144,341 research outputs found
Rapeseed: an alternative crop for Idaho
No full textBulletin no. 752 Moscow, Idaho :University of Idaho, College of Agriculture, Agriculture Experiment Station, 1994-02-01. Author(s): Melfi, J. A.; Withers, Russell V
Optimization of a new lead promoting the readthrough of the nonsense mutations for CFTR rescue in human CF cells
Full text linkOptimization of a new lead promoting the readthrough of the nonsense mutations for CFTR rescue in human CF cells
Laura Lentini, Raffaella Melfi, Sara Baldassano, Marco Tutone, Aldo Di Leonardo, Andrea Pace, Ivana Pibiri
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo
Background and rationale
Cystic Fibrosis patients with nonsense mutations in the CFTR gene have a more severe form of the disease. Nonsense mutations represent about 10% of the mutations that affect the CFTR gene and they are frequently associated to the classical F508 mutation (1). A potential treatment for this genetic alteration is to promote the translational readthrough of premature termination codons (PTCs) by Translational Read-Through-Inducing Drugs (TRIDs) (2-4).
Hypothesis and objectives
Our objective is to evaluate the functionality of the CFTR channel after treatment with a new molecule that we individuated in a precedent FFC project, and the activity of new lead molecules in cells stably expressing a nonsense-CFTR-mRNA (ns CFTR) in CF cellular model systems. We want also to study the supramolecular interactions among TRIDs, CFTR mRNA and the ribosomal A-site to identify the biological target and the mechanism of action.
Essential methods
QSAR, carried out on the basis of our preliminary results, will allow to achieve lead optimization and synthesize then a small library of analogs to be tested and compared to the Lead. We will mutagenize the CFTR cDNA by introducing the most diffuse nonsense mutations. Subsequently, FRT cells engineered with the vector expressing mutagenized nsCFTR, and nonsense-CF-human broncoepithelial cells will be grown in the air-liquid culture system to reproduce in vitro the epithelial organization. CFTR expression after treatments with our molecule will be evaluated by biomolecular techniques. CFTR activity will be revealed by specific CFTR-functionality assays. Finally, in vitro-in vivo (Zebrafish model) analyses of the safety profile for the set of synthesized molecules will complete the study.
Preliminary results
We screened the activity of several molecules synthetized by us in a precedent FFC project, identifying some molecules that showed high readthrough activity associated to the expression of the CFTR protein in ns CF immortalized cells.
Expected final results and their significance
We are confident that our findings will provide the validation of molecules with readthrough activity for the recovery of the CFTR function. Moreover, our pre-clinical study will assess the presence of toxic effects caused by the molecules in vivo.
References
1. Sermet-Gaudelus I, Boeck KD, Casimir GJ, Vermeulen F, Leal T, Mogenet A, Roussel D, Fritsch J, Hanssens L, Hirawat S, Miller NL, Constantine S, Reha A, Ajayi T, Elfring GL, Miller LL. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis., Am J RespirCrit Care Med. 2010 Nov 15;182(10):1262-72.
2. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I. Towards a rationale for the PTC124 (Ataluren) promoted read-through of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol. Pharm. 2014 11, 653-664.
3. Pibiri I, Lentini L, Melfi R, Gallucci G, Pace A, Spinello A, Barone G, Di Leonardo A. Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives European Journal of Medicinal Chemistry 06/2015; 101.
4. Nagel-Wolfrum K, Möller F, Penner I, Baasov T4, Wolfrum U. Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs), BioDrugs. 2016 Apr;30(2):49-74
Investigating CRISPR-CAS13b as a tool for the RNA editing of CFTR mRNA with premature stop codon
Full text linkBackground and Rationale
Some CF patients are compound heterozygous or homozygous for
nonsense mutations in the CFTR gene. Mutant CFTR gene coding for
transcripts with premature termination codons (PTCs) is responsible
for truncated CFTR protein and for a severe form of the disease. In a
precision medicine framework the “REPAIRv2” (RNA Editing for
Programmable A to I Replacement v2) tool, developed in the laboratory
of Dr. Feng Zhang (USA), seems a good alternative to restore
the full-length CFTR protein by editing its mRNA containing PTCs.
This new approach is based on the possibility of targeting a deaminase
enzyme (huADAR2) to a specific Adenosine, to be edited to Inosine
(G analogue), on the mutant RNA by a specific guide RNA
(gRNA), complementary to the target regions, and a Cas protein.
Hypothesis and objectives
We applied the new CRISPR/dCas13b based molecular tool of RNA
editing (REPAIRv2) to correct the premature stop codon UGA, changing
to UGG, in the H2bGFPopal and CFTRW1282X mRNAs with the
purpose of recovering the full-length proteins.Essential Methods
We designed and cloned the gRNAs needed to target the REPAIRv2
system to the Adenine to be modified. By site-directed mutagenesis
we introduced a premature stop codon, W1282X, in the CFTR cDNA.
Human HeLa cells expressing the H2BGFPopal mRNA, FRT cells expressing
CFTRW1282X and IB3.1 airway epithelial human cells
(CFTRΔ508/W12382X) were co-transfected with the plasmids coding
for the recombinant protein dCAS13b/ADAR2DD, and for the gRNAs.
Fluorescence microscopy was used to analyse the editing results.
Results
Direct fluorescence microscopy and immunofluorescence analyses
detecting the corrected proteins (H2BGFP and CFTR, respectively)
suggest that the REPAIRv2 system was able, in different cell lines, to
edit the H2BGFPopal and the CFTRW1282X mRNA. However, the rate
of editing does not seem high. Indeed, when RNA was purified from
transfected cell, retro-transcribed and amplified base correction was
not detectable by standard DNA sequencing and western blot.
Conclusions
Collectively, our results indicate that the REPAIRv2 tool is able to edit
the UGA premature stop codon present in the HeLa-H2BGFPopal
cells and in engineered FRTW1282X cells harbouring the UGA PTC in
the CFTR mRNA. Furthermore, the REPAIRv2 tool worked in the IB3.1
cells suggesting its ability to edit endogenous UGA premature stop
codon. Anyway, enhance the delivery of the plasmids as well increase/
stabilize the target mRNA to be edited, seem necessary to improve
the efficiency of REPAIRv2.
References
1. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J,
Zhang F.- RNA editing with CRISPR-Cas13. Science. 2017 Nov 24; 358
(6366):1019-1027)
2. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo
Piccionello A, Pibiri I. Toward a rationale for the PTC124 (Ataluren)
promoted readthrough of premature stop codons: a computational
approach and GFP-reporter cell-based assay. Mol Pharm. 2014 Mar
3;11(3):653-64.
Acknowledgment FFC#5/2018 funded by FFC and supported by Delegazione
FFC di Palerm
[Report to Chief J. E. Curry, by an unknown author #1]
No full textReport to Chief J. E. Curry, by an unknown author. The report contains a list of officers who gave depositions to the United States Attorney
[Report to Chief J. E. Curry, by an unknown author #2]
No full textReport to Chief J. E. Curry, by an unknown author. The report contains a list of officers who gave depositions to the United States Attorney
Robotic lobectomy: tips, pitfalls and troubleshooting
Full text linkThe robotic approach in thoracic surgery has rapidly gained popularity in recent years. As with the introduction of any new technology, this warrants not only adaptation of the operative technique itself, but also the evolution of appropriate troubleshooting strategies. A selected number of helpful tips and technical procedural manoeuvres have been compiled to prevent intraoperative problems, as well as to overcome challenging situations that can arise during robotic lobectomies. In robotic surgery, as opposed to open surgery or video-assisted thoracic surgery, these tips serve an important purpose for the operating surgeon, as well as the entire surgical team involved in the procedure. All the assembled recommendations have proved their effectiveness and have been successfully used by the authors in many procedures. Furthermore, these manoeuvres have been found to be of great importance in the training and proctoring of thoracic surgeons, fellows and residents (bed-side assistants). This guide of clearly arranged tips and troubleshooting strategies offers surgeons a useful tool to overcome difficult situations in robotic lobectomy and preferably improve the reproducibility and safety of their procedures
Evidence for the decay B0→J/ψω and measurement of the relative branching fractions of meson decays to J/ψη and J/ψη′
Full text linkFirst evidence of the B 0 → J / ψ ω decay is found and the B s 0 → J / ψ η and B s 0 → J / ψ η ′ decays are studied using a dataset corresponding to an integrated luminosity of 1.0 fb -1 collected by the LHCb experiment in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV. The branching fractions of these decays are measured relative to that of the B 0 → J / ψ ρ 0 decay:frac(B (B 0 → J / ψ ω), B (B 0 → J / ψ ρ 0)) = 0.89 ± 0.19 (stat) - 0.13 + 0.07 (syst),frac(B (B s 0 → J / ψ η), B (B 0 → J / ψ ρ 0)) = 14.0 ± 1.2 (stat) - 1.5 + 1.1 (syst) - 1.0 + 1.1 (frac(f d, f s)),frac(B (B s 0 → J / ψ η ′), B (B 0 → J / ψ ρ 0)) = 12.7 ± 1.1 (stat) - 1.3 + 0.5 (syst) - 0.9 + 1.0 (frac(f d, f s)), where the last uncertainty is due to the knowledge of f d / f s, the ratio of b-quark hadronization factors that accounts for the different production rate of B 0 and B s 0 mesons. The ratio of the branching fractions of B s 0 → J / ψ η ′ and B s 0 → J / ψ η decays is measured to befrac(B (B s 0 → J / ψ η ′), B (B s 0 → J / ψ η)) = 0.90 ± 0.09 (stat) - 0.02 + 0.06 (syst)
Measurement of the time-dependent CP asymmetry in B0 -> J/ψ KS0 decays
Full text linkThis Letter reports a measurement of the CP violation observables SJ/ψK0S and CJ/ψK0S in the decay channel B0→J/ψK0S performed with 1.0 fb−1 of pp collisions at s√=7 TeV collected by the LHCb experiment. The fit to the data yields SJ/ψK0S=0.73±0.07(stat)±0.04(syst) and CJ/ψK0S=0.03±0.09(stat)±0.01(syst). Both values are consistent with the current world averages and within
expectations from the Standard Model
Measurement of the ratio of prompt χ c to J / ψ production in pp collisions at √s = 7 TeV
Full text linkThe prompt production of charmonium χ c and J / ψ states is studied in proton-proton collisions at a centre-of-mass energy of √s = 7 TeV at the Large Hadron Collider. The χ c and J / ψ mesons are identified through their decays χ c → J / ψ γ and J / ψ → μ + μ - using 36 pb - 1 of data collected by the LHCb detector in 2010. The ratio of the prompt production cross-sections for χ c and J / ψ, σ (χ c → J / ψ γ) / σ (J / ψ), is determined as a function of the J / ψ transverse momentum in the range 2 < p T J / ψ < 15 GeV / c. The results are in excellent agreement with next-to-leading order non-relativistic expectations and show a significant discrepancy compared with the colour singlet model prediction at leading order, especially in the low p T J / ψ region
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