On the extent and role of the small proteome in the parasitic eukaryote Trypanosoma brucei

Background: Although technical advances in genomics and proteomics research have yielded a better understanding of the coding capacity of a genome, one major challenge remaining is the identification of all expressed proteins, especially those less than 100 amino acids in length. Such information can be particularly relevant to human pathogens, such as Trypanosoma brucei, the causative agent of African trypanosomiasis, since it will provide further insight into the parasite biology and life cycle. Results: Starting with 993 T. brucei transcripts, previously shown by RNA-Sequencing not to coincide with annotated coding sequences (CDS), homology searches revealed that 173 predicted short open reading frames in these transcripts are conserved across kinetoplastids with 13 also conserved in representative eukaryotes. Mining mass spectrometry data sets revealed 42 transcripts encoding at least one matching peptide. RNAi-induced down-regulation of these 42 transcripts revealed seven to be essential in insect-form trypanosomes with two also required for the bloodstream life cycle stage. To validate the specificity of the RNAi results, each lethal phenotype was rescued by co-expressing an RNAi-resistant construct of each corresponding CDS. These previously non-annotated essential small proteins localized to a variety of cell compartments, including the cell surface, mitochondria, nucleus and cytoplasm, inferring the diverse biological roles they are likely to play in T. brucei. We also provide evidence that one of these small proteins is required for replicating the kinetoplast (mitochondrial) DNA. Conclusions: Our studies highlight the presence and significance of small proteins in a protist and expose potential new targets to block the survival of trypanosomes in the insect vector and/or the mammalian host

An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

A Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated

Cytochrome oxidase subunit VI of Trypanosoma brucei is imported without a cleaved presequence and is developmentally regulated at both RNA and protein levels

Mitochondrial respiration in the African trypanosome undergoes dramatic developmental stage regulation. This requires co-ordinated control of components encoded by both the nuclear genome and the kinetoplast, the unusual mitochondrial genome of these parasites. As a model for understanding the co-ordination of these genomes, we have examined the regulation and mitochondrial import of a nuclear-encoded component of the cytochrome oxidase complex, cytochrome oxidase subunit VI (COXVI). By generating transgenic trypanosomes expressing intact or mutant forms of this protein, we demonstrate that COXVI is not imported using a conventional cleaved presequence and show that sequences at the N-terminus of the protein are necessary for correct mitochondrial sorting. Analyses of endogenous and transgenic COXVI mRNA and protein expression in parasites undergoing developmental stage differentiation demonstrates a temporal order of control involving regulation in the abundance of, first, mRNA and then protein. This represents the first dissection of the regulation and import of a nuclear-encoded protein into the cytochrome oxidase complex in these organisms, which were among the earliest eukaryotes to possess a mitochondrion

Tollip, an early regulator of the acute inflammatory response in the substantia nigra.

Tollip is a ubiquitously expressed protein, originally described as a modulator of the IL-1R/TLR-NF-κB signaling pathways. Although this property has been well characterized in peripheral cells, and despite some evidence of its expression in the central nervous system, the role of Tollip in neuroinflammation remains poorly understood. The present study sought to explore the implication of Tollip in inflammation in the substantia nigra pars compacta, the structure affected in Parkinson's disease. We first investigated Tollip distribution in the midbrain by immunohistochemistry. Then, we addressed TLR4-mediated response by intra-nigral injections of lipopolysaccharide (LPS), a TLR4 agonist, on inflammatory markers in Tollip knockout (KO) and wild-type (WT) mice. We report an unexpectedly high Tollip immunostaining in dopaminergic neurons of the mice brain. Second, intra-nigral injection of LPS led to increased susceptibility to neuroinflammation in Tollip KO compared to Tollip WT mice. This was demonstrated by a significant increase of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interferon gamma (IFN-γ) messenger RNA (mRNA) in the midbrain of Tollip KO mice upon LPS injection. Consistently, brain rAAV viral vector transduction with a nuclear factor kappa B (NF-κB)-inducible reporter gene confirmed increased NF-κB activation in Tollip KO mice. Lastly, Tollip KO mice displayed higher inducible NO synthase (iNOS) production, both at the messenger and protein level when compared to LPS-injected WT mice. Tollip deletion also aggravated LPS-induced oxidative and nitrosative damages, as indicated by an increase of 8-oxo-2'-deoxyguanosine and nitrotyrosine immunostaining, respectively. Altogether, these findings highlight a critical role of Tollip in the early phase of TLR4-mediated neuroinflammation. As brain inflammation is known to contribute to Parkinson's disease, Tollip may be a potential target for neuroprotection

ESPRESSO: The next European exoplanet hunter

The acronym ESPRESSO stems for Echelle SPectrograph for Rocky Exoplanets and Stable Spectroscopic Observations; this instrument will be the next VLT high resolution spectrograph. The spectrograph will be installed at the Combined-Coud\'e Laboratory of the VLT and linked to the four 8.2 m Unit Telescopes (UT) through four optical Coud\'e trains. ESPRESSO will combine efficiency and extreme spectroscopic precision. ESPRESSO is foreseen to achieve a gain of two magnitudes with respect to its predecessor HARPS, and to improve the instrumental radial-velocity precision to reach the 10 cm/s level. It can be operated either with a single UT or with up to four UTs, enabling an additional gain in the latter mode. The incoherent combination of four telescopes and the extreme precision requirements called for many innovative design solutions while ensuring the technical heritage of the successful HARPS experience. ESPRESSO will allow to explore new frontiers in most domains of astrophysics that require precision and sensitivity. The main scientific drivers are the search and characterization of rocky exoplanets in the habitable zone of quiet, nearby G to M-dwarfs and the analysis of the variability of fundamental physical constants. The project passed the final design review in May 2013 and entered the manufacturing phase. ESPRESSO will be installed at the Paranal Observatory in 2016 and its operation is planned to start by the end of the same year.Comment: 12 pages, figures included, accepted for publication in Astron. Nach

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work

Air Corrosivity in U.S. Outdoor-Air-Cooled Data Centers is Similar to That in Conventional Data Centers

There is a concern that environmental-contamination caused corrosion may negatively affect Information Technology (IT) equipment reliability. Nineteen data centers in the United States and two in India were evaluated using Corrosion Classification Coupons (CCC) to assess environmental air quality as it may relate IT equipment reliability. The data centers were of two basic types: closed and outside-air cooled. A closed data center provides cool air to the IT equipment using air conditioning in which only a small percent age of the recirculation air is make-up air continuously supplied from outside to meet human health requirements. An outside-air cooled data center uses outside air directly as the primary source for IT equipment cooling. Corrosion measuring coupons containing copper and silver metal strips were placed in both closed and outside-air cooled data centers. The coupons were placed at each data center (closed and outside-air cooled types) with the location categorized into three groups: (1) Outside - coupons sheltered, located near or at the supply air inlet, but located before any filtering, (2) Supply - starting just after initial air filtering continuing inside the plenums and ducts feeding the data center rooms, and (3) Inside located inside the data center rooms near the IT equipment. Each coupon was exposed for thirty days and then sent to a laboratory for a corrosion rate measurement analysis. The goal of this research was to investigate whether gaseous contamination is a concern for U.S. data center operators as it relates to the reliability of IT equipment. More specifically, should there be an increased concern if outside air for IT equipment cooling is used To begin to answer this question limited exploratory measurements of corrosion rates in operating data centers in various locations were undertaken. This study sought to answer the following questions: (1) What is the precision of the measurements (2) What are the approximate statistical distributions of copper and silver corrosion rates in the sampled data centers(3) To what extent are copper and silver corrosion measurements related (4) What is the relationship of corrosion rate measurements between outside-air cooled data centers compared to closed data centers (5) How do corrosivity measurements relate to IT equipment failure rates The data from our limited sample size suggests that most United States data center operators should not be concerned with environmental gaseous contamination causing high IT equipment failure rates even when using outside-air cooling. The research team recommends additional basic research on how environmental conditions, specifically gaseous contamination, affect electronic equipment reliability

HD 142527: quantitative disk polarimetry with SPHERE

We present high-precision photometry and polarimetry for the protoplanetary disk around HD142527, with a focus on determining the light scattering parameters of the dust. We re-reduced polarimetric differential imaging data of HD142527 in the VBB (735 nm) and H-band (1625 nm) from the ZIMPOL and IRDIS subinstruments of SPHERE/VLT. With polarimetry and photometry based on reference star differential imaging, we were able to measure the linearly polarized intensity and the total intensity of the light scattered by the circumstellar disk with high precision. We used simple Monte Carlo simulations of multiple light scattering by the disk surface to derive constraints for three scattering parameters of the dust: the maximum polarization of $P_{\rm max}$, the asymmetry parameter $g$, and the single-scattering albedo $\omega$. We measure a reflected total intensity of $51.4\pm1.5$ mJy and $206\pm12$ mJy and a polarized intensity of $11.3\pm0.3$ mJy and $55.1\pm3.3$ mJy in the VBB and H-band, respectively. We also find in the visual range a degree of polarization that varies between $28\%$ on the far side of the disk and $17\%$ on the near side. The disk shows a red color for the scattered light intensity and the polarized intensity, which are about twice as high in the near-infrared when compared to the visual. We determine with model calculations the scattering properties of the dust particles and find evidence for strong forward scattering ($g\approx 0.5-0.75$), relatively low single-scattering albedo ($\omega \approx 0.2-0.5$), and high maximum polarization ($P_{\rm max} \approx 0.5-0.75$) at the surface on the far side of the disk for both observed wavelengths. The optical parameters indicate the presence of large aggregate dust particles, which are necessary to explain the high maximum polarization, the strong forward-scattering nature of the dust, and the observed red disk color.Comment: 20 pages, 14 figure