil and gas exploration conditions.

In order to investigate the influence of minor Ru on the electrochemical behaviour and structural characteristics of passive films on the surface of Ti-6Al-4V alloys under various oil and gas exploration conditions, electrochemical techniques, X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM) and corrosion simulation tests were carried out. The results revealed that the oil and gas exploration conditions had a serious impact on the electrochemical behaviour and corrosion resistance of the tested alloys. The passivation film resistance and corrosion potential of the tested titanium alloys were significantly reduced with increasing acidity and temperature. With the addition of minor ruthenium, the potential of the passive film on the Ti-6Al-4V-0.11Ru alloy surface increased because of the high surface potential of the ruthenium element. The contents of metallic ruthenium and tetravalent titanium oxide TiO2 in the surface film of the Ti-6Al-4V-0.11Ru alloy both increased with increasing temperature, which led to increase the thickness, stability, corrosion resistance and repairability of the passive film on the surface of the Ti-6Al-4V-0.11Ru alloy being better than those qualities of Ti-6Al-4V. These results were also confirmed by corrosion simulation tests.

Yeast cell assay with a surface plasma resonance sensor at multiple penetration depths.

We demonstrated a novel yeast cell assay using a multiple depth surface plasmon resonance platform, which has three detection depths. In the study we first identified the minimal and maximum number of cells required for detection and then used the platform to conduct a drug potency study. The study showed that the rank order of potency for 3 drugs could be clearly determined and was the same regardless of the penetration depth used for the measurement. The rank order made sense based on their respective mechanism of action. However the data quality of the inhibition curve was best when measured at the 1500 nm depth. The dose dependent response was noticeable even after only 15 min treatment, which is much earlier than those from traditional methods. By comparing the response units obtained at 3 different penetration depths in various yeast cell assays, we have confirmed that simultaneously monitoring from multiple penetration depths likely offers advantages to better detect robust signals from yeast cells, which proves difficult with other optical sensors with a short penetration depth of < 300 nm. The assay could be broadly applicable to other rigid and spherical cell types for biology studies and for antibiotic discovery.

Modulating cell-cell communication with a high-throughput label-free cell assay.

A high-throughput label-free cell assay for modulating cell-cell communication is demonstrated with the Epic® system, a resonant waveguide grating sensor platform. Natural killer (NK) cells are known to be able to recognize abnormal cells (e.g., cancer cells and cells presenting intercellular adhesion molecule 1 [ICAM1] through cell surface receptors) and kill them. In this study, the effect of effecter cells NK92MI on two kinds of target cells, cervical cancer cells (HeLa) and Chinese hamster ovarian cells overexpressing ICAM1 (CHO-ICAM1), was examined. Living target cells' response to NK92MI cells was monitored in real time and measured as wavelength shift in picometers. The authors showed that the detectability of target cell response is affected by multiple factors: the ratio of effecter cells to target cells (E/T), the interaction time of the two types of cells, and the target cell type. For example, with the effecter cells NK92MI and the same incubation time of 16 h, a minimal E/T ratio of 1 is required to detect HeLa cell response, whereas an E/T of 0.5 is sufficient to detect CHO-ICAM1 cell response. The authors confirmed that NK92MI cell-mediated target cell cytotoxicity results in negative optical signals and is associated with apoptosis mainly through caspase pathways. Distinct optical signals could be generated with the pretreatment of the target cells with various known pharmaceutical reagents, making the assay useful for discovering new chemicals that may affect cell-cell communications.

Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells.

INTRODUCTION: Screening drugs against G protein-coupled receptors (GPCRs) - the single largest family of drug targets in the human genome - is still a major effort in pharmaceutical and biotech industries. Conventional cell-based assays generally measure a single cellular event, such as the generation of a second messenger or the relocation of a specific protein target. However, manipulation or engineering of cells is often a prerequisite for these technologies to achieve desired sensitivities. The present study is focused on the use of non-invasive and manipulation-free optical biosensors for assaying endogenous GPCRs in adherent cells. METHODS: Resonant waveguide grating (RWG) biosensor was applied to manifest ligand-induced dynamic mass redistribution (DMR) within the bottom portion of adherent cell layer. The DMR signatures mediated through the activation of several endogenous GPCRs in cells were characterized. Endogenous receptor panning was examined at cell system level by using a panel of agonists known to activate many GPCRs, and also at family receptor level by determining the efficacies of a set of family-specific agonists. RESULTS: Three major types of optical signatures were identified; each was correlated with the activation of a class of GPCRs, depending on the G protein with which the receptor is coupled (i.e., G(q), G(s) and G(i)). The characteristics of DMR signals, mostly the amplitude and kinetics of a DMR event, were dependent on the doses of agonists and the expression levels of endogenous receptors. All three classes of endogenous receptors were found in human epidermoid carcinoma A431 cells. Interestingly, the dose-dependent switching from one type of DMR signal to another was observed for several GPCR agonists examined. A small panel of P2Y receptor agonists exhibited distinct efficacies in three cell lines examined. DISCUSSIONS: The RWG biosensors were applicable to study the activation of endogenous GPCRs. Like second messengers or gene expression, the DMR signals obtained could be considered as novel and quantifiable physiological responses of living cells mediated through GPCRs and used for studying receptor biology.

Cellular functions of cholesterol probed with optical biosensors.

Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.

Optical biosensor provides insights for bradykinin B(2) receptor signaling in A431 cells.

The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B(2) receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B(2) specific antagonist HOE140, suggested that the optical signature is a direct result of B(2) receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B(2) receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B(2) signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B(2) receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B(2) signaling. The roles of endocytosis and cytoskeleton modulation in B(2) signaling were also demonstrated.

Probing cytoskeleton modulation by optical biosensors.

This paper reported the use of resonant waveguide grating biosensors for studying the cytoskeleton structure in cells. This was achieved by measuring the changes in mass within the bottom portion of cells upon exposure to saponin in the absence and presence of cytoskeleton modulators. Treatment of Chinese hamster ovary cells with saponin led to a dose-dependent and dynamic mass changes. When a higher concentration of saponin (> 60 microg/ml) was used, a net loss in mass was observed. This is probably resulted from the diffusion of soluble intracellular materials away from the bottom portion of cells after pore formation in the cell plasma membranes by saponin. The pretreatment of cells with actin disruption agents, cytochalasin B and latrunculin A, led to significantly increased loss in cell mass induced by either 75 or 125 microg/ml saponin. These results suggested that optical biosensors provide an attractive means to study the cytoskeleton structure and screen modulators that affect the cytoskeleton structure.

Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride.

Microarray technology is currently being used extensively in functional genomics research and modern drug discovery and development. Henceforward, tremendous application potential for this technology exists in the fields of clinical diagnostics and prognostics, pathology, and toxicology for high-throughput analysis of "disease" gene expression. However, the major hurdle now in this technology is not the performance of the arrays but rather the efficient reproducibility of the hybridization signal intensity in a fluorescence-based analysis. The sensitivity of fluorescence detection on an array is to a large extent limited by the amount of background signal arising due to nonspecifically bound probes and fluorescence that is intrinsically associated with the chip substrate and/or the attached target DNA, the so-called autofluorescence. Here, we describe a simple and efficient method to reduce autofluorescence from undetermined sources on coated glass slides with and without DNA arrays. This sodium borohydride-mediated reduction process resulted in significantly lower and more even background fluorescence. This in turn extended the dynamic range of detection and reduced the average coefficient of variation of fluorescent signal ratios on DNA microarrays in addition to improving the detection of genes that are expressed at a low level.

Microfluidic devices for fluidic circulation and mixing improve hybridization signal intensity on DNA arrays.

Reactions of biomolecules with surface mounted materials on microscope slides are often limited by slow diffusion kinetics, especially in low volumes where diffusion is the only means of mixing. This is a particular problem for reactions where only small amounts of analyte are available and the required reaction volume limits the analyte concentration. A low volume microfluidic device consisting of two interconnected 9 mm x 37.5 mm reaction chambers was developed to allow mixing and closed loop fluidic circulation over most of the surface of a microscope slide. Fluid samples are moved from one reaction chamber to the other by the rotation of a magnetic stirring bar that is driven by a standard magnetic stirrer. We demonstrate that circulation and mixing of different reagents can be efficiently accomplished by this closed loop device with solutions varying in viscosity from 1 to 16.2 centipoise. We also show by example of a microarray hybridization that the reaction efficiency can be enhanced 2-5 fold through fluid mixing under conditions where diffusion is rate limiting. For comparison, similar results were achieved with a disposable commercial device that covers only half of the reaction area of the closed loop device.

PRIMEGENS: robust and efficient design of gene-specific probes for microarray analysis.

MOTIVATION: DNA microarray is a powerful high-throughput tool for studying gene function and regulatory networks. Due to the problem of potential cross hybridization, using full-length genes for microarray construction is not appropriate in some situations. A bioinformatic tool, PRIMEGENS, has recently been developed for the automatic design of PCR primers using DNA fragments that are specific to individual open reading frames (ORFs). RESULTS: PRIMEGENS first carries out a BLAST search for each target ORF against all other ORFs of the genome to quickly identify possible homologous sequences. Then it performs optimal sequence alignment between the target ORF and each of its homologous ORFs using dynamic programming. PRIMEGENS uses the sequence alignments to select gene- specific fragments, and then feeds the fragments to the Primer3 program to design primer pairs for PCR amplification. PRIMEGENS can be run from the command line on Unix/Linux platforms as a stand-alone package or it can be used from a Web interface. The program runs efficiently, and it takes a few seconds per sequence on a typical workstation. PCR primers specific to individual ORFs from Shewanella oneidensis MR-1 and Deinococcus radiodurans R1 have been designed. The PCR amplification results indicate that this method is very efficient and reliable for designing specific probes for microarray analysis.

Isolation and characterization of metal-reducing thermoanaerobacter strains from deep subsurface environments of the Piceance Basin, Colorado.

Five bacterial strains were isolated from anaerobic enrichment cultures that had originated from inoculations with samples collected from the deep subsurface environments of the millions-of-years-old, geologically and hydrologically isolated Piceance Basin in Colorado. Small-subunit rRNA gene-based analyses indicated that all of these bacteria were closely related to Thermoanaerobacter ethanolicus, with similarities of 99.4 to 99.5%. Three isolates (X513, X514, and X561) from the five bacterial strains were used to examine physiological characteristics. These thermophilic bacteria were able to use acetate, glucose, hydrogen, lactate, pyruvate, succinate, and xylose as electron donors while reducing Fe(III), cobalt(III), chromium(VI), manganese(IV), and uranium(VI) at 60 degrees C. One of the isolates (X514) was also able to utilize hydrogen as an electron donor for Fe(III) reduction. These bacteria exhibited diverse mineral precipitation capabilities, including the formation of magnetite (Fe(3)O(4)), siderite (FeCO(3)), rhodochrosite (MnCO(3)), and uraninite (UO(2)). The gas composition of the incubation headspace and the ionic composition of the incubation medium exerted profound influences on the types of minerals formed. The susceptibility of the thermophilic Fe(III)-reducing cultures to metabolic inhibitors specific for ferric reductase, hydrogenase, and electron transport indicated that iron reduction by these bacteria is an enzymatic process.

Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors.

Changes in mRNA and protein expression profiles of Shewanella oneidenesis MR-1 during switch from aerobic to fumarate-, Fe(III)-, or nitrate-reducing conditions were examined using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In response to changes in growth conditions, 121 of the 691 arrayed genes displayed at least a two-fold difference in transcript abundance as determined by microarray analysis. Genes involved in aerobic respiration encoding cytochrome c and d oxidases and TCA cycle enzymes were repressed under anaerobic conditions. Genes induced during anaerobic respiration included those involved in cofactor biosynthesis and assembly (moaACE, ccmHF, nosD, cysG), substrate transport (cysUP, cysTWA, dcuB), and anaerobic energy metabolism (dmsAB, psrC, pshA, hyaABC, hydA). Transcription of genes encoding a periplasmic nitrate reductase (napBHGA), cytochrome c552, and prismane was elevated 8- to 56-fold in response to the presence of nitrate, while cymA, ifcA, and frdA were specifically induced three- to eightfold under fumarate-reducing conditions. The mRNA levels for two oxidoreductase-like genes of unknown function and several cell envelope genes involved in multidrug resistance increased two- to fivefold specifically under Fe(III)-reducing conditions. Analysis of protein expression profiles under aerobic and anaerobic conditions revealed 14 protein spots that showed significant differences in abundance on 2-D gels. Protein identification by mass spectrometry indicated that the expression of prismane, dihydrolipoamide succinyltransferase, and alcaligin siderophore biosynthesis protein correlated with the microarray data.