An Improved Transwell Design for Microelectrode Ion-Flux Measurements.

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures.

MPM motifs of the yeast SKT protein Trk1 can assemble to form a functional K+-translocation system.

The yeast Trk1 polypeptide, like other members of the Superfamily of K Transporters (SKT proteins) consists of four Membrane-Pore-Membrane motifs (MPMs A-D) each of which is homologous to a single K-channel subunit. SKT proteins are thought to have evolved from ancestral K-channels via two gene duplications and thus single MPMs might be able to assemble when located on different polypeptides. To test this hypothesis experimentally we generated a set of partial gene deletions to create alleles encoding one, two, or three MPMs, and analysed the cellular localisation and interactions of these Trk1 fragments using GFP tags and Bimolecular Fluorescence Complementation (BiFC). The function of these partial Trk1 proteins either alone or in combinations was assessed by expressing the encoding genes in a K+-uptake deficient strain lacking also the K-channel Tok1 (trk1,trk2,tok1Δ) and (i) analysing their ability to promote growth in low [K+] media and (ii) by ion flux measurements using "microelectrode based ion flux estimation" (MIFE). We found that proteins containing only one or two MPM motifs can interact with each other and assemble with a polypeptide consisting of the rest of the Trk system to form a functional K+-translocation system.

The S. cerevisiae cation translocation protein Trk1 is functional without its "long hydrophilic loop" but LHL regulates cation translocation activity and selectivity.

In Saccharomyces cerevisiae, K+-uptake under K+-limiting conditions is largely mediated by the cation translocation systems Trk1 and Trk2 belonging to the family of SKT proteins. They are related to two-transmembrane-domain (inward rectifying K-) channels but unlike the symmetrical tetrameric structure of K-channels, a single Trk contains four pore-forming domains (A-D) encoded on one polypeptide chain. Between domains A and B Trks contain large cytosolic regions dubbed "long hydrophilic loop" (LHL). LHLs are not homologous/similar to any other identified protein (domain) and also show little similarity between Trk1 and Trk2. Here we demonstrate that Trk1 is functional without LHL. However, in growth experiments NaCl sensitivity of Trk1[ΔLHL] expressing cells is increased under K+-limiting conditions compared to full-length Trk1. Non-invasive ion flux measurements showed that K+-influx through Trk1 and Trk1[ΔLHL] is decreased in the presence of surplus Na+ due to permeability of the proteins for both cations and competition between them. Trk1[ΔLHL] is less affected than full-length Trk1 because it is more selective for K+ over Na+. Furthermore, K+ re-uptake after starvation is delayed and decreased in Trk1[ΔLHL]. Thus, a role of LHL is regulating cation fluxes through Trk1 by (i) allowing also Na+ to pass if monovalent cations (mainly K+) are limiting and (ii) by accelerating/enhancing a switch from low to high affinity ion translocation. We propose that LHL could modulate Trk1 transport properties via direct influence on a transmembrane helix (M2A) which can switch between bent and straight conformation, thereby directly modifying the radius of the pore and selectivity filter.

Photoacoustic Sounds from Meteors.

Concurrent sound associated with very bright meteors manifests as popping, hissing, and faint rustling sounds occurring simultaneously with the arrival of light from meteors. Numerous instances have been documented with -11 to -13 brightness. These sounds cannot be attributed to direct acoustic propagation from the upper atmosphere for which travel time would be several minutes. Concurrent sounds must be associated with some form of electromagnetic energy generated by the meteor, propagated to the vicinity of the observer, and transduced into acoustic waves. Previously, energy propagated from meteors was assumed to be RF emissions. This has not been well validated experimentally. Herein we describe experimental results and numerical models in support of photoacoustic coupling as the mechanism. Recent photometric measurements of fireballs reveal strong millisecond flares and significant brightness oscillations at frequencies ≥40 Hz. Strongly modulated light at these frequencies with sufficient intensity can create concurrent sounds through radiative heating of common dielectric materials like hair, clothing, and leaves. This heating produces small pressure oscillations in the air contacting the absorbers. Calculations show that -12 brightness meteors can generate audible sound at ~25 dB SPL. The photoacoustic hypothesis provides an alternative explanation for this longstanding mystery about generation of concurrent sounds by fireballs.

The trajectory, structure and origin of the Chelyabinsk asteroidal impactor.

Earth is continuously colliding with fragments of asteroids and comets of various sizes. The largest encounter in historical times occurred over the Tunguska river in Siberia in 1908, producing an airburst of energy equivalent to 5-15 megatons of trinitrotoluene (1 kiloton of trinitrotoluene represents an energy of 4.185 × 10(12) joules). Until recently, the next most energetic airburst events occurred over Indonesia in 2009 and near the Marshall Islands in 1994, both with energies of several tens of kilotons. Here we report an analysis of selected video records of the Chelyabinsk superbolide of 15 February 2013, with energy equivalent to 500 kilotons of trinitrotoluene, and details of its atmospheric passage. We found that its orbit was similar to the orbit of the two-kilometre-diameter asteroid 86039 (1999 NC43), to a degree of statistical significance sufficient to suggest that the two were once part of the same object. The bulk strength--the ability to resist breakage--of the Chelyabinsk asteroid, of about one megapascal, was similar to that of smaller meteoroids and corresponds to a heavily fractured single stone. The asteroid broke into small pieces between the altitudes of 45 and 30 kilometres, preventing more-serious damage on the ground. The total mass of surviving fragments larger than 100 grams was lower than expected.

An anomalous basaltic meteorite from the innermost main belt.

Triangulated observations of fireballs allow us to determine orbits and fall positions for meteorites. The great majority of basaltic meteorites are derived from the asteroid 4 Vesta. We report on a recent fall that has orbital properties and an oxygen isotope composition that suggest a distinct parent body. Although its orbit was almost entirely contained within Earth's orbit, modeling indicates that it originated from the innermost main belt. Because the meteorite parent body would likely be classified as a V-type asteroid, V-type precursors for basaltic meteorites unrelated to Vesta may reside in the inner main belt. This starting location is in agreement with predictions of a planetesimal evolution model that postulates the formation of differentiated asteroids in the terrestrial planet region, with surviving fragments concentrated in the innermost main belt.

Photographic observations of Neuschwanstein, a second meteorite from the orbit of the Príbram chondrite.

Photographic observations of meteoroids passing through the atmosphere provide information about the population of interplanetary bodies in the Earth's vicinity in the size range from 0.1 m to several metres. It is extremely rare that any of these meteoroids survives atmospheric entry to be recovered as a meteorite on the ground. Príbram was the first meteorite (an ordinary chondrite) with a photographically determined orbit; it fell on 7 April 1959 (ref. 1). Here we report the fourth meteorite fall to be captured by camera networks. We determined the atmospheric trajectory and pre-atmospheric orbit of the object from the photographic records. One 1.75-kg meteorite--named Neuschwanstein and classified as an enstatite chondrite--was recovered within the predicted impact area. The bolide's heliocentric orbit is exceptional as it is almost identical to the orbit of Príbram, suggesting that we have discovered a 'stream' of meteoritic objects in an Earth-crossing orbit. The chemical classifications and cosmic-ray exposure ages of the two meteorites are quite different, however, which implies a heterogeneous stream.