Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1

BACKGROUND: Modulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many – if not all – molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase. RESULTS: Here we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates. CONCLUSION: The here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-016-0900-7) contains supplementary material, which is available to authorized users

Coexistence of two- and three-dimensional Shubnikov-de Haas oscillations in Ar^+ -irradiated KTaO_3

We report the electron doping in the surface vicinity of KTaO_3 by inducing oxygen-vacancies via Ar^+ -irradiation. The doped electrons have high mobility (> 10^4 cm^2/Vs) at low temperatures, and exhibit Shubnikov-de Haas oscillations with both two- and three-dimensional components. A disparity of the extracted in-plane effective mass, compared to the bulk values, suggests mixing of the orbital characters. Our observations demonstrate that Ar^+ -irradiation serves as a flexible tool to study low dimensional quantum transport in 5d semiconducting oxides

Dominant mobility modulation by the electric field effect at the LaAlO_3 / SrTiO_3 interface

Caviglia et al. [Nature (London) 456, 624 (2008)] have found that the superconducting LaAlO_3 / SrTiO_3 interface can be gate modulated. A central issue is to determine the principal effect of the applied electric field. Using magnetotransport studies of a gated structure, we find that the mobility variation is almost five times as large as the sheet carrier density. Furthermore, superconductivity can be suppressed at both positive and negative gate bias. These results indicate that the relative disorder strength strongly increases across the superconductor-insulator transition.Comment: 4 pages, 4 figure

Filtres numériques non linéaires φ-saturés

Nous proposons un nouveau type de filtres numériques extrayant un signal à variations brusques d'un bruit aléatoire d'amplitude limitée. Ces filtres sont obtenus en modifiant la sortie d'un filtre linéaire par une transformation non linéaire bornant supérieurement la différence entre la sortie et l'entrée . Ces filtres, faciles à réaliser, sont très efficaces . Nous proposons de les dénommer : filtres numériques non linéaires E-saturés . La partie linéaire est un des filtres usuellement employés pour la réduction de bruit . Nous envisageons successivement des filtres numériques non linéaires, non récursifs passe-bas, récursifs et de Kalman . Le filtre de Kalman est construit à partir d'un modèle statistique du signal . L'efficacité des filtres numériques non linéaires E-satured, non récursifs passe-bas, r de Kalman . Le filtre de Kalman est construit à partir d'un modèle statistique du signal . L'efficacité des filtres numériques non linéaires E-satured est illustrée sur des signaux simulés et leurs caractéristiques fonctionnelles moyennes sont présentées .A new type of nonlinear digital filters which aims at removing small-amplitude random noises from a signal containing large-amplitude abrupt changes is proposed . These filters are constructed by nonlinearizing linear digital filters with a simple nonlinear function, and the difference between the input and the output of these filters is limited to a certain finite value . These filters are very easy to be realized and work quite effectively. We name these nonlinear digital filters as E-separating nonlinear digital filters (E-separating filters for short) . The linear filters used in the nonlinearization are usual linear digital filters used for noise reduction . First, cseparating filters using a nonrecursive linear low-pass filter and a recursive one are proposed, and then an Eseparating filter using a Kalman filter is proposed . They are named as an E-filter, a recursive E-filter, and an EKalman filter, respectively . The E-Kalman filter can take the local statistical characteristics of the signal into consideration as well as the abrupt changes . Moreover, a generalized form of the E-separating filter is proposed. Finally, computer simulations show the effectiveness of these E-separating filters and their statistical caracteristics are presented

Thickness dependence of the mobility at the LaAlO_3 / SrTiO_3 interface

The electronic transport properties of a series of LaAlO_3 / SrTiO_3 interfaces were investigated, and a systematic thickness dependence of the sheet resistance and magnetoresistance was found for constant growth conditions. This trend occurs above the critical thickness of four unit cells, below which the LaAlO_3 / SrTiO_3 interface is not conducting. A dramatic decrease in mobility of the electron gas of nearly two orders of magnitude was observed with increasing LaAlO_3 thickness from five to 25 unit cells.Comment: 3 pages, 4 figures, submitted for publicatio

CISCO: Cooled Infrared Spectrograph and Camera for OHS on the Subaru Telescope

This paper describes a Cooled Infrared Spectrograph and Camera for OHS (CISCO), mounted on the Nasmyth focus of the Subaru telescope. It is primarily designed as a back-end camera of the OH-Airglow Suppressor (OHS), and is also used as an independent, general-purpose near-infrared camera/spectrograph. CISCO is based on a single 1024x1024 format HgCdTe HAWAII array detector, and is capable of either wide-field imaging of 1.8'x1.8' field-of-view or low-resolution spectroscopy from 0.9 to 2.4 um. The limiting magnitudes measured during test observations were found to be J=23.5mag and K'=22.4mag (imaging, 1" aperture, S/N=5, 1 hr exposure).Comment: 14 pages, 15 figures, accepted by PASJ. Figures with higher resolution are available at http://www.ioa.s.u-tokyo.ac.jp/~kmotohara/CISCO/ciscopap.tar.g

Defect-control of conventional and anomalous electron transport at complex oxide interfaces

Using low-temperature electrical measurements, the interrelation between electron transport, magnetic properties, and ionic defect structure in complex oxide interface systems is investigated, focusing on NdGaO3/SrTiO3 (100) interfaces. Field-dependent Hall characteristics (2–300 K) are obtained for samples grown at various growth pressures. In addition to multiple electron transport, interfacial magnetism is tracked exploiting the anomalous Hall effect (AHE). These two properties both contribute to a nonlinearity in the field dependence of the Hall resistance, with multiple carrier conduction evident below 30 K and AHE at temperatures ≲10  K. Considering these two sources of nonlinearity, we suggest a phenomenological model capturing the complex field dependence of the Hall characteristics in the low-temperature regime. Our model allows the extraction of the conventional transport parameters and a qualitative analysis of the magnetization. The electron mobility is found to decrease systematically with increasing growth pressure. This suggests dominant electron scattering by acceptor-type strontium vacancies incorporated during growth. The AHE scales with growth pressure. The most pronounced AHE is found at increased growth pressure and, thus, in the most defective, low-mobility samples, indicating a correlation between transport, magnetism, and cation defect concentratio

EasyClone: method for iterative chromosomal integration of multiple genes in <em>Saccharomyces cerevisiae</em>

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log(10) mean +/- 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out

Fabrication of Functionalized Double-Lamellar Multifunctional Envelope-Type Nanodevices Using a Microfluidic Chip with a Chaotic Mixer Array

Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>105) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency