The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant ...kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal.
Here we describe the treatment of Solanum lycopersicum seeds with 1% EMS and the development of a new mutated tomato population. To increase targeted mutant detection throughput an automated seed DNA extraction has been combined with novel mutation detection platforms for TILLING in plants. We have adapted two techniques used in human genetic diagnostics: Conformation Sensitive Capillary Electrophoresis (CSCE) and High Resolution DNA Melting Analysis (HRM) to mutation screening in DNA pools. Classical TILLING involves critical and time consuming steps such as endonuclease digestion reactions and gel electrophoresis runs. Using CSCE or HRM, the only step required is a simple PCR before either capillary electrophoresis or DNA melting curve analysis. Here we describe the development of a mutant tomato population, the setting up of two polymorphism detection platforms for plants and the results of the first screens as mutation density in the populations and estimation of the false-positives rate when using HRM to screen DNA pools.
These results demonstrate that CSCE and HRM are fast, affordable and sensitive techniques for mutation detection in DNA pools and therefore allow the rapid identification of new allelic variants in a mutant population. Results from the first screens indicate that the mutagen treatment has been effective with an average mutation detection rate per diploid genome of 1.36 mutation/kb/1000 lines.
The retinal phagocytic machinery resembles the one used by macrophages to clear apoptotic cells. However, in the retina, the permanent contact between photoreceptor outer segments (POS) and retinal ...pigment epithelial (RPE) cells requires a tight control of this circadian machinery. In addition to the known receptors synchronizing POS internalization, several others are expressed by RPE cells. Notably, scavenger receptor CD36 has been shown to intervene in the internalization speed. We thus investigated members of the scavenger receptor family class A SR-AI and MARCO and class B CD36, SR-BI and SR-B2/LIMP-2 using immunoblotting, immunohisto- and immunocytochemistry, lipid raft flotation gradients, phagocytosis assays after siRNA/antibody inhibition, RT-qPCR and western blot analysis along the light:dark cycle. All receptors were expressed by RPE cell lines and tissues and colocalized with POS, except SR-BI. All receptors were associated with lipid rafts, and even more upon POS challenge. SR-B2/LIMP-2 inhibition suggested a role in the control of the internalization speed similar to CD36. In vivo, MARCO and CD36 displayed rhythmic gene and protein expression patterns concomitant with the phagocytic peak. Taken together, our results indicate that CD36 and SR-B2/LIMP-2 play a direct regulatory role in POS phagocytosis dynamics, while the others such as MARCO might participate in POS clearance by RPE cells either as co-receptors or via an indirect process.
The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. We use this and ...the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. The S155 hydration site is evolutionarily conserved within the RRM domains. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes.
Gaia Data Release 2 Soubiran, C.; Jasniewicz, G.; Chemin, L. ...
Astronomy and astrophysics (Berlin),
08/2018, Letnik:
616
Journal Article, Web Resource
Recenzirano
Odprti dostop
Aims. The Radial Velocity Spectrometer (RVS) on board the ESA satellite mission Gaia has no calibration device. Therefore, the radial velocity zero point needs to be calibrated with stars that are ...proved to be stable at a level of 300 m s −1 during the Gaia observations. Methods. We compiled a dataset of ~71 000 radial velocity measurements from five high-resolution spectrographs. A catalogue of 4813 stars was built by combining these individual measurements. The zero point was established using asteroids. Results. The resulting catalogue has seven observations per star on average on a typical time baseline of 6 yr, with a median standard deviation of 15 m s −1 . A subset of the most stable stars fulfilling the RVS requirements was used to establish the radial velocity zero point provided in Gaia Data Release 2. The stars that were not used for calibration are used to validate the RVS data.
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods ...for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.
Abstract
Following the identification of SARS-CoV-2, screening for air travel helped mitigate spread, yet lessons learned from a case study of air travel within Canada display enhanced techniques to ...better identify infected individuals, informing future responsive screening. While international travel bans limit infectious spread beyond a country’s borders, such measures are hardly sustainable economically and infrequently address domestic travel. Here, we describe a case study from Canada, where a diagnostic laboratory at point of travel conducted real-time PCR-based detection of SARS-CoV-2 in support of existing interventions, including clinical and epidemiological questionnaires, and temperature checks. All mining workers departing from a populated urban area flying to one of two sites (Site A and B) in a remote northern Canadian region, which we deemed “at-risk”, because healthcare services are limited and vulnerable to epidemics. Data collected between June and November 2020 on 15,873 clinical samples, indicate that molecular diagnosis allowed for identification of 13 infected individuals, who would have otherwise been missed by using solely nonpharmaceutical interventions. Overall, no outbreaks, COVID-19-related or other, were detected at the point of travel up to December 2021 since the implementation of the laboratory, suggesting this screening process is an effective means to protect at-risk communities. The success of this study suggests a process more practical than travel bans or an unfocused screening of air travelers everywhere.
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is a promising treatment in relapsing B-cell lymphoma but is frequently associated with acute neurotoxicity. Neurologic long-term safety has ...not been thoroughly assessed.
All patients with consecutive refractory lymphoma admitted in our center for CAR T-cell therapy underwent neurologic examination, extensive neuropsychological assessment, and brain MRI (except 1 patient) and completed self-administrated questionnaires at baseline. The patients who remained disease-free at 2 years were re-evaluated similarly. All neurologic assessments were conducted by senior neurologists.
None of the 19 disease-free patients developed new neurologic deficits or MRI changes when compared with baseline. There was no difference in cognitive performances before and 2 years after, even for the 11 patients who had developed acute neurotoxicity after CAR T cells. In self-questionnaire assessments, cognitive complaint was stable, reported by 32% of the patients at 2 years. We observed a reduction in HADS anxiety scores 2 years after treatment when compared with baseline (median score: 7/21 vs 4/21,
= 0.01).
In conclusion, no significant neurocognitive or neurologic disorders were observed in this cohort of patients, 2 years after treatment with anti-CD19 CAR T cells.
A
bstract
Hasse diagrams (or phase diagrams) for moduli spaces of supersymmetric field theories have been intensively studied in recent years, and many tools to compute them have been developed. The ...moduli space of instantons, despite being well studied, has proven difficult to deal with. In this note we explore the Hasse diagram of this moduli space from several perspectives — using the partial Higgs mechanism, using brane systems and using quiver subtraction — having to refine previously developed techniques. In particular we introduce the new concept of
decorated quiver
, which allows to deal with a large class of unitary quivers, including those with adjoint matter.
Homologs of the mutagenic Escherichia coli DNA polymerase V (pol V) are encoded by numerous pathogens and mobile elements. We have used Rum pol (RumA'2B), from the integrative conjugative element ...(ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. Here, we show that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA'2B-RecA-ATP, with RecA being donated by each recipient bacteria. For R391, Rum Mut specific activities in vitro and mutagenesis rates in vivo depend on the phylogenetic distance of host-cell RecA from E. coli RecA. Rum pol is a highly conserved and effective mobile catalyst of rapid evolution, with the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance.
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