Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome.(A) The L1.3 ORF2p-3FLAG expression vector (pTMO2F3). The CMV promoter (black arrow) and 5 0 UTR augment L1 expression. Also indicated are the FLAG epitope tag, SV40 polyadenylation signal, and the EN, Z, RT, and cysteine-rich (C) domains. (B) The ORF2p-3FLAG complex. HEK293T cells were transfected with pAD500 (ORF2p-TAP) or pTMO2F3 (ORF2p-3FLAG). Left: the ORF2p-3FLAG complex was purified using anti-FLAG antibody-conjugated beads, separated on a denaturing polyacrylamide gel, and visualized by silver staining. Right: ORF2p-3FLAG was detected by western blot. Input, the whole-cell lysate used in the IP. Arrows, ORF2p-3FLAG. (C) Validation of proteins in the ORF2p-3FLAG complex. ORF2p-3FLAG complexes were purified (see B) from HEK293T cells transfected with a full-length (pTMF3, lane 2) or monocistronic (pTMO2F3, lane 4) L1 expression constructs. The ORF1p-1FLAG complex was purified from cells transfected with pJM101/L1.3FLAG (lane 5). The pJM101/L1.3 and pAD001 samples (lanes 1 and 3, respectively) served as negative controls. Input, western blot lanes of whole-cell lysate used in the IP. Red triangles, FLAG tags; SSB, single-strand break; NHEJ, non-homologous end-joining; NER, nucleotide excision repair. See also Figure S1 and Table S1.

No abstract

When the chondral lesion is assumed at the weight bearing area of the medial or lateral femoral condyle, the anterior portion of the sulcus terminalis (the groove for meniscus) in the middle section of the lateral condyle is considered to be the optimal donor site.

We have made three types of poly (DL-lactide-coglycolide) (PLG) scaffolds (porosity: scaffold I 80±0.9%, II 85±0.8%, III 92±0.7%; compression module determined with 10% strain: scaffold I 0.26 MPa, II 0.091 MPa, III 0.0047 MPa). Osteochondral defects made in the femoral condyle of rabbits were treated with these scaffolds and the possibilities of cartilage repair were investigated histologically. At post-operative weeks 6 and 12, histological scores in the groups of scaffolds II and III were significantly higher than the score in the group of scaffold I. Scaffolds II and III, which have higher porosity than scaffold I, allow better migration of bone marrow cells and better replacement of the scaffold with bone and cartilage than scaffold I. This study suggests that higher porosity allowing bone marrow cells to migrate to the scaffold is important in repairing osteochondral defects.Résumé Nous avons réalisé trois types de montage PLG (DL lactide co glycolide) avec des porosités différentes (montage I à 0,9%, montage II à 87 ,8%, montage III 0,7%). Avec le module de compression a été imposée une tension de 10% avec 0,26 MPa au niveau de la pièce I, 0,091 Mpa en montage II et 0,0047 MPa en montage III). Un defect ostéochondral réalisé dans le condyle fémoral d'un lapin a été traité par ces trois procédés avec une possibilité de réparation cartilagineuse. Cette possibilité a été évaluée histologiquement. A 6 et 12 semaines postopératoires, le score histologique dans les groupes II et III sont significativement plus élevés que dans le groupe I. Les pièces II et III qui ont une plus grande porosité que la I permettent une meilleure migration des cellules de la moelle osseuse et une meilleure réparation de l'os et du cartilage que dans le montage I. Cette étude nous permet de penser qu'une porosité plus importante permet la migration plus facile de ces cellules de moelle osseuse lors de la réparation des défects ostéochondraux.

A successful scaffold for use in tendon tissue engineering requires a high affinity for living organisms and the ability to maintain its mechanical strength until maturation of the regenerated tissue. We compared two types of poly(L-lactic acid) (PLLA) scaffolds for use in tendon regeneration, a plain-woven PLLA fabric (fabric P) with a smooth surface only and a double layered PLLA fabric (fabric D) with a smooth surface on one side and a rough (pile-finished) surface on the other side. These two types of fabric were implanted into the back muscles of rabbits and evaluated at three and six weeks after implantation. Histological examination showed collagen tissues were highly regenerated on the rough surface of fabric D. On the other hand, liner cell attachment was seen in the smooth surface of fabric P and fabric D. The total DNA amount was significantly higher in fabric D. Additionally, mechanical examination showed fabric P had lost its mechanical strength by six weeks after implantation, while the strength of fabric D was maintained. Fabric D had more cell migration on one side and less cell adhesion on the other side and maintained its initial strength. Thus, a novel form of double-layered PLLA fabric has the potential to be used as a scaffold in tendon regeneration.

We developed a new porous scaffold made from a synthetic polymer, poly(DL-lactide-co-glycolide) (PLG), and evaluated its use in the repair of cartilage. Osteochondral defects made on the femoral trochlear of rabbits were treated by transplantation of the PLG scaffold, examined histologically and compared with an untreated control group. Fibrous tissue was initially organised in an arcade array with poor cellularity at the articular surface of the scaffold. The tissue regenerated to cartilage at the articular surface. In the subchondral area, new bone formed and the scaffold was absorbed. The histological scores were significantly higher in the defects treated by the scaffold than in the control group (p < 0.05). Our findings suggest that in an animal model the new porous PLG scaffold is effective for repairing full-thickness osteochondral defects without cultured cells and growth factors.

In the presence of a catalytic amount of a rhodium(I) complex, allenenes undergo cycloisomerization reactions resulting in the selective formation of exo-alkylidenecarbocycles and heterocycles. In the catalytic system of rhodium complexes with triaryl phosphites, cyclic 1,4- or 1,5-dienes are formed in good to excellent yields in the formal exo-cyclization mode via the metallacycle intermediate having an exo-alkylidene moiety. In this cycloisomerization, (E)- and (Z)-allenenes are transformed stereospecifically to the corresponding cyclic (E)- and (Z)-1,4-dienes, respectively. On the other hand, the reactions under carbon monoxide atmosphere exclusively afford seven-membered-ring products through an endo-mode cyclization. The unusual cyclization involves an allylic C-H activation process. The allenene bearing a silicon substituent at the olefinic terminus incorporates carbon monoxide to give the corresponding [2+2+1] cycloaddition product. This result apparently indicates that the catalysis of the rhodium complex is explained in terms of the oxidative cyclization of an allenene to furnish the key exo-alkylidene metallacycle intermediate at the first stage of the catalysis.

Crystallographic pits were grown on Al͑100͒ at temperatures from 30 to 90°C, in solutions of HCl ϩ H 2 SO 4 . Pits grew during a galvanostatic anodic pulse for 5-100 ms that was preceded by a galvanostatic cathodic pulse of 50 ms. The anodic potential has a small peak for 1-2 ms and then remains constant. The cathodic pulse causes rapid pit nucleation so most pits nucleate within 5 ms, and pit passivation and pit growth are the dominant processes at longer times. It was determined that a substantial fraction of pits passivates during the pulse, so growth rates were calculated from the increase in largest pit size with pulse duration. The growth rate is constant at each temperature and follows an Arrhenius temperature dependence with an activation energy of 7.2 kcal mol Ϫ1 . There are significant differences between the growth of pits and the growth of etch tunnels. The activation energy for pit growth is one-half that for tunnel growth, and pit growth rates are greater than tunnel growth rates. It is proposed that the chemisorbed chloride complex postulated to be an intermediary for Al dissolution changes from one structure during pit growth to another, more stable, structure during tunnel growth.