Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F3 families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F3 sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance

An In-Depth Spectroscopic Analysis of the Blazhko Star RR Lyr. I. Characterisation of the star: abundance analysis and fundamental parameters

The knowledge of accurate stellar parameters is a keystone in several fields of stellar astrophysics, such as asteroseismology and stellar evolution. Although the fundamental parameters can be derived both from spectroscopy and multicolour photometry, the results obtained are sometimes affected by systematic uncertainties. In this paper, we present a self-consistent spectral analysis of the pulsating star RR Lyr, which is the primary target for our study of the Blazhko effect. We used high-resolution and high signal-to-noise ratio spectra to carry out a consistent parameter determination and abundance analysis for RR Lyr. We provide a detailed description of the methodology adopted to derive the fundamental parameters and the abundances. Stellar pulsation attains high amplitudes in RR Lyrae stars, and as a consequence the stellar parameters vary significantly over the pulsation cycle. The abundances of the star, however, are not expected to change. From a set of available high-resolution spectra of RR Lyr we selected the phase of maximum radius, at which the spectra are least disturbed by the pulsation. Using the abundances determined at this phase as a starting point, we expect to obtain a higher accuracy in the fundamental parameters determined at other phases. The set of fundamental parameters obtained in this work fits the observed spectrum accurately. Through the abundance analysis, we find clear indications for a depth-dependent microturbulent velocity, that we quantified. We confirm the importance of a consistent analysis of relevant spectroscopic features, application of advanced model atmospheres, and the use of up-to-date atomic line data for the determination of stellar parameters. These results are crucial for further studies, e.g., detailed theoretical modelling of the observed pulsations.Comment: 12 pages, accepted for publication in Astronomy & Astrophysic

Optimization and testing of dried antibody tube: The EuroFlow LST and PIDOT tubes as examples

Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots. In addition, the use of dried tubes may result in a reduced staining variability between different samples and thereby contributes to the generation of more robust data. Therefore, by using ready-to-use reagents in a dried single test tube format, the laboratory efficiency and quality will be improved

The Photodetector Array Camera and Spectrometer (PACS) on the Herschel Space Observatory

The Photodetector Array Camera and Spectrometer (PACS) is one of the three science instruments on ESA's far infrared and submillimetre observatory. It employs two Ge:Ga photoconductor arrays (stressed and unstressed) with 16x25 pixels, each, and two filled silicon bolometer arrays with 16x32 and 32x64 pixels, respectively, to perform integral-field spectroscopy and imaging photometry in the 60-210\mu\ m wavelength regime. In photometry mode, it simultaneously images two bands, 60-85\mu\ m or 85-125\mu\m and 125-210\mu\ m, over a field of view of ~1.75'x3.5', with close to Nyquist beam sampling in each band. In spectroscopy mode, it images a field of 47"x47", resolved into 5x5 pixels, with an instantaneous spectral coverage of ~1500km/s and a spectral resolution of ~175km/s. We summarise the design of the instrument, describe observing modes, calibration, and data analysis methods, and present our current assessment of the in-orbit performance of the instrument based on the Performance Verification tests. PACS is fully operational, and the achieved performance is close to or better than the pre-launch predictions

Catalog of Galactic Beta Cephei Stars

We present an extensive and up-to-date catalog of Galactic Beta Cephei stars. This catalog is intended to give a comprehensive overview of observational characteristics of all known Beta Cephei stars. 93 stars could be confirmed to be Beta Cephei stars. For some stars we re-analyzed published data or conducted our own analyses. 61 stars were rejected from the final Beta Cephei list, and 77 stars are suspected to be Beta Cephei stars. A list of critically selected pulsation frequencies for confirmed Beta Cephei stars is also presented. We analyze the Beta Cephei stars as a group, such as the distributions of their spectral types, projected rotational velocities, radial velocities, pulsation periods, and Galactic coordinates. We confirm that the majority of these stars are multiperiodic pulsators. We show that, besides two exceptions, the Beta Cephei stars with high pulsation amplitudes are slow rotators. We construct a theoretical HR diagram that suggests that almost all 93 Beta Cephei stars are MS objects. We discuss the observational boundaries of Beta Cephei pulsation and their physical parameters. We corroborate that the excited pulsation modes are near to the radial fundamental mode in frequency and we show that the mass distribution of the stars peaks at 12 solar masses. We point out that the theoretical instability strip of the Beta Cephei stars is filled neither at the cool nor at the hot end and attempt to explain this observation

Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes

Ribosomal surveillance pathways scan for ribosomes that are transiently paused or terminally stalled owing to structural elements in mRNAs or nascent chain sequences. Some stalls in budding yeast are sensed by the GTPase Hbs1, which loads Dom34, a catalytically inactive member of the archaeo-eukaryotic release factor 1 superfamily. Hbs1–Dom34 and the ATPase Rli1 dissociate stalled ribosomes into 40S and 60S subunits. However, the 60S subunits retain the peptidyl-tRNA nascent chains, which recruit the ribosome quality control complex that consists of Rqc1–Rqc2–Ltn1–Cdc48–Ufd1–Npl4. Nascent chains ubiquitylated by the E3 ubiquitin ligase Ltn1 are extracted from the 60S subunit by the ATPase Cdc48–Ufd1–Npl4 and presented to the 26S proteasome for degradation. Failure to degrade the nascent chains leads to protein aggregation and proteotoxic stress in yeast and neurodegeneration in mice. Despite intensive investigations on the ribosome quality control pathway, it is not known how the tRNA is hydrolysed from the ubiquitylated nascent chain before its degradation. Here we show that the Cdc48 adaptor Vms1 is a peptidyl-tRNA hydrolase. Similar to classical eukaryotic release factor 1, Vms1 activity is dependent on a conserved catalytic glutamine. Evolutionary analysis indicates that yeast Vms1 is the founding member of a clade of eukaryotic release factor 1 homologues that we designate the Vms1-like release factor 1 clade

NetCTLpan: pan-specific MHC class I pathway epitope predictions

Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates predictions of proteasomal cleavage, transporter associated with antigen processing (TAP) transport efficiency, and MHC class I binding affinity into a MHC class I pathway likelihood score and is an improved and extended version of NetCTL. The NetCTLpan method performs predictions for all MHC class I molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I ligands and cytotoxic T lymphocyte (CTL) epitopes. It has been reported that MHC molecules are differentially dependent on TAP transport and proteasomal cleavage. Here, we did not find any consistent signs of such MHC dependencies, and the NetCTLpan method is implemented with fixed weights for proteasomal cleavage and TAP transport for all MHC molecules. The predictive performance of the NetCTLpan method was shown to outperform other state-of-the-art CTL epitope prediction methods. Our results further confirm the importance of using full-type human leukocyte antigen restriction information when identifying MHC class I epitopes. Using the NetCTLpan method, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively, when compared to the NetMHCpan and NetCTL methods. The method and benchmark datasets are available at http://www.cbs.dtu.dk/services/NetCTLpan/

The CCR4-NOT Complex Physically and Functionally Interacts with TRAMP and the Nuclear Exosome

BACKGROUND: Ccr4-Not is a highly conserved multi-protein complex consisting in yeast of 9 subunits, including Not5 and the major yeast deadenylase Ccr4. It has been connected functionally in the nucleus to transcription by RNA polymerase II and in the cytoplasm to mRNA degradation. However, there has been no evidence so far that this complex is important for RNA degradation in the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In this work we point to a new role for the Ccr4-Not complex in nuclear RNA metabolism. We determine the importance of the Ccr4-Not complex for the levels of non-coding nuclear RNAs, such as mis-processed and polyadenylated snoRNAs, whose turnover depends upon the nuclear exosome and TRAMP. Consistently, mutation of both the Ccr4-Not complex and the nuclear exosome results in synthetic slow growth phenotypes. We demonstrate physical interactions between the Ccr4-Not complex and the exosome. First, Not5 co-purifies with the exosome. Second, several exosome subunits co-purify with the Ccr4-Not complex. Third, the Ccr4-Not complex is important for the integrity of large exosome-containing complexes. Finally, we reveal a connection between the Ccr4-Not complex and TRAMP through the association of the Mtr4 helicase with the Ccr4-Not complex and the importance of specific subunits of Ccr4-Not for the association of Mtr4 with the nuclear exosome subunit Rrp6. CONCLUSIONS/SIGNIFICANCE: We propose a model in which the Ccr4-Not complex may provide a platform contributing to dynamic interactions between the nuclear exosome and its co-factor TRAMP. Our findings connect for the first time the different players involved in nuclear and cytoplasmic RNA degradation