47 research outputs found
Dissecting molecular phenotypes through FACS-based pooled CRISPR screens
Pooled CRISPR screens are emerging as a powerful tool to dissect regulatory networks, by assessing how a protein responds to genetic perturbations in a highly multiplexed manner. A large number of genes are perturbed in a cell population through genomic integration of one single-guide RNA (sgRNA) per cell. A subset of cells with the phenotype of interest can then be enriched through fluorescence-activated cell sorting (FACS). SgRNAs with altered abundance after phenotypic enrichment allow identification of genes that either promote or attenuate the investigated phenotype. Here we provide detailed guidelines on how to design and execute a pooled CRISPR screen to investigate molecular phenotypes. We describe how to generate a custom sgRNA library and how to perform a FACS-based screen using readouts such as intracellular antibody staining or Flow-FISH to assess phosphorylation levels or RNA abundance. Through the variety of available perturbation systems and readout options many different molecular and cellular phenotypes can now be tackled with pooled CRISPR screens
Identification of X-chromosomal genes that drive global X-dosage effects in mammals
X-chromosomal genes contribute to sex differences, in particular during early development, whenboth X chromosomes are active in females. Here, double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting thedifferentiation-promoting MAP kinase (MAPK) signalling pathway and delaying differentiation. Toidentify the genetic basis of these sex differences, we have performed a series of CRISPR knockoutscreens in murine embryonic stem cells to comprehensively identify X-linked genes that cause thefemale pluripotency phenotype. We found multiple genes that act in concert, among which Klhl13plays a central role. We show that this E3 ubiquitin ligase substrate adaptor protein promotes pluripotency factor expression, delays differentiation and represses MAPK target genes, and weidentify putative substrates. We thus elucidate the mechanisms that drive sex-induced differences inpluripotent cells with implications for gender medicine in the context of induced pluripotent stem cellbased therapies
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
An approach to analyse the specific impact of rapamycin on mRNA-ribosome association
<p>Abstract</p> <p>Background</p> <p>Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background.</p> <p>Methods</p> <p>We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out). For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation.</p> <p>Results</p> <p>High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug.</p> <p>Conclusion</p> <p>The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of a particular drug in a living cell.</p
Sensitive and frequent identification of high avidity neo-epitope specific CD8 + T cells in immunotherapy-naive ovarian cancer.
Immunotherapy directed against private tumor neo-antigens derived from non-synonymous somatic mutations is a promising strategy of personalized cancer immunotherapy. However, feasibility in low mutational load tumor types remains unknown. Comprehensive and deep analysis of circulating and tumor-infiltrating lymphocytes (TILs) for neo-epitope specific CD8 <sup>+</sup> T cells has allowed prompt identification of oligoclonal and polyfunctional such cells from most immunotherapy-naive patients with advanced epithelial ovarian cancer studied. Neo-epitope recognition is discordant between circulating T cells and TILs, and is more likely to be found among TILs, which display higher functional avidity and unique TCRs with higher predicted affinity than their blood counterparts. Our results imply that identification of neo-epitope specific CD8 <sup>+</sup> T cells is achievable even in tumors with relatively low number of somatic mutations, and neo-epitope validation in TILs extends opportunities for mutanome-based personalized immunotherapies to such tumors
A precise architecture characterization of the Men planetary system
The bright star Men was chosen as the first target for a radial
velocity follow-up to test the performance of ESPRESSO, the new high-resolution
spectrograph at the ESO's Very-Large Telescope (VLT). The star hosts a
multi-planet system (a transiting 4 M planet at 0.07 au, and a
sub-stellar companion on a 2100-day eccentric orbit) which is
particularly appealing for a precise multi-technique characterization. With the
new ESPRESSO observations, that cover a time span of 200 days, we aim to
improve the precision and accuracy of the planet parameters and search for
additional low-mass companions. We also take advantage of new photometric
transits of Men c observed by TESS over a time span that overlaps with
that of the ESPRESSO follow-up campaign. We analyse the enlarged spectroscopic
and photometric datasets and compare the results to those in the literature. We
further characterize the system by means of absolute astrometry with Hipparcos
and Gaia. We used the spectra of ESPRESSO for an independent determination of
the stellar fundamental parameters. We present a precise characterization of
the planetary system around Men. The ESPRESSO radial velocities alone
(with typical uncertainty of 10 cm/s) allow for a precise retrieval of the
Doppler signal induced by Men c. The residuals show an RMS of 1.2 m/s,
and we can exclude companions with a minimum mass less than 2 M
within the orbit of Men c). We improve the ephemeris of Men c using
18 additional TESS transits, and in combination with the astrometric
measurements, we determine the inclination of the orbital plane of Men b
with high precision ( deg). This leads to the precise
measurement of its absolute mass M, and
shows that the planetary orbital planes are highly misaligned.Comment: Accepted for publication on A&
Revisiting Proxima with ESPRESSO
We aim to confirm the presence of Proxima b using independent measurements
obtained with the new ESPRESSO spectrograph, and refine the planetary
parameters taking advantage of its improved precision. We analysed 63
spectroscopic ESPRESSO observations of Proxima taken during 2019. We obtained
radial velocity measurements with a typical radial velocity photon noise of 26
cm/s. We ran a joint MCMC analysis on the time series of the radial velocity
and full-width half maximum of the cross-correlation function to model the
planetary and stellar signals present in the data, applying Gaussian process
regression to deal with stellar activity. We confirm the presence of Proxima b
independently in the ESPRESSO data. The ESPRESSO data on its own shows Proxima
b at a period of 11.218 0.029 days, with a minimum mass of 1.29
0.13 Me. In the combined dataset we measure a period of 11.18427 0.00070
days with a minimum mass of 1.173 0.086 Me. We find no evidence of
stellar activity as a potential cause for the 11.2 days signal. We find some
evidence for the presence of a second short-period signal, at 5.15 days with a
semi-amplitude of merely 40 cm/s. If caused by a planetary companion, it would
correspond to a minimum mass of 0.29 0.08 Me. We find that the FWHM of
the CCF can be used as a proxy for the brightness changes and that its gradient
with time can be used to successfully detrend the radial velocity data from
part of the influence of stellar activity. The activity-induced radial velocity
signal in the ESPRESSO data shows a trend in amplitude towards redder
wavelengths. Velocities measured using the red end of the spectrograph are less
affected by activity, suggesting that the stellar activity is spot-dominated.
The data collected excludes the presence of extra companions with masses above
0.6 Me at periods shorter than 50 days.Comment: 25 pages, 26 figure
ESPRESSO at VLT. On-sky performance and first results
Context. ESPRESSO is the new high-resolution spectrograph of ESO's Very Large Telescope (VLT). It was designed for ultra-high radial-velocity (RV) precision and extreme spectral fidelity with the aim of performing exoplanet research and fundamental astrophysical experiments with unprecedented precision and accuracy. It is able to observe with any of the four Unit Telescopes (UTs) of the VLT at a spectral resolving power of 140 000 or 190 000 over the 378.2 to 788.7 nm wavelength range; it can also observe with all four UTs together, turning the VLT into a 16 m diameter equivalent telescope in terms of collecting area while still providing a resolving power of 70 000. Aims: We provide a general description of the ESPRESSO instrument, report on its on-sky performance, and present our Guaranteed Time Observation (GTO) program along with its first results. Methods: ESPRESSO was installed on the Paranal Observatory in fall 2017. Commissioning (on-sky testing) was conducted between December 2017 and September 2018. The instrument saw its official start of operations on October 1, 2018, but improvements to the instrument and recommissioning runs were conducted until July 2019. Results: The measured overall optical throughput of ESPRESSO at 550 nm and a seeing of 0.65″ exceeds the 10% mark under nominal astroclimatic conditions. We demonstrate an RV precision of better than 25 cm s-1 during a single night and 50 cm s-1 over several months. These values being limited by photon noise and stellar jitter shows that the performance is compatible with an instrumental precision of 10 cm s-1. No difference has been measured across the UTs, neither in throughput nor RV precision. Conclusions: The combination of the large collecting telescope area with the efficiency and the exquisite spectral fidelity of ESPRESSO opens a new parameter space in RV measurements, the study of planetary atmospheres, fundamental constants, stellar characterization, and many other fields. Based on GTOs collected at the European Southern Observatory under ESO program(s) 1102.C-0744, 1102.C-0958 and 1104.C-0350 by the ESPRESSO Consortium