Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Tunable mechanical resonator with aluminum nitride piezoelectric

The electromechanical response of piezoelectrically-actuated AlN micromachined bridge resonators has been characterized using laser interferometry and electrical admittance measurements. We compare the response of microbridges with different dimensions and buckling (induced by the initial residual stress of the layers). The resonance frequencies are in good agreement with numerical simulations of the electromechanical behavior of the structures. We show that it is possible to perform a rough tuning of the resonance frequencies by allowing a determined amount of builtin stress in the microbridge during its fabrication. Once the resonator is made, a DC bias added to the AC excitation signal allows to fine-tune the frequency. Our microbridges yield a tuning factor of around 88 Hz/V for a 500 ?m-long microbridge.

A low cost and widely applicable methodology of digital imagen analysis of ores for mineral and envirommental engineering

Technological and environmental problems related to ore processing are a serious limitation for sustainable development of mineral resources, particularly for countries / companies rich in ores, but with little access to sophisticated technology, e.g. in Latin America. Digital image analysis (DIA) can provide a simple, unexpensive and broadly applicable methodology to assess these problems, but this methodology has to be carefully defined, to produce reproducible and relevant information.

Dual-Band Tunable Recursive Active Filter

This letter presents a novel recursive active filter topology that provides dual-band performance, with independent tuning capability in both bands. The dual-band operation is achieved by using two independent feedback lines. Additionally, linear phase shifters based on left-handed cells are included in these two branches in order to tune the center frequency of both pass bands.

Electrooptic characterization of tunable cylindrical liquid crystal lenses

In this work, one-dimensional arrays of cylindrical adaptive liquid crystal lenses were manufactured and characterized; and test devices were filled with nematic liquid crystal. Comb interdigitated electrodes were designed as a mask pattern for the control electrode on the top glass substrates. A radial graded refractive index along each microsized lens was achieved by fabricating a layer of high resistance sheet deposited as a control electrode. These tunable lenses were switched by applying amplitude and frequency optimized waveforms on the control electrode. Phase profiles generated by the radial electric field distribution on each lens were measured by a convectional interferometric technique.

Periodic time-domain modulation for the electrically tunable control of optical pulse train envelope and repetition rate multiplication

An electrically tunable system for the control of optical pulse sequences is proposed and demonstrated. It is based on the use of an electrooptic modulator for periodic phase modulation followed by a dispersive device to obtain the temporal Talbot effect. The proposed configuration allows for repetition rate multiplication with different multiplication factors and with the simultaneous control of the pulse train envelope by simply changing the electrical signal driving the modulator. Simulated and experimental results for an input optical pulse train of 10 GHz are shown for different multiplication factors and envelope shapes.

2D tunable graded index prism beam steering device based on nematic liquid crystals

Liquid crystal devices are being used in many non-display applications in order to construct small devices controlled by low voltage electronics without mechanical components. In this work, we present a novel liquid crystal device for laser beam steering. In this device the orientation of the liquid crystal molecules can be controlled. A change in the liquid crystal orientation results in a change of the refractive index. When a laser beam passes through the device, the beam will be deviated (Fig.1) and the device works a prism. The main difference between this device and a prism is that in the device the orientation profile of the liquid crystal molecules can be modified so that the laser beam can be deviated a required angle: the device is tuneable.

Electrically tunable delay for trains of optical pulses

A technique to implement an electrically tunable delay line with high bandwidth for trains of ultrashort optical pulses is presented. The system is based on the temporal self-imaging effect in fiber gratings and electrooptic modulation.

Tunable Fano resonance in symmetric multilayered gold nanoshells

Fano resonances (FRs) are produced when a discrete state is coupled with a continuum. In addition to fundamental scientific interests, FRs in plasmonic systems give rise to the so-called plasmon-induced transparency. In this work we have studied the evolution of dipole-dipole all-plasmonic FRs in symmetric multilayered nanoshells as the function of their geometrical parameters. We demonstrate that symmetry breaking is not mandatory for controlling the Fano resonance in such multilayered nanoshells. Generation of FRs in these symmetric nanostructures presents clear advantages over their asymmetric counterparts, as they are easier to fabricate and can be used in a wider range of technological applications.

Tunable Fano resonance in symmetric multilayered gold nanoshells

We have studied the evolution of dipole–dipole all-plasmonic Fano resonances (FRs) in symmetric multilayered nanoshells as a function of their geometrical parameters. We demonstrate that symmetry breaking is not mandatory for controlling the Fano resonance in such multilayer structures. By carefully selecting the geometrical parameters, the position of the FR can be tuned between 600 and 950 nm and its intensity can be increased up to four fold with respect to the non-optimized structures. Generation of FRs in such symmetric nanostructures presents clear advantages over their asymmetric counterparts, as they are easier to fabricate and can be used in a wider range of technological applications.

Wedged analog tunable beam steering device based on cholesteric liquid crystals

In this work we propose a novel cholesteric liquid crystal beam steering device based on the Kerr effect. The first version of the device consists of two ITO coated glass plates, with intentionally prepared electrodes, assembled together with a thickness gradient between both sides of the device. One side of the cell has two substrates at direct contact; the other side has separated substrates to form the wedge. The cell was filled with a cholesteric liquid crystal. The liquid crystal material is an innovative mixture called 1892E with extremely low viscosity doped with a ZLI chiral nematogen. The proposed beam steering device based on cholesteric liquid crystals has great potential for many photonic applications. Results describing the performance of the device and the properties of the selected liquid crystals are presented.

Tunable liquid crystal-photonic crystal fiber interferometer

In this work, we present a novel interferometer based on liquid crystal and photonic crystal fiber technology. The objective of this project is the development of a tunable (switchable) modal (Mach-Zehnder) interferometer for optical communications or sensing.

Thermally tunable polarization by nanoparticle plasmonic resonance in photonic crystal fibers

A photonic crystal fiber selectively filled with silver nanoparticles dispersed in polydimethylsiloxane has been numerically studied via finite elements analysis. These nanoparticles possess a localized surface plasmon resonance in the visible region which depends on the refractive index of the surrounding medium. The refractive index of polydimethylsiloxane can be thermally tuned leading to the design of polarization tunable filters. Filters found with this setup show anisotropic attenuation of the x-polarization fundamental mode around ?x = 1200dB/cm remarkably higher than the y-polarization mode. Moreover, high fiber birefringence and birefringence reversal is observed in the spectral region of the plasmon.