Vectors for therapeutic antisense sequences delivery and the modification of messenger RNA processing

Synthetic antisense oligoribonucleotides can be used to modulate gene splicing by masking key motifs on the pre-mRNA required for spliceosome assembly. Yet, intracellular expression of oligoribonucleotides generates only a transitory effect whereas stable delivery of antisense sequences can be achieved by linking them to chimeric small RNAs delivered and expressed by viral vectors. In the murine model of Duchenne Muscular Dystrophy a chimeric U7 snRNA (U7Dtex23) induces skipping of the mutated exon 23 and restores the Dystrophin mRNA reading frame. The main limitation of this approach remains the large amount of snRNA vector needed to be produced and administered to patients. To optimize this system we used self-complementary AAV vectors (scAAV) to express the U7snRNA shuttles. ScAAV vectors were tested in mouse myoblast cultures and we observed an increase in U7Dtex23 expression and in dystrophin exon 23 skipping compared to single-stranded AAV, highlighting the potential for this strategy to reduce the vector dose. Alternatively, we have used a muscle and heart-specific enhancer (MHCK) to drive the expression of U7Dtex23 cassettes delivered with AAV vectors and our results showed that MHCK improves chimeric U7snRNA expression and increases dystrophin exon 23 skipping in vitro and in vivo. However, additional U7snRNA species were produced following gene transfer, pointing at a possible limitation of the cellular processing machinery capability with saturating levels of U7 shuttles. We have also explored the possibility of using small nucleolar (sno) RNAs as novel molecular platforms for antisense delivery. We replaced the original antisense of MBII-52 snoRNA with the Dtex23 sequence and observed low levels of exon 23 skipping in AAV-transduced myotubes. While our observation validates the approach, the efficiency of skipping is still considerably lower than with the U7snRNA cassette. As a last approach, we engineered the human C/D box U24snoRNA to specifically target the methylation of an adenosine branch point in a luciferase reporter pre-mRNA in order to induce the skipping of the downstream exon. We were not able to observe any modulation of splicing using this strategy

International Network for Capacity Building for the Control of Emerging Viral Vector-Borne Zoonotic Diseases: Arbo-Zoonet

Arboviruses are arthropod-borne viruses, which include West Nile fever virus (WNFV), a mosquito-borne virus, Rift Valley fever virus (RVFV), a mosquito-borne virus, and Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne virus. These arthropod-borne viruses can cause disease in different domestic and wild animals and in humans, posing a threat to public health because of their epidemic and zoonotic potential. In recent decades, the geographical distribution of these diseases has expanded. Outbreaks of WNF have already occurred in Europe, especially in the Mediterranean basin. Moreover, CCHF is endemic in many European countries and serious outbreaks have occurred, particularly in the Balkans, Turkey and Southern Federal Districts of Russia. In 2000, RVF was reported for the first time outside the African continent, with cases being confirmed in Saudi Arabia and Yemen. This spread was probably caused by ruminant trade and highlights that there is a threat of expansion of the virus into other parts of Asia and Europe. In the light of global warming and globalisation of trade and travel, public interest in emerging zoonotic diseases has increased. This is especially evident regarding the geographical spread of vector-borne diseases. A multi-disciplinary approach is now imperative, and groups need to collaborate in an integrated manner that includes vector control, vaccination programmes, improved therapy strategies, diagnostic tools and surveillance, public awareness, capacity building and improvement of infrastructure in endemic regions

The jet of the BL Lac object PKS 0521 -365 in the near-IR : MAD adaptive optics observations

BL Lac objects are low--power active nuclei exhibiting a variety of peculiar properties that are caused by the presence of a relativistic jet and orientation effects. We present here adaptive optics near-IR images at high spatial resolution of the nearby BL Lac object PKS 0521-365, which is known to display a prominent jet both at radio and optical frequencies. The observations were obtained in Ks--band using the ESO multi-conjugated adaptive optics demonstrator at the Very Large Telescope. This allowed us to obtain images with 0.1 arcsec effective resolution. We performed a detailed analysis of the jet and its related features from the near-IR images, and combined them with images previously obtained with HST in the R band and by a re-analysis of VLA radio maps. We find a remarkable similarity in the structure of the jet at radio, near-IR, and optical wavelengths. The broad--band emission of the jet knots is dominated by synchrotron radiation, while the nucleus also exhibits a significant inverse Compton component. We discovered the near-IR counterpart of the radio hotspot and found that the near-IR flux is consistent with being a synchrotron emission from radio to X-ray. The bright red object (red-tip), detached but well aligned with the jet, is well resolved in the near-IR and has a linear light profile. Since it has no radio counterpart, we propose that it is a background galaxy not associated with the jet. The new adaptive optics near-IR images and previous observations at other frequencies allow us to study the complex environment around the remarkable BL Lac object PKS 0521-365. These data exemplify the capabilities of multi conjugate adaptive optics observations of extragalactic extended sources.Comment: accepted for publication in Astronomy and Astrophysics 9 pages. A & A 2009, in pres

On the radio and NIR jet of PKS 2155-304 and its close environment

PKS 2155-304 is one of the brightest BL Lac object in the sky and a very well studied target from radio to TeV bands. We report on high-resolution (~ 0.12 arcsec) direct imaging of the field of PKS 2155-304 using adaptive optics near-IR observations in J and Ks bands obtained with the ESO multi-conjugate adaptive optic demonstrator (MAD) at the Very Large Telescope. These data are complemented with archival VLA images at various frequencies to investigate the properties of the close environment of the source. We characterized the faint galaxies that form the poor group associated to the target. No radio emission is present for these galaxies, while an old radio jet at ~ 20 kpc from the nucleus of PKS 2155-304 and a jet-like structure of ~ 2 kpc (~ 1 arcsec) in the eastern direction are revealed. No counterparts of these radio jets are found in the NIR or in archival Chandra observations.Comment: 10 pages, 7 figures, accepted for publication in The Astronomical Journa

MAD Adaptive Optics Imaging of High Luminosity Quasars: A Pilot Project

We present near-IR images of five luminous quasars at z~2 and one at z~4 obtained with an experimental adaptive optics instrument at the ESO Very Large Telescope. The observations are part of a program aimed at demonstrating the capabilities of multi-conjugated adaptive optics imaging combined with the use of natural guide stars for high spatial resolution studies on large telescopes. The observations were mostly obtained under poor seeing conditions but in two cases. In spite of these non optimal conditions, the resulting images of point sources have cores of FWHM ~0.2 arcsec. We are able to characterize the host galaxy properties for 2 sources and set stringent upper limits to the galaxy luminosity for the others. We also report on the expected capabilities for investigating the host galaxies of distant quasars with adaptive optics systems coupled with future Extremely Large Telescopes. Detailed simulations show that it will be possible to characterize compact (2-3 kpc) quasar host galaxies for QSOs at z = 2 with nucleus K-magnitude spanning from 15 to 20 (corresponding to absolute magnitude -31 to -26) and host galaxies that are 4 mag fainter than their nuclei.Comment: 19 pages, 12 figures, accepted for pubblication in The Astronomical Journa

On-sky results of the adaptive optics MACAO for the new IR-spectrograph CRIRES at VLT

The adaptive optics MACAO has been implemented in 6 focii of the VLT observatory, in three different flavors. We present in this paper the results obtained during the commissioning of the last of these units, MACAO-CRIRES. CRIRES is a high-resolution spectrograph, which efficiency will be improved by a factor two at least for point-sources observations with a NGS brighter than R=15. During the commissioning, Strehl exceeding 60% have been observed with fair seeing conditions, and a general description of the performance of this curvature adaptive optics system is done.Comment: SPIE conference 2006, Advances in adaptive optics, 12 pages, 11 figure

Renewed global partnerships and redesigned roadmaps for rabies prevention and control

Canine rabies, responsible for most human rabies deaths, is a serious global public health concern. This zoonosis is entirely preventable, but by focusing solely upon rabies prevention in humans, this "incurable wound" persists at high costs. Although preventing human deaths through canine rabies elimination is feasible, dog rabies control is often neglected, because dogs are not considered typical economic commodities by the animal health sector. Here, we demonstrate that the responsibility of managing rabies falls upon multiple sectors, that a truly integrated approach is the key to rabies elimination, and that considerable progress has been made to this effect. Achievements include the construction of global rabies networks and organizational partnerships; development of road maps, operational toolkits, and a blueprint for rabies prevention and control; and opportunities for scaling up and replication of successful programs. Progress must continue towards overcoming the remaining challenges preventing the ultimate goal of rabies elimination

Resolving Stellar Populations outside the Local Group: MAD observations of UKS2323-326

We present a study aimed at deriving constraints on star formation at intermediate ages from the evolved stellar populations in the dwarf irregular galaxy UKS2323-326. These observations were also intended to demonstrate the scientific capabilities of the multi-conjugated adaptive optics demonstrator (MAD) implemented at the ESO Very Large Telescope as a test-bench of adaptive optics (AO) techniques. We perform accurate, deep photometry of the field using J and Ks band AO images of the central region of the galaxy. The near-infrared (IR) colour-magnitude diagrams clearly show the sequences of asymptotic giant branch (AGB) stars, red supergiants, and red giant branch (RGB) stars down to ~1 mag below the RGB tip. Optical-near-IR diagrams, obtained by combining our data with Hubble Space Telescope observations, provide the best separation of stars in the various evolutionary stages. The counts of AGB stars brighter than the RGB tip allow us to estimate the star formation at intermediate ages. Assuming a Salpeter initial mass function, we find that the star formation episode at intermediate ages produced ~6x10^5 M_sun of stars in the observed region.Comment: 4 pages, 4 figures, Accepted for publication in A&A Letter

MCAO near-IR photometry of the Globular Cluster NGC 6388: MAD observations in crowded fields

Deep photometry of crowded fields, such as Galactic Globular Clusters, is severely limited by the actual resolution of ground-based telescopes. On the other hand, the Hubble Space Telescope does not provide the near-infrared (NIR) filters needed to allow large color baselines. In this work we aim at demonstrating how ground based observations can reach the required resolution when using Multi-Conjugated Adaptive Optic (MCAO) devices in the NIR, such as the experimental infrared camera (MAD) available on the VLT. This is particularly important since these corrections are planned to be available on all ground--based telescopes in the near future. We do this by combining the infrared photometry obtained by MAD/VLT with ACS/HST optical photometry of our scientific target, the bulge globular cluster NGC 6388, in which we imaged two fields. In particular, we constructed color-magnitude diagrams with an extremely wide color baseline in order to investigate the presence of multiple stellar populations in this cluster. From the analysis of the external field, observed with better seeing conditions, we derived the deepest optical-NIR CMD of NGC 6388 to date. The high-precision photometry reveals that two distinct sub-giant branches are clearly present in this cluster. We also use the CMD from the central region to estimate the distance ((m-M)=15.33) and the reddening (E(B-V)=0.38) for this cluster. We estimate the age to be ~11.5+/- 1.5 Gyr. The large relative-age error reflects the bimodal distribution of the SGB stars. This study clearly demonstrates how MCAO correction in the NIR bands implemented on ground based telescopes can complement the high-resolution optical data from HST.Comment: 9 pages, 10 figures. Accepted for publication on A. &

Inhibition of somatosensory mechanotransduction by annexin A6

Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron-derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein. Annexin A6-deficient mice showed increased sensitivity to mechanical stimuli. Sensory neurons from these mice showed increased activity of the cation channel Piezo2, which mediates a rapidly adapting mechano-gated current linked to proprioception and touch, and a decrease in mechanically activated, slowly adapting currents. Conversely, overexpression of annexin A6 in sensory neurons inhibited rapidly adapting currents that were partially mediated by Piezo2. Furthermore, overexpression of annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis, a disease in which mechanically evoked pain is particularly problematic. These data suggest that annexin A6 can be exploited to inhibit chronic mechanical pain