Characterization of kinases involved in DNA damage response checkpoints after low and high doses of low-LET irradiation and detection of structural DNA variations after induction of complex DNA damage using Nanopore sequencing.

Αντικαρκινικές θεραπείες, όπως η ακτινοθεραπεία και η χημειοθεραπεία, δρουν κυρίως προκαλώντας βλάβες στο DNA των καρκινικών κυττάρων. Σε περίπτωση βλάβης του DNA, τα κύτταρα κινητοποιούν ένα πολύπλοκο σύστημα απόκρισης στις βλάβες DNA (DDR) για να αξιολογήσουν το είδος και το επίπεδο των βλαβών και να τις επιδιορθώσουν. Τα διάφορα σημεία ελέγχου στο κυτταρικό κύκλο διαδραματίζουν σημαντικό ρόλο κατά τη διάρκεια αυτής της απόκρισης, επομένως η στοχευμένη αναστολή των προαναφερθέντων μπορεί να οδηγήσει σε αυξημένη θνησιμότητα των καρκινικών κυττάρων και ενδεχομένως σε πιο αποτελεσματικές θεραπείες (Bartek and Lukas, 2001; Iliakis et al., 2003; Jackson and Zhou, 2004). Στη παρούσα εργασία, χρησιμοποιώντας δεδόμενα που παράγονται από κύτταρα που εκτίθενται σε χαμηλές και υψηλές δόσεις ιοντίζουσας ακτινοβολίας χαμηλού LET, που εισάγουν χαμηλό αριθμό δίκλωνων θραύσεων στο γονιδίωμα, επιβεβαιώνουμε τη ρύθμιση του σημείου ελέγχου G2/M κυρίως μέσω των κινασών ATR και ATM (αποτελέσματα που δημοσιεύθηκαν αρχικά από τους (Mladenov et al., 2019)). Παρέχουμε επίσης αποδείξεις ότι αυτές οι κινάσες αλληλεπιδρούν με το κυτταρικό κύκλο κυρίως μέσω της κινάσης Chk1. Στις υψηλές δόσεις παρουσιάζουμε προκαταρκτικά αποτελέσματα του κυρίαρχου ρόλου του Chk1 στην εποπτεία των σημείων ελέγχου. Μεγαλύτερος αριθμός δίκλωνων θραύσεων είναι γνωστό ότι προκαλεί ακόμα πιο σοβαρή κυτταρική βλάβη (Khanna and Jackson, 2001; van Gent et al., 2001). Ένας από τους παράγοντες επαγωγής πολλαπλών δίκλωνων θραύσεων (DSBs) θεωρείται ότι είναι ιοντίζουσα ακτινοβολία υψηλού LET. Ένα από τα χαρακτηριστικά της ακτινοβολίας υψηλού LET είναι η εισαγωγή στο γονιδίωμα σύνθετων αλλοιώσεων. Αυτή η πολυπλοκότητα παραδοσιακά θεωρείται ότι είναι ο συνδυασμός αλλοιώσεων χαμηλότερου επιπέδου όπως βλάβες της μίας αλυσίδας, μεταβολές ή απώλειες βάσης, θραύσεις μονής έλικας, δίκλωνες θραύσεις κλπ ή συνδυασμός των προαναφερθέντων τύπων βλαβών. Ο συνδυασμός των αλλοιώσεων χαμηλού επιπέδου με δίκλωνες θραύσεις αποτελεί τη κύρια απειλή για την ακεραιότητα του γονιδιώματος σύμφωνα με τη βιβλιογραφία (Campa et al., 2005, Gupta et al., 2009, Seluanov et al., 2010). Εδώ χρησιμοποιούμε ένα μοντέλο που βασίζεται στην υπόθεση ότι η αυξανόμενη πολυπλοκότητα της βλάβης του DNA προκύπτει από τη συνεγκατάσταση ή το συνδυασμό αυξημένου αριθμού DSBs σε ένα σχετικά μικρό εύρος αλληλουχίας (Iliakis et al., 2018; Mladenova et al., 2016). Οι κυτταρικοί κλώνοι που χρησιμοποιήθηκαν για αυτό το τμήμα της ανάλυσης προέρχονται από άνθρωπο και χάμστερ και δημιουργήθηκαν με σκοπό να μοντελοποιήσουν τις σύνθετες βλάβες DNA επαγόμενες μόνο από τις δίκλωνες θραύσεις DNA. Περιέχουν πολλαπλές γενετικά τροποποιημένες αλληλουχίες που δημιουργήθηκαν με τη βοήθεια πλασμιδιακού DNA κατασκευασμένες μέσω κατάλληλων θέσεων αναγνώρισης της μεγανουκλεάσης I-SceI , επακόλουθη έκφραση του ενζύμου προκαλεί διαφορετικό αριθμό DSBs σε προκαθορισμένες γεννετικές θέσεις. Έτσι, παρέχεται η δυνατότητα ποσοτικοποίησης της επίδρασης πολλαπλών DSBs σε ένα σχετικά μικρό τμήμα μιας αλληλουχίας και ταυτόχρονα εξαιρούνται άλλες μεταβλητές και άλλου τύπου βλάβες που εισέρχονται όταν χρησιμοποιείται ιοντίζουσα ακτινοβολία υψηλού LET. Ένας χαρακτηρισμός αυτών των κλώνων πραγματοποιήθηκε υλοποιώντας αλληλούχιση του DNA στην πλατφόρμα MinION από την ONT. Ο εντοπισμός και η ταυτοποίηση των θέσεων της I-SceI παρουσιάζεται ταυτόχρονα και σε κλώνους χάμστερ και σε ανθρώπινους. Επιπροσθέτως προτείνουμε ένα καινοτόμο μοντέλο για τη μελέτη επαγόμενων δομικών αλλοιώσεων. Τα αποτελέσματα αποκαλύπτουν περισσότερες τοποθεσίες ενσωμάτωσης της γενετικά τροποποιημένης αλληλουχίας σε σύγκριση με άλλες τεχνικές. Επιπλέον, στα αποτελέσματα της ανάλυσής μας, ήταν εμφανής η υποβάθμιση της λειτουργίας επιδιόρθωσης και αποκατάστασης των δίκλωνων θραύσεων καθώς και ο αυξημένες μετατοπίσεις σε τμήματα του γονιδιώματος.Cancer treatments such as radiotherapy and chemotherapy mainly act by inducing severe (complex) damage on DNA of cancer cells. In presence of DNA damage, cells implement their multifaceted DNA Damage Response (DDR) system to process the damage and repair it. Checkpoint control plays a major role during this response, therefore targeted inhibition of the aforesaid can lead to increased cell lethality and potentially to more efficient treatments (Bartek and Lukas, 2001; Iliakis et al., 2003; Jackson and Zhou, 2004). Here, using data generated by cells exposed to low and high doses of low LET ionizing radiation that introduce a low number of double strand breaks (DSBs) in the genome, we cross validate the regulation of the G2 checkpoint mainly by ATR and ATM (results first published by (Mladenov et al., 2019)). We also provide evidence that these kinases interphase with cell cycle machinery mainly through Chk1 kinase. At high doses, we present preliminary results of the dominant role of Chk1 in checkpoint control. High number of DSBs is known to cause even more severe cell damage (Khanna and Jackson, 2001; van Gent et al., 2001). One of the exogenous agents inducing multiple DSBs is the high Linear Energy Transfer (LET) ionizing radiation. One of the characteristics of high LET irradiation is the introduction of highly complex lesions. This complexity is traditionally thought to be the combination of lower level lesions like one stranded lesions, base changes or losses, SSBs, DSBs etc. or a combination of the previous mentioned types of damage. The combination of low level lesions with DSBs poses major threats to genome integrity according to literature (Campa et al., 2005; Gupta et al., 2009; Seluanov et al., 2010). Here we have used a model based on the hypothesis that increased complexity of DNA damage is manifested through the colocalization or combination of an increased number of DSBs in a relative small stretch of sequence (Iliakis et al., 2018; Mladenova et al., 2016). The cellular clones used for this part of the analysis are CHO and human clones that were created with the aim to model DSB-clusters. The I-SceI recognition sequence was inserted into cell lines using molecular biology techniques (Jasin, 1996) and clones as needed were selected and characterized. I-SceI recognition sequences introduced in a genome can then be cut, to generate from simple DSBs up to more complex DSB clusters. Constructs carrying different combinations of I-SceI sites were engineered at specific distances and orientations, subsequent transient expression of I-SceI induces the relevant number of DSBs in predetermined loci. Therefore these clones provide the opportunity to measure the effect of several DSBs within a relatively small part of a sequence, excluding postulations about other variables and types of DNA damage that arise when high LET IR is used. A characterization of these clones was performed using long-read sequencing with MinION platform by ONT. Localization and identification of the I-SceI sites is presented on both human and CHO clones and a novel model is proposed to study induced structural variations. Results reveal significantly more integrations sites in the clones tested in comparison to other techniques. In addition, compromised DSB repair as well as increased translocation formation were evident

The OSCAR-MP Consensus Criteria for Quality Assessment of Retinal Optical Coherence Tomography Angiography

BACKGROUND AND OBJECTIVES: Optical coherence tomography angiography (OCTA) is a noninvasive high-resolution imaging technique for assessing the retinal vasculature and is increasingly used in various ophthalmologic, neuro-ophthalmologic, and neurologic diseases. To date, there are no validated consensus criteria for quality control (QC) of OCTA. Our study aimed to develop criteria for OCTA quality assessment. METHODS: To establish criteria through (1) extensive literature review on OCTA artifacts and image quality to generate standardized and easy-to-apply OCTA QC criteria, (2) application of OCTA QC criteria to evaluate interrater agreement, (3) identification of reasons for interrater disagreement, revision of OCTA QC criteria, development of OCTA QC scoring guide and training set, and (4) validation of QC criteria in an international, interdisciplinary multicenter study. RESULTS: We identified 7 major aspects that affect OCTA quality: (O) obvious problems, (S) signal strength, (C) centration, (A) algorithm failure, (R) retinal pathology, (M) motion artifacts, and (P) projection artifacts. Seven independent raters applied the OSCAR-MP criteria to a set of 40 OCTA scans from people with MS, Sjogren syndrome, and uveitis and healthy individuals. The interrater kappa was substantial (κ 0.67). Projection artifacts were the main reason for interrater disagreement. Because artifacts can affect only parts of OCTA images, we agreed that prior definition of a specific region of interest (ROI) is crucial for subsequent OCTA quality assessment. To enhance artifact recognition and interrater agreement on reduced image quality, we designed a scoring guide and OCTA training set. Using these educational tools, 23 raters from 14 different centers reached an almost perfect agreement (κ 0.92) for the rejection of poor-quality OCTA images using the OSCAR-MP criteria. DISCUSSION: We propose a 3-step approach for standardized quality control: (1) To define a specific ROI, (2) to assess the occurrence of OCTA artifacts according to the OSCAR-MP criteria, and (3) to evaluate OCTA quality based on the occurrence of different artifacts within the ROI. OSCAR-MP OCTA QC criteria achieved high interrater agreement in an international multicenter study and is a promising QC protocol for application in the context of future clinical trials and studies

Azimuthal anisotropy of charged jet production in root s(NN)=2.76 TeV Pb-Pb collisions

We present measurements of the azimuthal dependence of charged jet production in central and semi-central root s(NN) = 2.76 TeV Pb-Pb collisions with respect to the second harmonic event plane, quantified as nu(ch)(2) (jet). Jet finding is performed employing the anti-k(T) algorithm with a resolution parameter R = 0.2 using charged tracks from the ALICE tracking system. The contribution of the azimuthal anisotropy of the underlying event is taken into account event-by-event. The remaining (statistical) region-to-region fluctuations are removed on an ensemble basis by unfolding the jet spectra for different event plane orientations independently. Significant non-zero nu(ch)(2) (jet) is observed in semi-central collisions (30-50% centrality) for 20 <p(T)(ch) (jet) <90 GeV/c. The azimuthal dependence of the charged jet production is similar to the dependence observed for jets comprising both charged and neutral fragments, and compatible with measurements of the nu(2) of single charged particles at high p(T). Good agreement between the data and predictions from JEWEL, an event generator simulating parton shower evolution in the presence of a dense QCD medium, is found in semi-central collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Forward-central two-particle correlations in p-Pb collisions at root s(NN)=5.02 TeV

Two-particle angular correlations between trigger particles in the forward pseudorapidity range (2.5 2GeV/c. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B. V.Peer reviewe

Event-shape engineering for inclusive spectra and elliptic flow in Pb-Pb collisions at root(NN)-N-S=2.76 TeV

Peer reviewe

Pseudorapidity and transverse-momentum distributions of charged particles in proton-proton collisions at root s=13 TeV

The pseudorapidity (eta) and transverse-momentum (p(T)) distributions of charged particles produced in proton-proton collisions are measured at the centre-of-mass energy root s = 13 TeV. The pseudorapidity distribution in vertical bar eta vertical bar <1.8 is reported for inelastic events and for events with at least one charged particle in vertical bar eta vertical bar <1. The pseudorapidity density of charged particles produced in the pseudorapidity region vertical bar eta vertical bar <0.5 is 5.31 +/- 0.18 and 6.46 +/- 0.19 for the two event classes, respectively. The transverse-momentum distribution of charged particles is measured in the range 0.15 <p(T) <20 GeV/c and vertical bar eta vertical bar <0.8 for events with at least one charged particle in vertical bar eta vertical bar <1. The evolution of the transverse momentum spectra of charged particles is also investigated as a function of event multiplicity. The results are compared with calculations from PYTHIA and EPOS Monte Carlo generators. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Elliptic flow of muons from heavy-flavour hadron decays at forward rapidity in Pb-Pb collisions at root s(NN)=2.76TeV

The elliptic flow, v(2), of muons from heavy-flavour hadron decays at forward rapidity (2.5 <y <4) is measured in Pb-Pb collisions at root s(NN)= 2.76TeVwith the ALICE detector at the LHC. The scalar product, two- and four-particle Q cumulants and Lee-Yang zeros methods are used. The dependence of the v(2) of muons from heavy-flavour hadron decays on the collision centrality, in the range 0-40%, and on transverse momentum, p(T), is studied in the interval 3 <p(T)<10 GeV/c. A positive v(2) is observed with the scalar product and two-particle Q cumulants in semi-central collisions (10-20% and 20-40% centrality classes) for the p(T) interval from 3 to about 5GeV/c with a significance larger than 3 sigma, based on the combination of statistical and systematic uncertainties. The v(2) magnitude tends to decrease towards more central collisions and with increasing pT. It becomes compatible with zero in the interval 6 <p(T)<10 GeV/c. The results are compared to models describing the interaction of heavy quarks and open heavy-flavour hadrons with the high-density medium formed in high-energy heavy-ion collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V.Peer reviewe