Calibration Kit Research Articles

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 μM and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.

30.3: Comparison of On‐Screen Display‐based and ICC Profile‐based Calibration for OLED Displays

AbstractThe tone reproduction curves of an OLED display were measured and modeled as a function of three on‐screen display (OSD) settings. The optimal OSD settings, derived from the model, outperformed two ICC profile‐based color calibration kits by reducing the calibration errors from 2.89% to 0.25% and the quantization errors from 9.28% to 0%.

Characterization of SiCN Ceramic Material Dielectric Properties at High Temperatures for Harsh Environment Sensing Applications

A novel method is presented in this paper to precisely characterize the dielectric properties of silicon carbon nitride (SiCN) ceramic materials at high temperatures for wireless passive sensing applications. This technique is based on a high quality factor (Q) dielectrically loaded cavity resonator, which allows for accurate characterization of both dielectric constant and loss tangent. The dielectric properties of SiCN ceramics are characterized from 25 °C to 1000 °C. Two different metallization processes are implemented for the measurements with the highest temperatures of 500 °C and 1000 °C, respectively. A custom-made thru-reflect-line calibration kit is used to maximize the measurement accuracy at every temperature point. It is observed that the dielectric constant and loss tangent of the SiCN sample without Boron doping increase from 3.707 to 3.883 and from 0.0038 to 0.0213, respectively, when the temperature is raised from 25 °C to 500 °C, and for the SiCN with Boron doping (SiBCN), the dielectric constant and loss tangent increase from 4.817 to 5.132 and from 0.0020 to 0.0186, respectively, corresponding to the temperature ranging from 25 °C to 1000 °C. Experimental uncertainties for extracted er and tanδ are no more than 0.0004 and 0.0001, respectively. The temperature dependency of Si(B)CN dielectric properties, as well as the dielectrically loaded cavity resonator structure, provides the basis for the development of wireless passive temperature sensors for high-temperature applications.

THE ROUND-ENDED DESIGN AND MEASUREMENT OF ALL SYMMETRIC EDGE-COUPLED BANDPASS FILTER

The round-corner design of an all symmetric edge-coupled bandpass fllter (BPF) is presented. The manufacturing tolerances and its efiects of frequency shift on the design of the edge-coupled are investigated. Consequently, the round-ended design method is proposed in order to compensate the open-end efiect in the half- wavelength resonator section with the round-ended corners rather than decrementing the lengths in a conventional way, and an experience formula and a corresponding procedure are devised for the design of such fllters. The widths of all the half-wavelength resonators are set equal to avoid discontinuities in the interior of the fllter. The fllter is realized on a ceramic fllled soft substrate with dielectric constant of 6.2. For obtaining the de-embedded measured results at the device plane an in-house customized through-re∞ect-line (TRL) calibration kit is produced. Three kinds of fllters at difierent center frequencies are manufactured, and their measured results are in good agreement with the simulated ones after calibration.

Thru‐reflect‐line calibration for substrate integrated waveguide devices with tapered microstrip transitions

One of the main problems when exciting or measuring substrate integrated waveguide (SIW) devices lies in the need of a good interconnection with planar structures. In this reported work, the negative effects produced by the connectors and the tapered microstrip-to-SIW transitions are de-embedded from the measurements of the SIW structure by a thru-reflect-line calibration with an adequate and cheap SIW calibration kit.

반사 펄스의 주파수 해석을 이용한 광대역 3.5 mm 동축형 단일 포트 벡터 회로망 분석법

본 논문에서는 샘플링 오실로스코프를 이용한 시간 영역의 해석을 통해 초고주파 소자 및 안테나의 반사 계수를 평가하는 방법을 소개한다. 20 GHz 급의 펄스 입력 신호에 대한 반사 신호를 측정한 후, 이를 푸리에 변환하여 반사 계수를 추출하였다. 초고주파 반사파 회로망의 보정에 필요한 3가지 오차 계수들은 3.5 mm calibration kit을 이용하여 유도하였다. 또한, 반사파 측정을 위하여 결합기를 사용한 경우의 오차 계수를 유도하고, 이를 적용하여 얻은 반사 계수와 벡터 회로망 분석기로 측정한 결과를 비교하였다. This paper presents a method to measure reflection coefficients of microwave devices or antennas based on time domain analysis with sampling oscilloscopes. The reflection coefficients were extracted by the Fourier transformation of echo pulses from devices with respect to the 20 GHz incident pulse signals. The three-error terms, which are commonly used for the correction of a microwave network, were determined using a 3.5 mm calibration kit. In addition, a modified error-correction model associated with a directional coupler for reflection coefficient measurements is introduced. The results were compared with those of measured with a commercial vector network analyzer.

Development of a Calibration Phantom Set for MRI Temperature Imaging System Quality Assurance

Magnetic resonance imaging (MRI) temperature imaging systems need to be routinely calibrated to guarantee accurate temperature results and qualified MRI. No independent physical temperature calibration phantom (TCP) set is currently available. An economical TCP set was developed to routinely ensure the quality of MRI temperature imaging system. The novel TCP was constructed using a heating unit, temperature sensor, and MRI phantom liquid. A specialized heating unit was developed using carbon fibers. The TCP set design was an integration of the TCP, temperature measurement unit, display unit, and control unit. The proposed MRI calibration kit, which is a combination of the TCP set and standard MRI phantom, was used in the MRI thermometry calibration and MRI quality calibration. The TCP set provided an efficient, accurate, and homogeneous temperature map as the reference standard temperature for calibration. Accuracy and heating efficiency of the TCP set were 1°C and 1°C/minute, respectively. Calibration of the MRI thermometry and MRI quality were implemented successfully. The proposed TCP set is completely compatible with the MRI system and can be used to calibrate MRI thermometry and MRI quality to ensure the quality performance of the MRI temperature imaging system.

Wireless communicating nodes at 60 GHz integrated on flexible substrate for short-distance instrumentation in aeronautics and space

This paper presents the research done at LAAS-CNRS and in the context of “NANOCOMM” project. This project aims to demonstrate the potential of nanotechnology for the development of reconfigurable, ultra-sensitive, low consumption, and easy installation sensor networks with high performance in terms of reliability in line with the requirements of aeronautics and space. Each node of the sensor network is composed of nano-sensors, transceiver, and planar antenna. In this project, three-dimensional (3D) heterogeneous integration of these different components, on flexible polyimide substrate, is planned. Two types of sensors are selected for this project: strain gauges are used for the structure health monitoring (SHM) application and electrochemical cells are used to demonstrate the ability to detect frost phenomenon. After processing, sensors data are processed and transmitted to the reader unit using an ultra-wide band (UWB) transceiver. (digital baseband and radiofrequency (RF) head). The design and implementation of reconfigurable wireless communication architectures are provided according to the application requirements using nanoscale 65 nm CMOS technology. It is proposed to integrate on flexible substrate the transceiver using the flip-chip technique. A 60 GHz planar antenna is connected to the transceiver for the wireless data transmission. This paper is focused on the 3D integration techniques and the technological process used for the realization of such communicating nano-objects on polyimide substrate. The first assembly tests were carried out. Tests of interconnections quality and electrical contacts (Daisy Chain, calibration kit, etc.) were also performed with good results. A bumps contact resistance of 15 mΩ is measured.

Calibration Protocol for Broadband Near-Field Microwave Microscopy

In this paper, we describe how to define and build a set of known loads to be used in near-field microwave microscopy. Such loads are necessary to set up a microwave calibration kit, enabling local quantitative measurements by the microscope. The proposed protocol is validated through the microscopy system we have recently developed that combines a scanning tunneling microscope and a 70-GHz vector network analyzer.

70.2: Virtual Display: A Platform for Evaluating Display Color Calibration Kits

AbstractWe propose a method of evaluating the performance of color calibration kits for LCD monitors. Routine color calibration is imperative for critical applications that rely on color fidelity such as digital pathology and professional graphics. However, the commercially available products vary greatly in price and performance with no available evaluation standard. We propose the concept of Virtual Display, a universal display platform that emulates tone reproduction curves. A field programmable gate array board was used to process the video signals based on a preprogrammed look‐up table, which contains the tone reproduction curves of the target display. A spectroradiometer‐based and a colorimeter‐based color calibration kits were challenged by 8 virtual displays that have problematic tone reproduction curves. The results show that both color calibration kits offer only one‐dimensional lookup tables for each channel, which might be insufficient. We also spectrally characterized three real displays of different grades and derived spectral models for them. By using the virtual display and spectral models, evaluation of color calibration kits can be greatly simplified and standardized without the burden of optical measurement on target displays.

Mixed Dimers of Insulin-degrading Enzyme Reveal a Cis Activation Mechanism

Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity. The mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. This effect shows that substrate or small peptide activation occurs through a cis effect. A mixed dimer composed of one wild-type subunit and the other subunit containing a mutation that neither permits substrate binding nor catalysis (H112Q) exhibits the same turnover number per active subunit as wild-type IDE. In contrast, a mixed dimer in which one subunit contains the wild-type sequence and the other contains a mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. This indicates a negative trans effect of substrate binding at the active site. On the other hand, activation in trans is observed with extended substrates that occupy both the active and distal sites. Comparison of the binding of an amyloid β peptide analog to wild-type IDE and to the Y609F mutant showed no difference in affinity, indicating that Y609 does not play a significant role in substrate binding at the distal site.

Submillimeter Wave <formula formulatype="inline"> <tex Notation="TeX">${\rm S}$</tex></formula>-Parameter Characterization of Integrated Membrane Circuits

We demonstrate S-parameter characterization of membrane circuits in the WR-03 frequency band (220-325 GHz) utilizing thru-reflect-line (TRL) -calibration technique. The TRL calibration kit design features 3 μm thick GaAs membrane circuits packaged in E-plane split waveguide blocks with the reference planes inside the membrane circuit structure. A 300 GHz membrane ring resonator filter circuit has been characterized by applying the proposed calibration kit, showing good agreement with simulations.

Ultrasonic Enhanced Applications in Proteomics Workflows: single probe versus multiprobe.

Ultrasonic Enhanced Applications in Proteomics Workflows: single probe versus multiprobe.

Investigation of the pH gradient formation and cathodic drift in microchip isoelectric focusing with imaged UV detection

This paper reports the protein analysis by using microchip IEF carried on an automated chip system. We herein focused on two important topics of microchip IEF, the pH gradient and cathodic drift. The computer simulation clarified that the EOF could delay the establishment of pH gradient and move the carrier ampholytes (CAs) to cathode, which probably caused a cathodic drift to happen. After focusing, the peak positions of components in a calibration kit with broad pI were plotted against their pI values to know the actual pH gradient in a microchannel varying time. It was found that the formed pH gradient was stable, not decayed after readily steady state, and migrated to cathode at a rate of 10.0 μm/s that determined by the experimental conditions such as chip material, internal surface coating and field strength. The theoretical pH gradient was parallel with the actual pH gradient, which was demonstrated in two types of microchip with different channel lengths. No compression of pH gradient was observed when 2% w/v hydroxypropyl methyl cellulose was added in sample and electrolytes. The effect of CAs concentration on current and cathodic drift was also explored. With the current automatic chip system, the calculated peak capacity was 23-48, and the minimal pI difference was 0.20-0.42 for the used single channel microchip with the effective length of 40.5 mm. The LOD for the analysis of CA-I and CA-II was around 0.32 μg/mL by using normal imaged UV detection, the detected amount is ca. 0.07 ng.

Nicotinic Receptor Subunit α5 Modifies Assembly, Up-regulation, and Response to Pro-inflammatory Cytokines

In the mammalian brain high affinity nicotine-binding sites are composed of at least the alpha4 and beta2 subunits. Additional nicotinic acetylcholine receptor subunits that are often co-expressed with alpha4+beta2 include alpha5. The introduction of alpha5 into 293 cells expressing alpha4+beta2 strongly favors assembly of alpha4+alpha5+beta2 receptors, increases constitutive ligand binding density as measured using [(3)H]epibatidine, but reduces the magnitude of up-regulation in response to chronic nicotine. In contrast, when beta4 is substituted for beta2, alpha5 interferes with the assembly of these receptors, demonstrating an important role for the beta subunit in this process. When cells co-express alpha4+alpha5+beta2+beta4, over 50% of the subunit associations include all four subunits, but they fail to be detected using [(3)H]epibatidine binding. However, complexes of alpha4+alpha5+beta2 do preferentially emerge from these subunit mixtures, and these mixtures bind ligand. In previous studies of alpha4+beta2+beta4 co-expression by 293 cells, the inflammatory cytokines IL-1beta and TNFalpha influenced the outcome of receptor assembly (Gahring, L. C., Days, E. L., Kaasch, T., González de Mendoza, M., Owen, L., Persiyanov, K., and Rogers, S. W. (2005) J. Neuroimmunol. 166, 88-101). When alpha5 is included in this subunit mixture, and cells are exposed to either inflammatory cytokine, subunit association is no longer altered. These findings suggest that alpha5 is an influential modulator of alpha4+beta2 nicotinic acetylcholine receptor assembly and stabilizes their expression in response to fluctuations in external conditions.

Bowtie filter and water calibration in the improvement of cone beam CT image quality

Objective To evaluate the improvement of cone beam CT (CBCT) image quality by using bewtie filter (F_1) and water calibration. Methods First the multi-level gain calibration of the detector panel with the method of Cal_2 calibration was performed, and the CT images of CATPHAN503 with F_0 and bowtic filter were collected, respectively. Then the detector panel using water calibration kit was calibrated, and images were acquired again. Finally, the change of image quality after using F_1 and (or) water calibration method was observed. The observed indexes included low contrast visibility, spatial uniformity, ring artifact, spatial resolution and geometric accuracy. Results Comparing with the traditional combination of F_0 filter and Cal_2 calibration, the combination of bowtie filter F_1 and water calibration improves low contrast visibility by 13.71%, and spatial uniformity by 54. 42%. Water calibration removes ring artifacts effectively. However, none of them improves spatial resolution and geometric accuracy. Conclusions The combination of F_1 and water calibration improves CBCT image quality effectively. This improvement is aid to the registration of CBCT images and localization images. Key words: Bowtie filter; Water calibration; Image quality; Tomograpby, X-ray computed, cone beam

Biochemical and Biophysical Characterization of the Mg2+-induced 90-kDa Heat Shock Protein Oligomers

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.

Alanine-shaving Mutagenesis to Determine Key Interfacial Residues Governing the Assembly of a Nano-cage Maxi-ferritin

The fundamental process of protein self-assembly is governed by protein-protein interactions between subunits, which combine to form structures that are often on the nano-scale. The nano-cage protein, bacterioferritin from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for an alanine-shaving mutagenesis study to discover key amino acid residues at symmetry-related protein-protein interfaces that control protein stability and self-assembly. By inspection of these interfaces and "virtual alanine scanning," nine mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, dynamic light scattering, native PAGE, and temperature-dependent CD. Many of the selected amino acids act as hot spot residues. Four of these (Arg-30, which is located at the two-fold axis, and Arg-61, Tyr-114, and Glu-128, which are located at the three-fold axis), when individually mutated to alanine, completely shut down detectable solution formation of 24-mer, favoring a cooperatively folded dimer, suggesting that they may be oligomerization "switch residues." Furthermore, two residues, Arg-30 and Arg-61, when changed to alanine form mutants that are more thermodynamically stable than the native protein. This investigation into the structure and energetics of this self-assembling nano-cage protein not only can act as a jumping off point for the eventual design of novel protein nano-structures but can also help to understand the role that structure plays on the function of this important class of proteins.

Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (K(D) = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (K(av)) measured with gel permeation chromatography, a high hydrodynamic radius (R(h)) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional (15)N-(1)H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a beta-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a beta-strand-rich structure, suggestive of the known beta-zipper mode of NTD binding.

Structural Characterization of Clusterin-Chaperone Client Protein Complexes

Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-transferase (GST) complexes were generated in vitro, and their structures studied using size exclusion chromatography (SEC), ELISA, SDS-PAGE, dynamic light scattering (DLS), bisANS fluorescence, and circular dichroism spectrophotometry (CD). Densitometry of Coomassie Blue-stained SDS-PAGE gels indicated that all three HMW CLU-client protein complexes had an approximate mass ratio of 1:2 (CLU:client protein). SEC indicated that all three clients formed complexes with CLU>or=4x10(7) Da; however, DLS estimated HMW CLU-FGN to have a diameter of 108.57+/-18.09 nm, while HMW CLU-CS and HMW CLU-GST were smaller with estimated diameters of 51.06+/-6.87 nm and 52.61+/-7.71 nm, respectively. Measurements of bisANS fluorescence suggest that the chaperone action of CLU involves preventing the exposure to aqueous solvent of hydrophobic regions that are normally exposed by the client protein during heat-induced unfolding. CD analysis indicated that, depending on the individual client protein, CLU may interact with a variety of intermediates on protein unfolding pathways with different amounts of native secondary structure. In vivo, soluble complexes like those studied here are likely to serve as vehicles to dispose of otherwise dangerous aggregation-prone misfolded extracellular proteins.