Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

<p>Abstract</p> <p>Background</p> <p>Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using <it>Arabidopsis </it>mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and <it>in vivo </it>protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.</p> <p>Results</p> <p>In this report, we present a new procedure that we call the Tape-<it>Arabidopsis </it>Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-<it>Arabidopsis </it>Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions <it>in vivo</it>, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr.</p> <p>Conclusion</p> <p>The protoplasts generated by this new Tape-<it>Arabidopsis </it>Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.</p

Formation of an SIR-Nucleosome Filament in vitro and Its Modulation by O-Acetyl-ADP-Ribose

It is not possible to clearly visualize how chromatin condenses to heterochromatin in vivo. However, in an in vitro system for Saccharomyces cerevisiae, the requirements for heterochromatin filament formation mirror those found in vivo. Here we report that the nucleosomes and the Sir2, Sir3 and Sir4 proteins, which are required for in vitro filament assembly, are also components of these filaments, confirming that the filaments are SIR-nucleosome filaments. We show the individual localization patterns of the Sir proteins on this SIR-nucleosome filament, and demonstrate that the metabolite, AAR, plays a specific and essential role in promoting the formation of this SIR-nucleosome filament

Optoelectronic properties of poly(fluorene) co‐polymer light‐emitting devices on a plastic substrate *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92131/1/1.2150379.pd

Almost sure state estimation with H2-type performance constraints for nonlinear hybrid stochastic systems

This paper is concerned with the problem of almost sure state estimation for general nonlinear hybrid stochastic systems whose coefficients only satisfy local Lipschitz conditions. By utilizing the stopping time method combined with martingale inequalities, a theoretical framework is established for analyzing the so-called almost surely asymptotic stability of the addressed system. Within such a theoretical framework, some sufficient conditions are derived under which the estimation dynamics is almost sure asymptotically stable and the upper bound of estimation error is also determined. Furthermore, a suboptimal state estimator is obtained by solving an optimization problem in the H2 sense. According to the obtained results, for a class of special nonlinear hybrid stochastic systems, the corresponding conditions reduce to a set of matrix inequalities for the purpose of easy implementation. Finally, two numerical simulation examples are used to demonstrate the effectiveness of the results derived.This work was supported in part by the National Natural Science Foundation of China under Grants 61134009 and 61329301, the Royal Society of the UK, and the Alexander von Humboldt Foundation of Germany

Impaired Osteoblast Function in GPRC6A Null Mice

GPRC6A is a widely expressed orphan G protein–coupled receptor that senses extracellular amino acids, osteocalcin, and divalent cations in vitro. GPRC6A null (GPRC6A−/−) mice exhibit multiple metabolic abnormalities including osteopenia. To investigate whether the osseous abnormalities are a direct function of GPRC6A in osteoblasts, we examined the function of primary osteoblasts and bone marrow stromal cell cultures (BMSCs) in GPRC6A−/− mice. We confirmed that GPRC6A−/− mice exhibited a decrease in bone mineral density (BMD) associated with reduced expression of osteocalcin, ALP, osteoprotegerin, and Runx2-II transcripts in bone. Osteoblasts and BMSCs derived from GPRC6A−/− mice exhibited an attenuated response to extracellular calcium-stimulated extracellular signal-related kinase (ERK) activation, diminished alkaline phosphatase (ALP) expression, and impaired mineralization ex vivo. In addition, siRNA-mediated knockdown of GPRC6A in MC3T3 osteoblasts also resulted in a reduction in extracellular calcium-stimulated ERK activity. To explore the potential relevance of GPRC6A function in humans, we looked for an association between GPRC6A gene polymorphisms and BMD in a sample of 1000 unrelated American Caucasians. We found that GPRC6A gene polymorphisms were significantly associated with human spine BMD. These data indicate that GRPC6A directly participates in the regulation of osteoblast-mediated bone mineralization and may mediate the anabolic effects of extracellular amino acids, osteocalcin, and divalent cations in bone. © 2010 American Society for Bone and Mineral Research

Discovery of six high-redshift quasars with the Lijiang 2.4m telescope and the Multiple Mirror Telescope

Quasars with redshifts greater than 4 are rare, and can be used to probe the structure and evolution of the early universe. Here we report the discovery of six new quasars with $i$-band magnitudes brighter than 19.5 and redshifts between 2.4 and 4.6 from the YFOSC spectroscopy of the Lijiang 2.4m telescope in February, 2012. These quasars are in the list of $z>3.6$ quasar candidates selected by using our proposed $J-K/i-Y$ criterion and the photometric redshift estimations from the SDSS optical and UKIDSS near-IR photometric data. Nine candidates were observed by YFOSC, and five among six new quasars were identified as $z>3.6$ quasars. One of the other three objects was identified as a star and the other two were unidentified due to the lower signal-to-noise ratio of their spectra. This is the first time that $z>4$ quasars have been discovered using a telescope in China. Thanks to the Chinese Telescope Access Program (TAP), the redshift of 4.6 for one of these quasars was confirmed by the Multiple Mirror Telescope (MMT) Red Channel spectroscopy. The continuum and emission line properties of these six quasars, as well as their central black hole masses and Eddington ratios, were obtained.Comment: 7 pages, 2 figures, published in Research in Astronomy and Astrophysics (RAA) as a lette