In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8

Background: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods: A method is described to massively expand bone marrow–derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

Table-mounted ring retractor for consistent visualization in endoscopy-assisted anterior reconstruction of burst fractures of the thoracolumbar junction

The authors tested an autoclavable external ring retractor, fixed to the operation table, for the endoscopic reconstruction of anterior column injuries of the thoracolumbar junction. It served as a retractor for the diaphragm, and offered a stable support for the scope and other instruments, making an assistant superfluous. Moreover, it allowed bimanual manipulation. Of course, the two-dimensional image, provided by the scope, necessitated proper eye-hand coordination. Twenty-eight consecutive patients underwent either a monosegmental (n = 10) or a bisegmental (n = 18) anterior stabilization in the area Th11L1. Three portals were necessary, but an assistant was not needed. The overall (mean +/- SD) operating time was 196 +/- 56 min, the blood loss was 804 +/- 719 mL. Intraoperatively, one epidural bleeding and a single screw cut-out occurred. All complications were managed endoscopically. Postoperatively, evacuation of a haemothorax (n = 1) was necessary. In all patients, wounds and fractures healed uneventfully. The combination of the endoscopic technique and the retractor system was feasible, successful, safe, and time efficient. Moreover, it allowed for anterior instrumentation of thoracolumbar fractures by a single surgeon. It became the standard approach in the authors' department.

Transverse-momentum spectra of electroweak bosons near threshold at NNLO

We obtain the next-to-next-to-leading order corrections to transverse-momentum spectra of W, Z and Higgs bosons near the partonic threshold. In the threshold limit, the electroweak boson recoils against a low-mass jet and all radiation is either soft, or collinear to the jet or the beam directions. We extract the virtual corrections from known results for the relevant two-loop four-point amplitudes and combine them with the soft and collinear two-loop functions as defined in Soft-Collinear Effective Theory. We have implemented these results in a public code PeTeR and present numerical results for the threshold resummed cross section of W and Z bosons at next-to-next-to-next-to-leading logarithmic accuracy, matched to next-to-leading fixed-order perturbation theory. The two-loop corrections lead to a moderate increase in the cross section and reduce the scale uncertainty by about a factor of two. The corrections are significantly larger for Higgs production.

The long arm of time pressure at work: Cognitive failure and commuting near-accidents

This study tests whether cognitive failures mediate effects of work-related time pressure and time control on commuting accidents and near-accidents. Participants were 83 employees (56% female) who each commuted between their regular place of residence and place of work using vehicles. The Workplace Cognitive Failure Scale (WCFS) asked for the frequency of failure in memory function, failure in attention regulation, and failure in action execution. Time pressure and time control at work were assessed by the Instrument for Stress Oriented Task Analysis (ISTA). Commuting accidents in the last 12 months were reported by 10% of participants, and half of the sample reported commuting near-accidents in the last 4 weeks. Cognitive failure significantly mediated the influence of time pressure at work on near-accidents even when age, gender, neuroticism, conscientiousness, commuting duration, commuting distance, and time pressure during commuting were controlled for. Time control was negatively related to cognitive failure and neuroticism, but no association with commuting accidents or near-accidents was found. Time pressure at work is likely to increase cognitive load. Time pressure might, therefore, increase cognitive failures during work and also during commuting. Hence, time pressure at work can decrease commuting safety. The result suggests a reduction of time pressure at work should improve commuting safety.

Solar wind reflection from the lunar surface: The view from far and near

The Moon appears bright in the sky as a source of energetic neutral atoms (ENAs). These ENAs have recently been imaged over a broad energy range both from near the lunar surface, by India's Chandrayaan-1 mission (CH-1), and from a much more distant Earth orbit by NASA's Interstellar Boundary Explorer (IBEX) satellite. Both sets of observations have indicated that a relatively large fraction of the solar wind is reflected from the Moon as energetic neutral hydrogen. CH-1's angular resolution over different viewing angles of the lunar surface has enabled measurement of the emission as a function of angle. IBEX in contrast views not just a swath but a whole quadrant of the Moon as effectively a single pixel, as it subtends even at the closest approach no more than a few degrees on the sky. Here we use the scattering function measured by CH-1 to model global lunar ENA emission and combine these with IBEX observations. The deduced global reflection is modestly larger (by a factor of 1.25) when the angular scattering function is included. This provides a slightly updated IBEX estimate of AH=0.11±0.06 for the global neutralized albedo, which is ˜25% larger than the previous values of 0.09±0.05, based on an assumed uniform scattering distribution.

Proton entry into the near-lunar plasma wake for magnetic field aligned flow

We report the first observation of protons in the near-lunar (100-200 km from the surface) and deeper (near anti-subsolar point) plasma wake when the interplanetary magnetic field (IMF) and solar wind velocity (vsw) are parallel (aligned flow; angle between IMF and vsw≤10°). More than 98% of the observations during aligned flow condition showed the presence of protons in the wake. These observations are obtained by the Solar Wind Monitor sensor of the Sub-keV Atom Reflecting Analyser experiment on Chandrayaan-1. The observation cannot be explained by the conventional fluid models for aligned flow. Back tracing of the observed protons suggests that their source is the solar wind. The larger gyroradii of the wake protons compared to that of solar wind suggest that they were part of the tail of the solar wind velocity distribution function. Such protons could enter the wake due to their large gyroradii even when the flow is aligned to IMF. However, the wake boundary electric field may also play a role in the entry of the protons into the wake.

A Near-Real-Time Automatic Orbit Determination System for COSMIC and Its Follow-On Satellite Mission: Analysis of Orbit and Clock Errors on Radio Occultation

The COSMIC-2 mission is a follow-on mission of the Constellation Observing System for Meteorology, Ionosphere, and Climate (COSMIC) with an upgraded payload for improved radio occultation (RO) applications. The objective of this paper is to develop a near-real-time (NRT) orbit determination system, called NRT National Chiao Tung University (NCTU) system, to support COSMIC-2 in atmospheric applications and verify the orbit product of COSMIC. The system is capable of automatic determinations of the NRT GPS clocks and LEO orbit and clock. To assess the NRT (NCTU) system, we use eight days of COSMIC data (March 24-31, 2011), which contain a total of 331 GPS observation sessions and 12 393 RO observable files. The parallel scheduling for independent GPS and LEO estimations and automatic time matching improves the computational efficiency by 64% compared to the sequential scheduling. Orbit difference analyses suggest a 10-cm accuracy for the COSMIC orbits from the NRT (NCTU) system, and it is consistent as the NRT University Corporation for Atmospheric Research (URCA) system. The mean velocity accuracy from the NRT orbits of COSMIC is 0.168 mm/s, corresponding to an error of about 0.051 μrad in the bending angle. The rms differences in the NRT COSMIC clock and in GPS clocks between the NRT (NCTU) and the postprocessing products are 3.742 and 1.427 ns. The GPS clocks determined from a partial ground GPS network [from NRT (NCTU)] and a full one [from NRT (UCAR)] result in mean rms frequency stabilities of 6.1E-12 and 2.7E-12, respectively, corresponding to range fluctuations of 5.5 and 2.4 cm and bending angle errors of 3.75 and 1.66 μrad .

Walking near a conformal fixed point

We investigate the 2-d O(3) model with a q-term as a toy model for slowly walking 4-d non-Abelian gauge theories. Using the very efficient meron-cluster algorithm, an accurate investigation of the scale dependence of the renormalized coupling is carried out for different values of the vacuum angle q. Approaching q = p, the infrared dynamics of the 2-d O(3) model is determined by a non-trivial conformal fixed point. We provide evidence for a slowly walking behavior near the fixed point and we perform a finite-size scaling analysis of the mass gap.

Topoclimatological case-study of Alpine pastures near the Albula Pass in the eastern Swiss Alps

Alpine grasslands are an important source of fodder for the cattle of Alpine farmers. Only during the short summer season can these pastures be used for grazing. With the anticipated climate change, it is likely that plant production – and thus the fodder basis for the cattle – will be influenced. Investigating the dependence of biomass production on topoclimatic factors will allow us to better understand how anticipated climate change may influence this traditional Alpine farming system. Because small-scale topoclimatological variations of the main meteorological variables: temperature, humidity, precipitation, shortwave incoming radiation and wind speed are not easily derived from available long-term climate stations in mountainous terrain, it was our goal to investigate the topoclimatic variations over the pastures belonging to the Alp Weissenstein research station north of the Albula Pass in the eastern Swiss Alps. We present a basic assessment of current topoclimatic conditions as a site characterization for ongoing ecological climate change studies. To be able to link short-term studies with long-term climate records, we related agrometeorological measurements with those of surrounding long-term sites run by MeteoSwiss, both on valley bottoms (Davos, Samedan), and on mountain tops (Weissfluhjoch, Piz Corvatsch). We found that the Davos climate station north of the study area is most closely correlated with the local climate of Alp Weissenstein, although a much closer site (Samedan) exists on the other side of the Albula Pass. Mountain top stations, however, did not provide a convincing approximation for the climate at Alp Weissenstein. Direct comparisons of near-surface measurements from a set of 11 small weather stations distributed over the domain where cattle and sheep are grazed indicate that nocturnal minimum air temperature and minimum vapor pressure deficit are mostly governed by the altitudinal gradient, whereas daily maxima – including also wind speed – are more strongly depending on vegetation cover and less on the altitude.

Molecular mechanism by which the Escherichia coli nucleoid occlusion factor, SlmA, keeps cytokinesis in check

Cell division or cytokinesis is one of the most fundamental processes in biology and is essential for the propagation of all living species. In Escherichia coli, cell division occurs by ingrowth of the membrane envelope at the cell center and is orchestrated by the FtsZ protein. FtsZ self-assembles into linear protofilaments in a GTP dependent manner to form a cytoskeletal scaffold called the Z-ring. The Z-ring provides the framework for the assembly of the division apparatus and determines the site of cytokinesis. The total amount of FtsZ molecules in a cell significantly exceeds the concentration required for Z-ring formation. Hence, Z-ring formation must be highly regulated, both temporally and spatially. In particular, the assembly of Z-rings at the cell poles and over chromosomal DNA must be prevented. These inhibitory roles are played by two key regulatory systems called the Min and nucleoid occlusion (NO) systems. In E. coli, Min proteins oscillate from pole to pole; the net result of this oscillatory process is the formation of a zone of FtsZ inhibition at the cell poles. However, the replicated nucleoid DNA near the midcell must also be protected from bisection by the Z-ring which is ensured by NO. A protein called SlmA was shown to be the effector of NO in E. coli. SlmA was identified in a screen designed to isolate mutations that were lethal in the absence of Min, hence the name SlmA (synthetic lethal with a defective Min system). Furthers SlmA was shown to bind DNA and localize to the nucleoid fraction of the cell. Additionally, light scattering experiments suggested that SlmA interacts with FtsZ-GTP and alters its polymerization properties. Here we describe studies that reveal the molecular mechanism by which SlmA mediates NO in E. coli. Specifically, we determined the crystal structure of SlmA, identified its DNA binding site specificity, and mapped its binding sites on the E. coli chromosome by chromatin immuno-precipitation experiments. We went on to determine the SlmA-FtsZ structure by small angle X-ray scattering and examined the effect of SlmA-DNA on FtsZ polymerization by electron microscopy. Our combined data show how SlmA is able to disrupt Z-ring formation through its interaction with FtsZ in a specific temporal and spatial manner and hence prevent nucleoid guillotining during cell division.

Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C-ring assembly flexion.

The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of approximately 1,280 flagellar motors, a approximately 3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.