Protein-trap version 2.1: screening for expressed proteins in mammalian cells based on their localizations.

BACKGROUND: "Protein-trap" is a method that allows epitope-tagging of endogenous proteins. This method allows for the identification of endogenously expressed proteins that exhibit specific localization of interest. This method has been recently reported for its application in the study of Drosophila development by using a relatively large epitope, green-fluorescent-protein (GFP). RESULT: Herein, we report a new "protein-trap" vector for mammalian cells. This new method utilizes a much smaller epitope-tag and also allows for drug-selection prior to the epitope-tagging. Pre-selection by drug resulted in the highly efficient protein-trapping frequency. CONCLUSION: The "protein-trap" method based on this new vector is expected to serve as a complimentary approach to the previously reported GFP-based method

Study of Time Evolution of Thermal and Non-Thermal Emission from an M-Class Solar Flare

We conduct a wide-band X-ray spectral analysis in the energy range of 1.5-100 keV to study the time evolution of the M7.6 class flare of 2016 July 23, with the Miniature X-ray Solar Spectrometer (MinXSS) CubeSat and the Reuven Ramaty High Energy Solar Spectroscopic Imager (RHESSI) spacecraft. With the combination of MinXSS for soft X-rays and RHESSI for hard X-rays, a non-thermal component and three-temperature multi-thermal component -- "cool" ($T \approx$ 3 MK), "hot" ($T \approx$ 15 MK), and "super-hot" ($T \approx$ 30 MK) -- were measured simultaneously. In addition, we successfully obtained the spectral evolution of the multi-thermal and non-thermal components with a 10 s cadence, which corresponds to the Alfv\'en time scale in the solar corona. We find that the emission measures of the cool and hot thermal components are drastically increasing more than hundreds of times and the super-hot thermal component is gradually appearing after the peak of the non-thermal emission. We also study the microwave spectra obtained by the Nobeyama Radio Polarimeters (NoRP), and we find that there is continuous gyro-synchrotron emission from mildly relativistic non-thermal electrons. In addition, we conducted a differential emission measure (DEM) analysis by using Atmospheric Imaging Assembly (AIA) onboard the Solar Dynamics Observatory (SDO) and determine that the DEM of cool plasma increases within the flaring loop. We find that the cool and hot plasma components are associated with chromospheric evaporation. The super-hot plasma component could be explained by the thermalization of the non-thermal electrons trapped in the flaring loop.Comment: 20 pages, 12 figures, 1 tables. Accepted for publication in Ap

Chromospheric Anemone Jets as Evidence of Ubiquitous Reconnection

The heating of the solar chromosphere and corona is a long-standing puzzle in solar physics. Hinode observations show the ubiquitous presence of chromospheric anemone jets outside sunspots in active regions. They are typically 3 to 7 arc seconds = 2000 to 5000 kilometers long and 0.2 to 0.4 arc second = 150 to 300 kilometers wide, and their velocity is 10 to 20 kilometers per second. These small jets have an inverted Y-shape, similar to the shape of x-ray anemone jets in the corona. These features imply that magnetic reconnection similar to that in the corona is occurring at a much smaller spatial scale throughout the chromosphere and suggest that the heating of the solar chromosphere and corona may be related to small-scale ubiquitous reconnection.Comment: 10 pages, 5 figure

A two-domain elevator mechanism for sodium/proton antiport

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets2. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli1, 3, for which both electron microscopy and crystal structures are available4, 5, 6. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein1, 4. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur7. The only reported NhaA crystal structure so far is of the low pH inactivated form4. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding1, 8, 9 directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second3, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general

Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death

The purinergic signalling system, which uses purines and pyrimidines as chemical transmitters, and purinoceptors as effectors, is deeply rooted in evolution and development and is a pivotal factor in cell communication. The ATP and its derivatives function as a 'danger signal' in the most primitive forms of life. Purinoceptors are extraordinarily widely distributed in all cell types and tissues and they are involved in the regulation of an even more extraordinary number of biological processes. In addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion, there is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body. In this article, we focus on the latter in the immune/defence system, in stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption, as well as in cancer. Cell Death and Disease (2010) 1, e9; doi:10.1038/cddis.2009.11; published online 14 January 201

Fixed-time artificial insemination in beef cattle

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background: The study was designed to test the effect of fixed-time artificial insemination (fixed-AI) after the slightly modified Ovsynch protocol on the pregnancy rate in beef cattle in Finnish field conditions. The modification was aimed to optimize the number of offsprings per AI dose. Methods: Ninety Charolais cows and heifers were entered into the program an average of 1.8 times. Thus, 164 animal cases were included. Animals were administered 10-12 μg of buserelin. Seven days later animals without a corpus luteum (CL) were rejected (20.7%) while the remaining 130 cases with a CL were administered prostaglandin F2α, followed 48 h later with a second injection of buserelin (8-10 μg). Fixed-AI was performed 16-20 hours after the last injection. Results: The pregnancy rate was 51.5 % (67/130). The pregnancy rate after a short interval (50-70 d) from calving to entering the program was significantly higher than that after a long interval (>70 d). Conclusion: This protocol seems to give acceptable pregnancy results in beef herds and its effect on saving labour is notable