5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs

Temporal separation of replication and recombination requires the intra-S checkpoint

In response to DNA damage and replication pausing, eukaryotes activate checkpoint pathways that prevent genomic instability by coordinating cell cycle progression with DNA repair. The intra-S-phase checkpoint has been proposed to protect stalled replication forks from pathological rearrangements that could result from unscheduled recombination. On the other hand, recombination may be needed to cope with either stalled forks or double-strand breaks resulting from hydroxyurea treatment. We have exploited fission yeast to elucidate the relationship between replication fork stalling, loading of replication and recombination proteins onto DNA, and the intra-S checkpoint. Here, we show that a functional recombination machinery is not essential for recovery from replication fork arrest and instead can lead to nonfunctional fork structures. We find that Rad22-containing foci are rare in S-phase cells, but peak in G2 phase cells after a perturbed S phase. Importantly, we find that the intra-S checkpoint is necessary to avoid aberrant strand-exchange events during a hydroxyurea block

Editoriale

The editorial analyzes the history of the scientific method, up to its crisis in the field ofhuman sciences. Then, it analyzes the current situation in the theory of education, whichis characterized by a methodological pluralism and the need for a principle of unitary understanding.The current educational historiography mirrors the multiplicity of approachesand methods in this field. The analysis and study of the sources, which are oftenquite unusual, have been conducted either with well known methodologies (such as narrativeand qualitative methods) or more recents and experimental ones (such as structuraland quantitative methods). These latter, in particular, can prove to be effective but requirea great deal of critical attention. Among the current study objects of the special educationfield, the theme of disability assumes many facets. Subsequently, it supports a reflectionon the need for a conscious adoption of targeted authoritative methodologicalparadigms, inspired by the rigor of research, as well as on ethical issues related to the directparticipation of persons with disabilities. Moreover, the link between epistemologicaland methodological aspects of pedagogical research is outlined. The variety of pathsof pedagogical research methods brings to the fore some epistemological, methodological,ethical and political issues that deserve attention to avoid the pitfalls that threatenpedagogical research.L’editoriale esamina la storia dell’idea di metodo scientifico, fino alla sua crisi odierna nel campo delle scienze umane e analizza la situazione attuale della pedagogia generale, caratterizzata dal pluralismo metodologico e dall’esigenza di un principio di comprensione unitaria. La prospettiva storico-educativa è oggi aperta a una pluralità di approcci e di metodi: l’analisi e l’interpretazione delle fonti – spesso inusuali – sono state condotte ora con metodologie collaudate, come quelle di tipo narrativo e qualitativo, ora di tradizione più recente, come quelle di tipo quantitativo e strutturale. Si tratta di metodologie che spesso hanno dato buona prova, ma che richiedono vigilanza critica, soprattutto nei modelli più innovativi. Nell’ambito della pedagogia speciale, il tema della disabilità può assumere numerose sfaccettature, alla luce delle quali si avanza una riflessione sulla necessità di un’adozione consapevole di paradigmi metodologici mirati, autorevoli, ispirati al rigore alla ricerca, nonché sulle questioni, etiche e progettuali, relative alla diretta partecipazione delle persone con disabilità. Il legame tra aspetti epistemologici e metodologici della ricerca pedagogica viene inoltre tratteggiato. La varietà di percorsi e declinazioni della ricerca pedagogica riporta in primo piano alcune questioni epistemologiche, metodologiche, etiche e politiche che meritano costante e vigile attenzione per evitare le insidie che minacciano la ricerca pedagogica

Inhalation therapy in the next decade : Determinants of adherence to treatment in asthma and COPD

Peer reviewedPublisher PD

Impact of different exposure models and spatial resolution on the long-term effects of air pollution.

Abstract Long-term exposure to air pollution has been related to mortality in several epidemiological studies. The investigations have assessed exposure using various methods achieving different accuracy in predicting air pollutants concentrations. The comparison of the health effects estimates are therefore challenging. This paper aims to compare the effect estimates of the long-term effects of air pollutants (particulate matter with aerodynamic diameter less than 10 μm, PM10, and nitrogen dioxide, NO2) on cause-specific mortality in the Rome Longitudinal Study, using exposure estimates obtained with different models and spatial resolutions. Annual averages of NO2 and PM10 were estimated for the year 2015 in a large portion of the Rome urban area (12 × 12 km2) applying three modelling techniques available at increasing spatial resolution: 1) a chemical transport model (CTM) at 1km resolution; 2) a land-use random forest (LURF) approach at 200m resolution; 3) a micro-scale Lagrangian particle dispersion model (PMSS) taking into account the effect of buildings structure at 4 m resolution with results post processed at different buffer sizes (12, 24, 52, 100 and 200 m). All the exposures were assigned at the residential addresses of 482,259 citizens of Rome 30+ years of age who were enrolled on 2001 and followed-up till 2015. The association between annual exposures and natural-cause, cardiovascular (CVD) and respiratory (RESP) mortality were estimated using Cox proportional hazards models adjusted for individual and area-level confounders. We found different distributions of both NO2 and PM10 concentrations, across models and spatial resolutions. Natural cause and CVD mortality outcomes were all positively associated with NO2 and PM10 regardless of the model and spatial resolution when using a relative scale of the exposure such as the interquartile range (IQR): adjusted Hazard Ratios (HR), and 95% confidence intervals (CI), of natural cause mortality, per IQR increments in the two pollutants, ranged between 1.012 (1.004, 1.021) and 1.018 (1.007, 1.028) for the different NO2 estimates, and between 1.010 (1.000, 1.020) and 1.020 (1.008, 1.031) for PM10, with a tendency of larger effect for lower resolution exposures. The latter was even stronger when a fixed value of 10 μg/m3 is used to calculate HRs. Long-term effects of air pollution on mortality in Rome were consistent across different models for exposure assessment, and different spatial resolutions

Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site

The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins

A Newly Identified Essential Complex, Dre2-Tah18, Controls Mitochondria Integrity and Cell Death after Oxidative Stress in Yeast

A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H2O2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex

Operationalizing mild cognitive impairment criteria in small vessel disease: The VMCI-Tuscany Study

Introduction Mild cognitive impairment (MCI) prodromic of vascular dementia is expected to have a multidomain profile. Methods In a sample of cerebral small vessel disease (SVD) patients, we assessed MCI subtypes distributions according to different operationalization of Winblad criteria and compared the neuroimaging features of single versus multidomain MCI. We applied three MCI diagnostic scenarios in which the cutoffs for objective impairment and the number of considered neuropsychological tests varied. Results Passing from a liberal to more conservative diagnostic scenarios, of 153 patients, 5% were no longer classified as MCI, amnestic multidomain frequency decreased, and nonamnestic single domain increased. Considering neuroimaging features, severe medial temporal lobe atrophy was more frequent in multidomain compared with single domain. Discussion Operationalizing MCI criteria changes the relative frequency of MCI subtypes. Nonamnestic single domain MCI may be a previously nonrecognized type of MCI associated with SVD

Checkpoint de réplication et instabilité génomique chez la levure Schizosaccharomyces pombe et leur éventuelle implication dans le cancer du sein

Au cours de la réplication, la synthèse d'ADN est coordonnée avec la progression du cycle cellulaire par l'action de systèmes de contrôle (checkpoints) qui vérifient le déroulement correct de la réplication. Lorsque plusieurs fourches de réplication s'arrêtent, le checkpoint de réplication est activé ce qui permet le redémarrage correct des fourches une fois la cause de l'arrêt de la synthèse éliminé. L'hydroxyurée (HU) inhibe l'activité de la ribonucléotide réductase et dérégule la concentration de désoxyribonucléotides triphosphate. Ceci provoque un ralentissement drastique de la progression des fOUfches de réplication dans une souche sauvage de S. pombe. Ce ralentissement active la protéine Cds1, kinase centrale du checkpoint de réplication. Dans une première étude, j'ai montré qu'en présence d'HU, le checkpoint de réplication prévient la fragmentation de l'ADN spécifique de la phase S et dépendante de l'endonucléase Mus81. En cas de stress réplicatif, le checkpoint de la réplication protège la fourche de réplication et participe ainsi au maintien de la stabilité génomique. Dans une seconde étude, je me suis attaché à comprendre l'implication de ces protéines de checkpoints et de réparation des dommages de l'ADN dans la genèse de cellules souches tumorales chimiorésistantes. Cette étude a été menée en utilisant un modèle de xénogreffe de tumeurs de sein de type basal-like. Mon travail a tout d'abord consisté à la mise en place des outils nécessaires à l'isolement et à la caractérisation des cellules souches aussi bien au niveau génomique que transcriptomique. Les premiers résultats expérimentaux sont en cours d'analyse.During replication, DNA synthesis is coordinated to cell cycle progression through the action of checkpoints. When several replication forks are blocked, the replication checkpoint is activated and blocks cell cycle until replicative stress disappears allowing proper replication forks restart. Hydroxyurea (HU) inhibits the activity of ribonucleotide reductase and decreases the pool of triphosphate deoxyribonucleotides. This leads to a drastic slow down of the progression of replication forks in a wild type strain of S. pombe. This delay activates Cds1, the central kinase of the replication checkpoint. ln the first part of my thesis, 1 have shown that, in presence of HU, the replication checkpoint prevents S-phase specific and Mus811dependent DNA fragmentation. Under replicative stress, the replication checkpoint protects replication fork and therefore participates to the maintenance of genomic stability. The aim of the second part was to elucidate the implication of checkpoint and DNA repair proteins in the emergence of tumor stem cells. The well-characterized model of xenograft of basal-like breast tumors was used. 1 first established the technical tools required for the isolation of tumor stem cells and their characterisation at the genomic and transcriptionallevels. The first experimental results are currently under analysis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Single-stranded DNA binding proteins unwind the newly synthesized double-stranded DNA of model miniforks.

International audienceSingle-stranded DNA binding (SSB) proteins are essential proteins of DNA metabolism. We characterized the binding of the bacteriophage T4 SSB, Escherichia coli SSB, human replication protein A (hRPA), and human hSSB1 proteins onto model miniforks and double-stranded-single-stranded (ds-ss) junctions exposing 3' or 5' ssDNA overhangs. T4 SSB proteins, E. coli SSB proteins, and hRPA have a different binding preference for the ss tail exposed on model miniforks and ds-ss junctions. The T4 SSB protein preferentially binds substrates with 5' ss tails, whereas the E. coli SSB protein and hRPA show a preference for substrates with 3' ss overhangs. When interacting with ds-ss junctions or miniforks, the T4 SSB protein, E. coli SSB protein, and hRPA can destabilize not only the ds part of a ds-ss junction but also the daughter ds arm of a minifork. The T4 SSB protein displays these unwinding activities in a polar manner. Taken together, our results position the SSB protein as a potential key player in the reversal of a stalled replication fork and in gap repair-mediated repetitive sequence expansion