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Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the alpha-tubulin mRNA 5' untranslated region (5' UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic alpha-tubulin 5' UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of alpha-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to alpha-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized α-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.

Ixodes scapularis ticks transmit many pathogens, including Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Vaccines directed against arthropod proteins injected into the host during tick engorgement could prevent numerous infectious diseases. Salp14, a salivary anticoagulant, poses a key target for such intervention. Salp14 is the prototypic member of a family of potential I. scapularis anticoagulants, expressed and secreted in tick saliva during tick feeding. RNA interference was used to assess the role of Salp14 in tick feeding. Salp14 and its paralogs were silenced, as demonstrated by the reduction of mRNA and protein specific for these antigens. Tick salivary glands lacking Salp14 had reduced anticoagulant activity, as revealed by a 60-80% reduction of anti-factor Xa activity. Silencing the expression of salp14 and its paralogs also reduced the ability of I. scapularis to feed, as demonstrated by a 50-70% decline in the engorgement weights. Because ticks have several anticoagulants, it is likely that the expression of multiple anticoagulants in I. scapularis saliva would have to be ablated simultaneously to abolish tick feeding. These studies demonstrate that RNA interference can silence I. scapularis genes and disrupt their physiologic function in vivo, and they identify vaccine candidates that can alter vector engorgement.

In Trypanosoma brucei gambiense, the Ca2+ binding protein calmodulin is encoded by three identical tandemly repeated genes. The transcripts of these genes consist of several RNA species similar in size. A 35-nucleotide spliced leader sequence is present at the 5′ end of each mRNA but is not encoded by DNA contiguous to these genes. We have identified two different sites for the fusion of the leader to the mRNA. These results strongly support the idea that a novel, possibly discontinuous, transcription mechanism is used by these parasites.

In animals and protozoa gene-specific double-stranded RNA triggers the degradation of homologous cellular RNAs, the phenomenon of RNA interference (RNAi). RNAi has been shown to represent a novel paradigm in eukaryotic biology and a powerful method for studying gene function. Here we discuss RNAi in terms of its mechanism, its relationship to other post-transcriptional gene silencing phenomena in plants and fungi, its connection to retroposon silencing and possibly to translation, and its biological role. Among the organisms where RNAi has been demonstrated the protozoan parasite Trypanosoma brucei represents the most ancient branch of the eukaryotic lineage. We provide a synopsis of what is currently known about RNAi in T. brucei and outline the recent advances that make RNAi the method of choice to disrupt gene function in these organisms.

We investigate high resolution imaging polarimetry of HD 169142 taken in the R' and I' bands with the SPHERE/ZIMPOL instrument for an accurate quantitative measurement of the radiation scattered by the circumstellar disk. We observe a strong dependence of the disk polarimetry on the atmospheric turbulences, which strongly impact the AO performance. With our non-coronagraphic data we can analyze the polarimetric signal of the disk simultaneously with the strongly variable stellar PSF, correct for the convolution effects to determine the intrinsic polarization of the disk with high precision. We also extract the disk intensity signal and derive the fractional polarization. We compare the scattered flux from the inner and outer disk rings with the corresponding thermal dust emissions measured in the IR and estimate the ratio between scattered and absorbed radiation. We obtain ratios between the integrated disk polarization flux and total system flux of 0.43% for the R' band and 0.55% for the I' band. This indicates a reddish color for the light reflection by the dust. The inner disk ring contributes about 75% to the total disk flux. The obtained fractional polarization for the bright inner disk ring is 23.6% for the I' band and similar for the R' band. The ratio between scattered disk flux and star flux is about 2.3%. This is much smaller than the derived IR excess of 17.6% for the disk components observed in scattered light. This indicates that only a small fraction of the radiation illuminating the disk is scattered; most is absorbed and reemitted in the IR. We conclude that accurate, quantitative measurements of the scattered light from circumstellar disks are possible with ground based high contrast AO systems, if the PSF convolution effects are properly taken into account, and this provides important new constraints on the properties of the scattering dust.

We carried out very deep VLT/SPHERE imaging polarimetry of the nearby system Eps Eri based on 38.5 hours of integration time with a 600 - 900 nm broadband filter to search for polarized scattered light from a planet or from circumstellar dust using AO, coronagraphy, high precision differential polarimetry, and angular differential imaging. We have improved several data reduction and post-processing techniques and also developed new ones to further increase the sensitivity of SPHERE/ZIMPOL. The data provide unprecedented contrast limits, but no significant detection of a point source or an extended signal from circumstellar dust. For each observing epoch, we obtained a point source contrast for the polarized intensity between \\(2\\cdot 10^{-8}\\) and \\(4\\cdot 10^{-8}\\) at the expected separation of the planet Eps Eri b of 1'' near quadrature phase. The polarimetric contrast limits are about six to 50 times better than the intensity limits because polarimetric imaging is much more efficient in speckle suppression. Combining the entire 14-month data set to the search for a planet moving on a Keplerian orbit with the K-Stacker software further improves the contrast limits by a factor of about two, to about \\(8 \\cdot 10^{-9}\\) at 1''. This would allow the detection of a planet with a radius of about 2.5 Jupiter radii. The surface brightness contrast limits achieved for the polarized intensity from an extended scattering region are about 15 mag arcsec\\(^{-2}\\) at 1'', or up to 3 mag arcsec\\(^{-2}\\) deeper than previous limits. For Eps Eri, these limits exclude the presence of a narrow dust ring and they constrain the dust properties. This study shows that the polarimetric contrast limits for reflecting planets with SPHERE/ZIMPOL can be improved to a level \\(<10^{-8}\\) simply by collecting more data over many nights and using the K-Stacker software.

African trypanosomes change their antigenicity by successively expressing different members of a group of related but highly diverse proteins, the variant surface glycoproteins (VSGs). We describe a synthetic oligonucleotide that can prime specifically reverse transcription of VSG mRNA out of total trypanosome poly(A)+RNA. The specificity of this priming was verified by cDNA sequence analysis of the transcription products and by the demonstration of variant-specific hybridization of the individual cDNAs to cellular RNA. The oligonucleotide primer also was used as a probe for the conserved sequence found on these VSG mRNAs in trypanosome genomic DNA libraries. A large number of primer-positive clones were detected in a Trypanosoma gambiense genomic library, but very few positive signals were found in a library of Trypanosoma congolense genomic DNA.

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.

SPHERE (Beuzit et al,. 2019) has now been in operation at the VLT for more than 5 years, demonstrating a high level of performance. SPHERE has produced outstanding results using a variety of operating modes, primarily in the field of direct imaging of exoplanetary systems, focusing on exoplanets as point sources and circumstellar disks as extended objects. The achievements obtained thus far with SPHERE (~200 refereed publications) in different areas (exoplanets, disks, solar system, stellar physics...) have motivated a large consortium to propose an even more ambitious set of science cases, and its corresponding technical implementation in the form of an upgrade. The SPHERE+ project capitalizes on the expertise and lessons learned from SPHERE to push high contrast imaging performance to its limits on the VLT 8m-telescope. The scientific program of SPHERE+ described in this document will open a new and compelling scientific window for the upcoming decade in strong synergy with ground-based facilities (VLT/I, ELT, ALMA, and SKA) and space missions (Gaia, JWST, PLATO and WFIRST). While SPHERE has sampled the outer parts of planetary systems beyond a few tens of AU, SPHERE+ will dig into the inner regions around stars to reveal and characterize by mean of spectroscopy the giant planet population down to the snow line. Building on SPHERE's scientific heritage and resounding success, SPHERE+ will be a dedicated survey instrument which will strengthen the leadership of ESO and the European community in the very competitive field of direct imaging of exoplanetary systems. With enhanced capabilities, it will enable an even broader diversity of science cases including the study of the solar system, the birth and death of stars and the exploration of the inner regions of active galactic nuclei.