A high-throughput methodology allowing for the simultaneous analysis of the biological properties of multiple samples was necessary to complete this large-scale investigation. Recently, we successfully incorporated next-generation sequencing into our site-specific mutagenesis assay, 35 allowing for a concurrent analysis of multiple lesions in multiple cell strains. 36 In the present study, we applied the same methodology to investigate polymerase bypass and mutagenesis mediated by the four BD-dA adducts (Fig. 1B) in various repair/ replication backgrounds (Fig. 2). The adducts hindered replication to various degrees but, counter to our previous in vitro results with human polymerases, 25, 26 none of the adducts were significantly mutagenic in the repair/replication backgrounds investigated. Sometimes, the lack of significant mutagenicity of a DNA adduct in vivo is attributable to DNA repair systems that counter the effect of an otherwise mutagenic adduct. We found, however, that neither AlkB, MutM nor MutY appeared to play a role in the repair of the BD-dA lesions since the mutagenicities and genotoxicities of the lesions were not significantly different in the presence or absence of these repair enzymes. Our observations suggest that as yet
the same point (dif/terC), clearly showing that the phenomenon is spatially and mechanistically independent of replication ter- mination (Dimude, Midgley-Smith and Rudolph 2018 ).
RecA ChIP-seq analysis in the terminus of wild type cells was performed to check if any DSBs could be detected in the chro- mosome terminus under the growth conditions where terminus DNA loss was observed in the recBC mutant. Such RecA bind- ing would be expected according to this first model, as RecBCD action would lead to RecA loading following encounter with an appropriately oriented Chi site. However, no RecA binding could be detected arguing against the first model and, more surpris- ingly, arguing against any model proposing that the terminus- localized DSBs (or DSEs) are formed in wild type cells. Instead, the observation made it clear that these terminus-localized DNA breaks are only generated in the recBC mutants (Sinha et al. 2017 ). This dual realization that the location of DNA loss observed in a recBC mutant did not correspond to the location of replica- tion termination and that the terminus breaks are only gener- ated in the recBC mutant led to the proposal of a second model (Sinha et al. 2018 ). In this second model (Fig. 4 ), randomly located breakage of a DNA replication fork leads to the formation of a sigma-replicating chromosome. The broken fork cannot be repaired in a recBC mutant, so the sigma-replicating chromo- some persists (Fig. 4 cde). This then causes a problem when the cell tries to segregate its replicated chromosomes and divide (Fig. 4 e). The terminus region of one of the chromosomes (the linear tail of the sigma structure) becomes trapped as the sep- tum attempts to close and this attempted cell division leads to chromosome breakage (Fig. 4 f). The initial break of the replica- tion fork is randomly located on the chromosome and so cannot be detected in a population of cells (Fig. 4 c). It is independent of recBC but persists in a recBC mutant. The subsequent break occurs in the dif/terC region but only if the cell is a recBC mutant, because only in the absence of replication fork repair by RecBCD does the sigma-replicating chromosome persist and get trapped in the closing septum at cell division (Fig. 4 e-f). This subsequent
Unrepaired O 6 -methylguanine lesions induced by the alkylating chemotherapy agent
temozolomide lead to replication-associated single-ended DNA double-strand breaks (seDSBs) that are repaired predominantly through RAD51-mediated homologous recombination (HR). Here, we show that loss of the pre-mRNA splicing and DNA repair protein XAB2 leads to increased temozolomide sensitivity in glioblastoma cells, which reflects abortive HR due to Ku retention on resected seDSBs. XAB2-dependent Ku eviction also occurred at seDSBs generated by the topoisomerase I poison campthotecin and operated in parallel to an ATM-dependent pathway previously described. Although Ku retention elicited by loss of XAB2 did not prevent RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased engagement of non- homologous-end-joining in S/G2 and genetic instability. Overexpression of RAD51 or the single-stranded DNA annealing factor RAD52 rescued the XAB2 defects. RAD52 depletion led to severe temozolomide sensitivity, whereas a synthetic lethality interaction was
We estimate that ~5-10% of cells in an asynchronous population have a conflict between the transcription andreplication machineries at a rRNA operon. This estimate is based on an ~50-100-fold greater association of helicase at oriC in a synchronous population at the time of replication initiation 20 than at one of the ten rRNA operons, assuming that there are similar crosslinking efficiencies at oriC and rrn loci. The co-directional conflicts between replicationand transcription likely account for a significant fraction of endogenous events requiring repair of stalled replication forks 1 , 26 , and may even account for some of the sensitivity to rapid growth conditions of priA mutants defective in replication restart 25 , 30 . Since inability to repair a stalled fork would prevent completion of a replication cycle and the production of viable progeny, there is strong selective pressure to avoid such
evidence that BIR occurs in human cells and that Rad52 promotes replication restart after HU treatment also support this model [ 25 , 26 ]. However, the potential importance of homologous recombination and end-joining mechanisms in the repair of broken forks cannot be ruled out.
Finally, because ARTEMIS and XPF-ERCC1 are structure-specific endonucleases, it is tempting to speculate that they might independently and specifically target several unusual DNA structures such as hairpins or cruciform structures at repeated sequences, R-loops at transcription sites or regressed forks. This hypothesis would explain the additive effects of depletion of ARTEMIS and XPF that we observed in our experiments. Interestingly, it has been shown that most of DSB arising from DNA replication stress are located within DNA repetitive sequences prone to form secondary structures such as hairpins but also at active transcription sites [ 27 ]. These genomic locations could be separately targeted by ARTEMIS and XPF-ERCC1 since ARTEMIS is known to cleave efficiently hairpins such as those present during VDJ recombination and XPF-ERCC1 has been shown to cleave DNA at active tran- scription sites that accumulate R-loops [ 28 ].
A replicated dictionary (or table) is a popular example, where all dictionary operations (insertion and deletion) with different keys commute with each other [Wuu and Bernstein 1984; Mishra et al. 1989].
5.2.3 Semantic scheduling: Canonical ordering. Ramsey and Csirmaz  formally study optimistic replication in a file system. For every possible pair of concurrent opera- tions, they define a rule that specifies how they interact and may be ordered (non-concurrent operations are applied in their happens-before order.) For instance, they allow creating two files /a/b and /a/c in any order, even though they both update the same directory. Or, if one user modifies a file, and another deletes its parent directory, it marks them as con- flicting and asks the users to repair them manually. Ramsey and Csirmaz  prove that this algebra in fact keeps a file system consistent and converges the state of replicas.
and explant derived BMSCs, providing further evidence that cells in bone have the potential to dedifferentiate into a more primitive and undefined phenotype.
In an important observation, we found a strong influence of age of BMSCs on the corresponding biological properties including their number, stemness, expression of cell surface markers and differentiation potency. Reduced migration potential due to decreased response to chemoattractants might also contribute to inferior repair outcomes in older individuals. Ageing also induces changes in chondrocytes including senescence, reduced response to growth factors, accumulation of AGE and oxidative damage . The resultant disturbance in the cartilage homeostasis makes the cartilage matrix vulnerable to further damage. Reduction in cell number and senescence in existing progenitor cell population are prevalent in older individuals. In addition to poor telomerase activity, the genes involved in DNA replication, cell cycle, mitosis and DNA repair are significantly downregulated with increasing age . As a result of accumulation of DNA damages, cells enter senescent phase. According to Hayflick model of senescence, once the cells reach senescent limit, they switch from a rapidly dividing phenotype to a slow dividing one accompanied by a loss in differentiation potential . There is a decline in anti-oxidants with increasing age which leads to escalation of levels of reactive oxygen species . Moreover, the levels of growth factors decrease which might cause a shortfall in factors required critically for differentiation. Finally, Xin et al proposed that migratory ability of BMSCs towards chemoattractants reduces with increasing age . This is an important consideration since it might lead to reduced migration and recruitment of BMSCs towards cartilage lesion in older individuals. The data from our study is consistent with the earlier findings of Payne et al and Zheng et al where a reduced chondrogenic potential was observed in older rats [220, 221]. This was confirmed in chondrogenic and osteogenic differentiation assays. Older animals had negligible Col-II and Saf-O and Alizarin Red-S staining denoting poor cartilaginous and osseous matrix. On the other hand, BMSCs from young animals synthesized a matrix rich in cartilaginous and osseous proteins.
A number of DNA repair pathways protect us from the
deleterious e ﬀects of DNA damage. The importance of
these mechanisms is highlighted by the existence of genetic disorders in which impaired DNA repair mechanisms pre- dispose patients to cancer and early onset of aging. A major advance in our understanding of these DNA repair mechanisms has been to uncover the tangled connection existing between these systems and other fundamental cellular processes such as DNA replicationand transcription. These cellular processes are not only highly connected with DNA repair pathways but they also share common factors with them. This complexity leads to new hypothesis about the cause of the phenotypes displayed by patients suﬀering from DNA repair disorders and may even force us to re-evaluate the place of the repair factors in cellular homeostasis.
FIG. 2. (a) Histogram Nt r of the replication timing ratio t r at
the 83 putative oris. (b) Histogram Nt r of the difference t r
in replication timing ratio between the borders and the center of the 38 replication domains of length L 1 Mbp. (c) Percentage of border-center pairs such that t r > 0 ( 䉱) and mean value
3.2 The correlates of light-trip behaviour
We have established that light trips are made within a shorter time span than regular trips, but this distinction has consequences on other dimensions of the trip profile; light trips are characterised (by definition) by less items bought (5.9 versus 31.6) and higher average price per item (29.8 FF versus 14.4 FF). More systematically, as each trip can be considered either “light” (coded 1) or “regular” (coded 0), we apply logistic regressions to the data. The base model (Table 2a) reveals that all variables are significant. The probability of getting a light trip is lower for larger families, higher income households, and more loyal shoppers. This probability is also higher when the previous trip was a light trip ; this suggest that light-trip behaviour is not (just) a random process. Our conclusion is similar to Kahn and Schmittlein (1989) as well as Leszczyc and Timmermans (1997), while extending their results to the impact of distance and (some) psychographics on light-trip behaviour (Table 2b). Indeed, light trips are also associated with visits to closer stores and with shoppers reporting a more favourable attitude toward promotions (we expected a negative relation).
In the single-SNP analysis and considering a general codom- inant model for each SNP effect, one SNP, rs512625, showed association with psoriasis that reached the 1% significance level classically required for replication (Table 2). Heterozygotes for this SNP had a decreased risk for psoriasis as compared to the reference group of homozygotes for the most frequent allele (OR = 0.61 [0.42; 0.89]) (Table 3). Moreover, a dominant model was rejected against the general codominant model (p = 0.02). When examining early-onset psoriasis, we found association with another SNP, rs628977, at the 1% level (Table 2). Homozygotes for the minor T allele had a higher risk for early-onset psoriasis as compared to the reference group (OR = 2.52 [1.31; 4.86]) (Table 3). A recessive model for rs628977 (which fits the data, p = 0.55) increased the significance (p = 0.003). Two polymor- phisms in the PSORS1 gene, rs3131000 and rs3130559, were associated with psoriasis (p,0.01; Supplementary, Table S1). None of these two SNPs was significantly associated with early- onset psoriasis.
Pull-off test is a convenient method for evaluating both the mechanical integrity of the concrete surface prior to repairand the repair bond strength. A relia- ble evaluation of these properties can be obtained, provided that a minimum number of tests are per- formed, with adequate equipment. When specifying a pull-off strength limit in the field, the value should be increased (by 10 to 15 %) to take into account the potential reduction due to testing misalignment. Bond strength of concrete repairs depends on a number of parameters. It has been shown that in ab- sence of substrate-induced damage, tensile bond strength increases with the substrate coarseness. Still, the most important parameter apparently re- mains the mechanical integrity of the substrate. In that regard, it must be stressed that the use of im- pacting methods such as jack hammering leaves sig- nificant damage at the surface, which can easily outweigh the benefits of an increased roughness. Other potentially important parameters upon repair bond development are the substrate moisture condi- tioning and state of carbonation at the time of cast- ing. The results obtained in the present study show that optimum moisture saturation levels for repair bond strength would lie somewhere between 55 to 90 %. Carbonation may in turn have only a slight impact on bond strength for an otherwise sound, properly prepared concrete substrate surface.
In designing new treatments to regenerate articular cartilage, biomaterials that do not need to be seeded with cultured cells have the advantage of being clinically practical and cost-efficient. However, to be effective, biomaterial-only implants must be able to guide mesen- chymal stem cells with chondrogenic potential from the bone marrow to above the osteochondral junction and into the cartilage lesion. Biomaterials have previously been successfully implanted into osteochondral defects, but re- producible long-term clinical benefits have yet to be demonstrated. A tubular implant composed of electrospun poly(D,L-lactide-co-glycolide) (PLG) inserted in large (5 mm wide, 5 mm deep) osteochondral defects in a rabbit model generated good cartilage repair in most defects at 24 weeks post-operative . Subchondral implants made of PLG copolymer, calcium sulfate, polyglycolide fibers and surfactant have demonstrated clinical benefit, with complete pain alleviation and resumption of functional ac- tivity after 24 months of continued rehabilitation in a case study ; although other studies reported that PLG persists and inhibits subchondral bone regeneration 6 to 12 months post-operative [5,6]. In a rabbit model, filling the drill hole with a chemically cross-linked chitosan hydrogel was previously shown to block cell infiltration and bone repair . Altogether, these data suggest that the porosity and degradation kinetics of biomaterials implanted in subchondral bone are key elements in the induction of reproducible osteochondral repair.
Replication mode of Constant Timing Regions
One possible mechanism for explaining why an equal proportion of rightward and leftward moving forks replicate a
CTR is that it does not contain origins and is passively replicated from an outside origin that is activated equally often on its right or its left side (Figure S3A). Given a mean fork velocity of 0.68 kb/ min (40 kb/h) this mechanism could only apply to short enough CTRs (,300 kb) to replicate within a 7–8 h S phase in HeLa cells. At the 100 kb scale, CTRs,300 kb and .300 kb cover 19.7% and 21.2% of the genome, respectively (Figure 4C). This mechanism predicts that i) the edges of small CTRs would replicate asynchronously in non-adjacent S-phase compartments whereas their centers would replicate synchronously in mid-S phase; ii) that small CTRs lying between 150 and 300 kb would replicate rather in mid-S phase. A previous study of Hela cells replication timing determined that about 20% of the ENCODE regions present a pan-S replication profile . However, we reported that in HeLa cells only 7.4% of all genomic sequences replicate with such a pan-S profile . Although a significant correlation was observed between these two studies (Pearson, R = 0.77, P,10 215 ), the differences may result from the use of microarray hybridization and cell synchronisation by drug treatment in the first study vs. massive sequencing and no drug treatment in our study. Furthermore, we have found that the TR 50
Hanspal, M., Smockova, Y., Uong, Q. 1998. Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages. Blood. 92, 2940- 2950.
Hara, M., Yuasa, S., Shimoji, K., Onizuka, T., Hayashiji, N., Ohno, Y., Arai, T., Hattori, F., Kaneda, R., Kimura, K., Makino, S., Sano, M., Fukuda, K. 2011. G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation. J. Exp. Med. 208, 715-727. Hashimoto, D., Chow, A., Noizat, C., Teo, P., Beasley, M.B., Leboeuf, M., Becker, C.D., See, P., Price, J., Lucas, D., Greter, M., Mortha, A., Boyer, S.W., Forsberg, E.C., Tanaka, M., van, R.N., Garcia-Sastre, A., Stanley, E.R., Ginhoux, F., Frenette, P.S., Merad, M. 2013. Tissue-Resident Macrophages Self- Maintain Locally throughout Adult Life with Minimal Contribution from Circulating Monocytes. Immunity. 38, 792-804.
In young patients with full body growth as well as adults with pectus deformities and congenital or acquired cardiomyopathies, the surgical strategy may differ. In this case no spontaneous improvement is expect- ed  and concomitant cardiac surgery and pectus repair may be rec- ommended. Concomitant surgery requires optimal operative exposure during cardiac procedure without any added bleeding risk. An optimal chest-wall stabilization in the postoperative period is also mandatory, usually by means of a device that enables eventual urgent postoperative reentry and cardiac resuscitation maneuvers  . Several authors have followed this approach with good postoperative results in coronary ar- tery bypass grafting  , aortic root surgery  and acute type A aortic dissection  . In addition, concomitant pectus repair may be recom- mended as it improves cardiovascular function by facilitating heart ﬁlling  and potentially improves postoperative outcome.
biochemical composition up to 28 days, and cell viability and density up to 14 days, during refrigerated storage.
Cartilage biomechanical properties, determined in indentation testing (Black, Shadle, Parsons, & Brighton, 1979; Kennedy, Tordonado, & Duma, 2007) and confined compression (Willett, Whiteside, Wild, Wyss, & Anastassiades, 2005) have been shown to degrade following freezing. However, several studies (Kempson, Spivey, Swanson, & Freeman, 1971; Kiefer et al., 1989; Swann, 1988) and anecdotal observations (Athanasiou, Rosenwasser, Buckwalter, Malinin, & Mow, 1991; Elmore, Sokoloff, Norris, & Carmeci, 1963) suggest that no changes in indentation properties occur due to freezing. Confined compression tests of osteochondral plugs, before and after storage at -20ºC immersed in PBS, resulted in statistically significant decreases in aggregate modulus (Willett, et al., 2005). Kennedy et al. (2007) reported that cartilage indentation stiffness decreased in whole bovine joints either frozen at -20ºC or flash-frozen to - 80ºC. However, creep tests of individual condyles after freezing at 0ºC resulted in no change in instantaneous shear modulus but a statistically significant decrease in relaxed shear modulus and increase in creep rate, indicating that the ability to resist sustained loading was compromised (Black, et al., 1979; Parsons & Black, 1977). Similarly, indentation testing performed on osteochondral cores at physiological stress levels showed no effect due to freezing at -80ºC with or without DMSO as a protectant (Kiefer, et al., 1989), and whole human femoral heads tested prior to and following freezing at -20ºC did not result in changes in the response to indentation testing (Kempson, et al., 1971). In summary, several studies have reported substantial losses in biomechanical properties in articular cartilage subjected to a single freeze-thaw cycle, while others report the contrary; that the biomechanical properties of cartilage are not altered as a result of freezing.