Identification of gliadin-binding peptides by phage display

BACKGROUND: Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products.RESULTS: Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin.CONCLUSIONS: We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated

Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes

Background: Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved beta-glucan-, antioxidant-and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda. Results: Here a population of 2600 mutagenised M2 lines, producing 2550 M3 seed lots were obtained. The M2 population was initially evaluated by visual inspection and a number of different phenotypes were seen ranging from dwarfs to giants, early flowering to late flowering, leaf morphology and chlorosis. Phloroglucinol/HCl staining of M3 seeds, obtained from 1824 different M2 lines, revealed a number of potential lignin mutants. These were later confirmed by quantitative analysis. Genomic DNA was prepared from the M2 population and the mutation frequency was determined. The estimated mutation frequency was one mutation per 20 kb by RAPD-PCR fingerprinting, one mutation per 38 kb by MALDI-TOF analysis and one mutation per 22.4 kb by DNA sequencing. Thus, the overall mutation frequency in the population is estimated to be one mutation per 20-40 kb, depending on if the method used addressed the whole genome or specific genes. During the investigation, 6 different mutations in the phenylalanine ammonia-lyase (AsPAL1) gene and 10 different mutations in the cellulose synthase-like (AsCslF6) beta-glucan biosynthesis gene were identified. Conclusion: The oat TILLING population produced in this work carries, on average, hundreds of mutations in every individual gene in the genome. It will therefore be an important resource in the development of oat with specific characters. The population (M5) will be available for academic research via Nordgen http://www.nordgen.org as soon as enough seeds are obtained

Substrates of the \u3cem\u3eArabidopsis thaliana\u3c/em\u3e Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance

Wideband Amplifier Design for STO Technology

Spin Torque Oscillator (STO) is a promising technology for microwave and radar applications due to its large tunability, miniature size, high operation frequency, high integration level, etc. However, the technology comes also with issues and challenges,such as low output power and spectrum impurity. For instance, in order to apply the STO technology into communication systems, an amplifier is required to compensate the STO’s low output power.     This thesis presents an amplifier for promising Magnetic Tunnel Junction (MTJ) STO devices. The motional resistance of different MTJ STO devices varies from several Ohms to hundreds Ohms, which makes the design challenging. This thesis focuses first on extracting the amplifier requirements using the state-of-the-art MTJ STO devices. The operation frequency of MTJ STO is in the range of 4-8GHzwith a -40～-60 dBm output power. Therefore, a wideband amplifier with 45-65 dB gain is required. Then based on the amplifier requirements, an amplifier topology is proposed, which is composed of two types of input balun-LNA stages depending onthe motional resistance of the STO, a broadband limiting amplifier and an outputbuffer. CG-CS architecture is suitable for the input balun-LNA in the small motional resistance case and cascoded-CS architecture is suitable for the large motional resistance case. The limiting amplifier and the output buffer are the common circuits shared by two cases via switches.     The wideband amplifier for STO is implemented using a 65nm CMOS process with 1.2 V supply and it exhibits 52.36 dB gain with 1.34-11.8 GHz bandwidth insmall motional resistance case and 59.29 dB gain with 1.171-8.178 GHz bandwidth in large motional resistance case. The simulation results show that the amplifier has very low power consumption and meets the linearity and noise performance requirements

CMOS High Frequency Circuits for Spin Torque Oscillator Technology

Spin torque oscillator (STO) technology has a unique blend of features, including but not limited to octave tunability, GHz operating frequency, and nanoscaled size, which makes it highly suitable for microwave and radar applications. This thesis studies the fundamentals of STOs, utilizes the state-of-art STO's advantages, and proposes two STO-based microwave systems targeting its microwave applications and measurement setup, respectively. First, based on an investigation of possible STO applications, the magnetic tunnel junction (MTJ) STO shows a great suitability for microwave oscillator in multi-standard multi-band radios. Yet, it also imposes a large challenge due to its low output power, which limits it from being used as a microwave oscillator. In this regard, different power enhancement approaches are investigated to achieve an MTJ STO-based microwave oscillator. The only possible approach is to use a dedicated CMOS wideband amplifier to boost the output power of the MTJ STO. The dedicated wideband amplifier, containing a novel Balun-LNA, an amplification stage and an output buffer, is proposed, analyzed, implemented, measured and used to achieve the MTJ STO-based microwave oscillator. The proposed amplifier core consumes 25.44 mW from a 1.2 V power supply and occupies an area of 0.16 mm2 in a 65 nm CMOS process. The measurement results show a S21 of 35 dB, maximum NF of 5 dB, bandwidth of 2 GHz - 7 GHz. This performance, as well as the measurement results of the proposed MTJ STO-based microwave oscillator, show that this microwave oscillator has a highly-tunable range and is able to drive a PLL. The second aspect of this thesis, firstly identifies the major difficulties in measuring the giant magnetoresistance (GMR) STO, and hence studying its dynamic properties. Thereafter, the system architecture of a reliable GMR STO measurement setup, which integrates the GMR STO with a dedicated CMOS high frequency IC to overcome these difficulties in precise characterization of GMR STOs, is proposed. An analysis of integration methods is given and the integration method based on wire bonding is evaluated and employed, as a first integration attempt of STO and CMOS technologies. Moreover, a dedicated high frequency CMOS IC, which is composed of a dedicated on-chip bias-tee, ESD diodes, input and output networks, and an amplification stage for amplifying the weak signal generated by the GMR STO, is proposed, analyzed, developed, implemented and measured. The proposed dedicated high frequency circuits for GMR STO consumes 14.3 mW from a 1.2 V power supply and takes a total area of 0.329 mm2 in a 65 nm CMOS process. The proposed on-chip bias-tee presents a maximum measured S12 of -20 dB and a current handling of about 25 mA. Additionally, the proposed dedicated IC gives a measured gain of 13 dB with a bandwidth of 12.5 GHz - 14.5 GHz. The first attempt to measure the (GMR STO+IC) pair presents no RF signal at the output. The possible cause and other identified issues are given.QC 20140114</p

Spin Torque Oscillator Modeling, CMOS Design and STO-CMOS Integration

Spin torque oscillators (STOs) are microwave oscillators with an attractive blend of features, including a more-than-octave tunability, GHz operating frequencies, nanoscale size, nanosecond switching speed and full compatibility with CMOS technology. Over the past decade, STOs' physical phenomena have been explored to a greater extent, their performance has been further improved, and STOs have already shown great potential for a wide range of applications, from microwave sources and detectors to neuromorphic computing. This thesis is devoted to promoting the STO technology towards its applications, by means of implementing the STO's electrical model, dedicated CMOS integrated circuits (ICs), and STO-CMOS IC integration. An electrical model, which can capture magnetic tunnel junction (MTJ) STO's characteristics, while enabling system- and circuit-level designs and performance evaluations, is of great importance for the development of MTJ STO-based applications. A comprehensive and compact analytical model, which is based on macrospin approximations and can fulfill the aforementioned requirements, is proposed. This model is fully implemented in Verilog-A, and can be used for efficient simulations of various MTJ STOs. Moreover, an accurate phase noise generation approach, which ensures a reliable model, is proposed and successfully used in the Verilog-A model implementation. The model is experimentally validated by three different MTJ STOs under different bias conditions. CMOS circuits, which can enhance the limited output power of MTJ STOs to levels that are required in different applications, are proposed, implemented and tested. A novel balun-low noise amplifier (LNA), which can offer sufficient gain, bandwidth and linearity for MTJ STO-based magnetic field sensing applications, is proposed. Additionally, a wideband amplifier, which can be connected to an MTJ STO to form a highly-tunable microwave oscillator in a phase-locked loop (PLL), is also proposed. The measurement results demonstrate that the proposed circuits can be used to develop MTJ STO-based magnetic field sensing and microwave source applications. The investigation of possible STO-CMOS IC integration approaches demonstrates that the wire-bonding-based integration is the most suitable approach. Therefore, a giant magnetoresistance (GMR) STO is integrated with its dedicated CMOS IC, which provides the necessary functions, using the wire-bonding-based approach. The RF characterization of the integrated GMR STO-CMOS IC system under different magnetic fields and DC currents shows that such an integration can eliminate wave reflections. These findings open the possibility of using GMR STOs in magnetic field sensing and microwave source applications.QC 20151112</p

Wideband Amplifier Design for Magnetic Tunnel JunctionBased Spin Torque Oscillators

Spin torque oscillator (STO) is a novel current-control-oscillator (CCO), based on two spintronic effects: spin momentum transfer torque and magneto-resistance (MR). It features large tunability, miniature size, high integration level, high quality factor, high operation frequency, etc., which makes it a promising technology for microwave and radar applications. However, the STO is still an immature technology, which requires intensive research for improving the spectrum purity and the output power performance [1]. This paper proposes a wideband amplifier targeting magnetic tunnel junction (MTJ) type of STO device, which compensates the low output power of the STO.     The MTJ STO devices can cover a large part of ultra-wideband (UWB) from 3 - 8 GHz and provide an output power from -60 dBm to -40 dBm by tuning the bias DC current and the magnetic field [2][3]. One important and potential application of STO device is a local oscillator (LO) in an RF transceiver. To achieve this task, the amplifier requires a gain of 45 - 65 dB. In addition, the source impedance of different MTJ STO devices varies from a dozen to several hundred Ohms, which makes the amplifier design challenging. An universal amplifier, which fulfills the extracted design requirements, is proposed. It is composed of two types of Balun-LNAs depending on the MR of STO devices as the input stages, a broadband limiting amplifier chain and an output buffer. A combination of a common source (CS) stage and a cross-coupled common gate (CG) stage is employed for the input Balun-LNA in the low impedance case while a cascoded CS stage is used in the high impedance case. The output of both LNAs is connected to a limiting amplifier chain, which provides enough voltage gain. An output buffer is used as the output stage to convert the balanced output to single-ended output and to match the output impedance to 50 Ohms.     The proposed wideband amplifier for MTJ STO is implemented in a 65nm CMOS process with   1.2 V supply. In the band of interest, it exhibits 55 dB gain with a maximum noise figure (NF) of    4.5 dB in the small MR case, and a 59 dB gain with a maximum NF of 3 dB in the large MR case. Besides the low noise performance and the high gain, the simulation results of the proposed amplifier also show that it has low power consumption and moderate impedance matching in the frequency range of 3 - 8 GHz, which is suitable for MTJ STO applications.QC 20130115</p

Research and Development of High-Performance High-Damping Rubber Materials for High-Damping Rubber Isolation Bearings: A Review

At present, high-damping rubber materials, widely used in the field of engineering seismic isolation, generally have the problems such as narrow effective damping temperature range, low damping loss factor and strong temperature dependence, which lead to prominent dependence of temperature and load conditions of the isolation performance of high-damping rubber isolation bearings. Research and development of high-performance high-damping rubber materials with broad effective damping temperature range, high damping loss factor and weak temperature dependence are very urgent and necessary to ensure the safety of the seismic isolation of engineering structures. This paper mainly reviews the recent progress in the research and development of high-damping rubber materials using nitrile butadiene rubber (NBR), epoxidized natural rubber (ENR), ethylene propylene diene rubber (EPDM), butyl rubber (IIR), chlorinated butyl rubber (CIIR), and bromine butyl rubber (BIIR). This is followed by a review of vulcanization and filler reinforcement systems for the improvement of damping and mechanical properties of high-damping rubber materials. Finally, it further reviews the constitutive models describing the hyperelasticity and viscoelasticity of rubber materials. In view of this focus, four key issues are highlighted for the development of high-performance high-damping rubber materials used for high-damping rubber isolation bearings

An Inductorless Wideband Balun-LNA for Spin Torque Oscillator-based Field Sensing

This paper presents a wideband inductorless Balun-LNA targeting spin torque oscillator-based magnetic field sensing applications. The LNA consistsof a CS stage combined with a cross-coupled CG stage, which offers wideband matching, noise/distortion cancellation and gain boosting, simultaneously. The Balun-LNA is implemented in a 65 nm CMOS technology, and it is fully ESD-protected and packaged. Measurement results show a bandwidth of 2 GHz - 7 GHz, a voltage gain of 20 dB, an IIP3 of +2 dBm, and a maximum NF of 5 dB. The LNA consumes 3.84 mW from a 1.2 V power supply and occupies a total silicon area of 0.0044 mm2. The measurement results demonstrate that the proposed Balun-LNA is highly suitable for the STO-based field sensing applications.QC 20150210</p