Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

<p>Abstract</p> <p>Background</p> <p>Gene expression in lipopolysaccharide (LPS)-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR) using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or ACTB (beta-actin) as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system.</p> <p>Results</p> <p>Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B), B2M (beta-2-microglobulin) and PPIA (cyclophilin A) as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein) and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha) and IL10 (interleukin 10) expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH.</p> <p>Conclusions</p> <p>Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.</p

New Extinction and Mass Estimates of the Low-mass Companion 1RXS 1609 B with the Magellan AO System: Evidence of an Inclined Dust Disk

We used the Magellan adaptive optics system to image the 11 Myr substellar companion 1RXS 1609 B at the bluest wavelengths to date (z' and Ys). Comparison with synthetic spectra yields a higher temperature than previous studies of $T_\mathrm{eff}=2000\pm100\mathrm{K}$ and significant dust extinction of $A_V=4.5^{+0.5}_{-0.7}$ mag. Mass estimates based on the DUSTY tracks gives 0.012-0.015 Msun, making the companion likely a low-mass brown dwarf surrounded by a dusty disk. Our study suggests that 1RXS 1609 B is one of the 25% of Upper Scorpius low-mass members harboring disks, and it may have formed like a star and not a planet out at 320 AU.Comment: 5 pages, 4 figures; accepted to ApJ

New Extinction and Mass Estimates from Optical Photometry of the Very Low Mass Brown Dwarf Companion CT Chamaeleontis B with the Magellan AO System

We used the Magellan adaptive optics (MagAO) system and its VisAO CCD camera to image the young low mass brown dwarf companion CT Chamaeleontis B for the first time at visible wavelengths. We detect it at r', i', z', and Ys. With our new photometry and Teff~2500 K derived from the shape its K-band spectrum, we find that CT Cha B has Av = 3.4+/-1.1 mag, and a mass of 14-24 Mj according to the DUSTY evolutionary tracks and its 1-5 Myr age. The overluminosity of our r' detection indicates that the companion has significant Halpha emission and a mass accretion rate ~6*10^-10 Msun/yr, similar to some substellar companions. Proper motion analysis shows that another point source within 2" of CT Cha A is not physical. This paper demonstrates how visible wavelength AO photometry (r', i', z', Ys) allows for a better estimate of extinction, luminosity, and mass accretion rate of young substellar companions.Comment: Accepted for publication in ApJ; 6 figure

Unraveling spatiotemporal variability of arbuscular mycorrhizal fungi in a temperate grassland plot

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Goldmann, K., Boeddinghaus, R. S., Klemmer, S., Regan, K. M., Heintz-Buschart, A., Fischer, M., Prati, D., Piepho, H., Berner, D., Marhan, S., Kandeler, E., Buscot, F., & Wubet, T. Unraveling spatiotemporal variability of arbuscular mycorrhizal fungi in a temperate grassland plot. Environmental Microbiology, 22(3),(2020): 873-888, doi:10.1111/1462-2920.14653.Soils provide a heterogeneous environment varying in space and time; consequently, the biodiversity of soil microorganisms also differs spatially and temporally. For soil microbes tightly associated with plant roots, such as arbuscular mycorrhizal fungi (AMF), the diversity of plant partners and seasonal variability in trophic exchanges between the symbionts introduce additional heterogeneity. To clarify the impact of such heterogeneity, we investigated spatiotemporal variation in AMF diversity on a plot scale (10 × 10 m) in a grassland managed at low intensity in southwest Germany. AMF diversity was determined using 18S rDNA pyrosequencing analysis of 360 soil samples taken at six time points within a year. We observed high AMF alpha‐ and beta‐diversity across the plot and at all investigated time points. Relationships were detected between spatiotemporal variation in AMF OTU richness and plant species richness, root biomass, minimal changes in soil texture and pH. The plot was characterized by high AMF turnover rates with a positive spatiotemporal relationship for AMF beta‐diversity. However, environmental variables explained only ≈20% of the variation in AMF communities. This indicates that the observed spatiotemporal richness and community variability of AMF was largely independent of the abiotic environment, but related to plant properties and the cooccurring microbiome.We thank the managers of the three Exploratories, Kirsten Reichel‐Jung, Swen Renner, Katrin Hartwich, Sonja Gockel, Kerstin Wiesner, and Martin Gorke for their work in maintaining the plot and project infrastructure; Christiane Fischer and Simone Pfeiffer for giving support through the central office, Michael Owonibi and Andreas Ostrowski for managing the central data base, and Eduard Linsenmair, Dominik Hessenmöller, Jens Nieschulze, Ernst‐Detlef Schulze, Wolfgang W. Weisser and the late Elisabeth Kalko for their role in setting up the Biodiversity Exploratories project. The work has been funded by the DFG Priority Program 1374 ‘Infrastructure‐Biodiversity‐Exploratories’ (BU 941/22‐1, BU 941/22‐3, KA 1590/8‐2, KA 1590/8‐3). Field work permits were issued by the responsible state environmental office of Baden‐Württemberg (according to § 72 BbgNatSchG). Likewise, we kindly thank Beatrix Schnabel, Melanie Günther and Sigrid Härtling for 454 sequencing in Halle. AHB gratefully acknowledges the support of the German Centre for Integrative Biodiversity Research (iDiv) Halle‐Jena‐Leipzig funded by the German Research Foundation (FZT 118). Authors declare no conflict of interests

New Spatially Resolved Observations of the T Cha Transition Disk and Constraints on the Previously Claimed Substellar Companion

We present multi-epoch non-redundant masking observations of the T Cha transition disk, taken at the VLT and Magellan in H, Ks, and L' bands. T Cha is one of a small number of transition disks that host companion candidates discovered by high-resolution imaging techniques, with a putative companion at a position angle of 78 degrees, separation of 62 mas, and contrast at L' of 5.1 mag. We find comparable binary parameters in our re-reduction of the initial detection images, and similar parameters in the 2011 L', 2013 NaCo L', and 2013 NaCo Ks data sets. We find a close-in companion signal in the 2012 NaCo L' dataset that cannot be explained by orbital motion, and a non-detection in the 2013 MagAO/Clio2 L' data. However, Monte-carlo simulations show that the best fits to the 2012 NaCo and 2013 MagAO/Clio2 followup data may be consistent with noise. There is also a significant probability of false non-detections in both of these data sets. We discuss physical scenarios that could cause the best fits, and argue that previous companion and scattering explanations are inconsistent with the results of the much larger dataset presented here.Comment: 25 pages, 22 figures, accepted for publication in Ap

Directly Imaged L-T Transition Exoplanets in the Mid-Infrared

Gas-giant planets emit a large fraction of their light in the mid-infrared ($\gtrsim$3$\mu$m), where photometry and spectroscopy are critical to our understanding of the bulk properties of extrasolar planets. Of particular importance are the L and M-band atmospheric windows (3-5$\mu$m), which are the longest wavelengths currently accessible to ground-based, high-contrast imagers. We present binocular LBT AO images of the HR 8799 planetary system in six narrow-band filters from 3-4$\mu$m, and a Magellan AO image of the 2M1207 planetary system in a broader 3.3$\mu$m band. These systems encompass the five known exoplanets with luminosities consistent with L$\rightarrow$T transition brown dwarfs. Our results show that the exoplanets are brighter and have shallower spectral slopes than equivalent temperature brown dwarfs in a wavelength range that contains the methane fundamental absorption feature (spanned by the narrowband filters and encompassed by the broader 3.3$\mu$m filter). For 2M1207 b, we find that thick clouds and non-equilibrium chemistry caused by vertical mixing can explain the object's appearance. For the HR 8799 planets, we present new models that suggest the atmospheres must have patchy clouds, along with non-equilibrium chemistry. Together, the presence of a heterogeneous surface and vertical mixing presents a picture of dynamic planetary atmospheres in which both horizontal and vertical motions influence the chemical and condensate profiles.Comment: Accepted to Ap

Magellan Adaptive Optics first-light observations of the exoplanet beta Pic b. II. 3-5 micron direct imaging with MagAO+Clio, and the empirical bolometric luminosity of a self-luminous giant planet

Young giant exoplanets are a unique laboratory for understanding cool, low-gravity atmospheres. A quintessential example is the massive extrasolar planet $\beta$ Pic b, which is 9 AU from and embedded in the debris disk of the young nearby A6V star $\beta$ Pictoris. We observed the system with first light of the Magellan Adaptive Optics (MagAO) system. In Paper I we presented the first CCD detection of this planet with MagAO+VisAO. Here we present four MagAO+Clio images of $\beta$ Pic b at 3.1 $\mu$m, 3.3 $\mu$m, $L^\prime$, and $M^\prime$, including the first observation in the fundamental CH$_4$ band. To remove systematic errors from the spectral energy distribution (SED), we re-calibrate the literature photometry and combine it with our own data, for a total of 22 independent measurements at 16 passbands from 0.99--4.8 $\mu$m. Atmosphere models demonstrate the planet is cloudy but are degenerate in effective temperature and radius. The measured SED now covers $>$80\% of the planet's energy, so we approach the bolometric luminosity empirically. We calculate the luminosity by extending the measured SED with a blackbody and integrating to find log($L_{bol}$/$L_{Sun}$) $= -3.78\pm0.03$. From our bolometric luminosity and an age of 23$\pm$3 Myr, hot-start evolutionary tracks give a mass of 12.7$\pm$0.3 $M_{Jup}$, radius of 1.45$\pm$0.02 $R_{Jup}$, and $T_{eff}$ of 1708$\pm$23 K (model-dependent errors not included). Our empirically-determined luminosity is in agreement with values from atmospheric models (typically $-3.8$ dex), but brighter than values from the field-dwarf bolometric correction (typically $-3.9$ dex), illustrating the limitations in comparing young exoplanets to old brown dwarfs.Comment: Accepted to ApJ. 27 pages, 22 figures, 19 table

Dynacortin facilitates polarization of chemotaxing cells

<p>Abstract</p> <p>Background</p> <p>Cell shape changes during cytokinesis and chemotaxis require regulation of the actin cytoskeletal network. Dynacortin, an actin cross-linking protein, localizes to the cell cortex and contributes to cortical resistance, thereby helping to define the cell shape changes of cytokinesis. Dynacortin also becomes highly enriched in cortical protrusions, which are sites of new actin assembly.</p> <p>Results</p> <p>We studied the effect of dynacortin on cell motility during chemotaxis and on actin dynamics <it>in vivo </it>and <it>in vitro</it>. Dynacortin enriches with the actin, particularly at the leading edge of chemotaxing cells. Cells devoid of dynacortin do not become as polarized as wild-type control cells but move with similar velocities as wild-type cells. In particular, they send out multiple pseudopods that radiate at a broader distribution of angles relative to the chemoattractant gradient. Wild-type cells typically only send out one pseudopod at a time that does not diverge much from 0° on average relative to the gradient. Though <it>dynacortin</it>-deficient cells show normal bulk (whole-cell) actin assembly upon chemoattractant stimulation, dynacortin can promote actin assembly <it>in vitro</it>. By fluorescence spectroscopy, co-sedimentation and transmission electron microscopy, dynacortin acts as an actin scaffolder in which it assembles actin monomers into polymers with a stoichiometry of 1 Dyn₂:1 actin under salt conditions that disfavor polymer assembly.</p> <p>Conclusion</p> <p>Dynacortin contributes to cell polarization during chemotaxis. By cross-linking and possibly stabilizing actin polymers, dynacortin also contributes to cortical viscoelasticity, which may be critical for establishing cell polarity. Though not essential for directional sensing or motility, dynacortin is required to establish cell polarity, the third core feature of chemotaxis.</p