Mu transpososome and RecBCD nuclease collaborate in the repair of simple Mu insertions

The genome of transposable phage Mu is packaged as a linear segment, flanked by several hundred base pairs of non-Mu DNA. The linear ends are held together and protected from nucleases by the phage N protein. After transposition into the Escherichia coli chromosome, the flanking DNA (FD) is degraded, and the 5-bp gaps left in the target are repaired to generate a simple Mu insertion. Our study provides insights into this repair pathway. The data suggest that the first event in repair is removal of the FD by the RecBCD exonuclease, whose entry past the N-protein block is licensed by the transpososome. In vitro experiments reveal that, when RecBCD is allowed entry into the FD, it degrades this DNA until it arrives at the transpososome, which presents a barrier for further RecBCD movement. RecBCD action is required for stimulating endonucleolytic cleavage within the transpososome-protected DNA, leaving 4-nt flanks outside both Mu ends. This end product of collaboration between the transpososome and RecBCD resembles the intermediate products of Tn7 and retroviral and retrotransposon transposition, and may hint at a common gap-repair mechanism in these diverse transposons.The repair of transposon insertions is the least understood aspect of transposon biology. Transposable phage Mu provides an excellent system to study this process because of its high transposition frequency. Mu is a temperate phage, which uses transposition not only to integrate into its Escherichia coli host to generate prophages but also to amplify its genome during the lytic cycle, where transposition is coupled to replication (Fig. 1A) (1–4).Open in a separate windowFig. 1.Two transposition pathways during the Mu life cycle. The chemical steps of single-stranded DNA cleavage at Mu ends followed by strand transfer (ST) of the cleaved ends to phosphodiester bonds spaced 5 bp apart on the target are the same in both the lytic (A) and infection (B) phases of transposition. During the lytic phase, Mu transposition is intramolecular, and the ST intermediate is resolved by target-primed replication through Mu (A). During the infection phase, transposition is intermolecular, and the Ɵ ST intermediate is resolved by removal of the flanking DNA and repair of the 5-bp gaps left in the target (B). The FD is covalently closed in A but noncovalently closed by phage N protein (oval) bound to the tips of the FD in B. The target DNA flanking Mu is red in all of the figures. Arrowheads indicate the 3′ ends.This study focuses on the transposition event that occurs immediately after infection, which is followed by repair rather than replication of the Mu insertion. The infecting Mu genome is linear, and is linked to several hundred base pairs of non–Mu-flanking DNA. The tips of this DNA are held together by an injected phage protein, N, which converts it into a noncovalently closed circular form and protects the DNA against exonucleases (5–7). After integration, the flanking DNA (FD) is removed (8), and the 5-bp gaps generated in the target are repaired to yield a simple Mu insertion (Fig. 1B) (9). Unlike the noncovalently closed configuration of the infecting Mu genome, replicating Mu is initially part of a covalently closed circular E. coli chromosome (Fig. 1A). At the end of the lytic phase, Mu replicas are packaged such that host DNA linked to either side of the insertion is included in the phage head; this is the source of the FD in the infecting Mu genome.The chemical steps of Mu transposition during the replication and repair pathways are the same, namely the MuA transposase nicks Mu DNA at each end and then joins the nicked ends to target DNA cleaved 5 bp apart (Fig. 1) (2, 8). However, the resulting branched strand-transfer intermediate is resolved alternatively by target-primed replication during the lytic phase (Fig. 1A) (10) and by FD removal and limited replicative repair during the infection phase (Fig. 1B) (8, 11). The alternate fates of a similar strand-transfer joint have long been a matter of speculation. Although much has been learned about the proteins that promote the transition from transposition to replication (10, 12, 13), little is known about those that assist in repair, other than that the gap-filling E. coli polymerase PolA is not required and that the double-strand break (DSB) repair machinery is somehow involved (14).We show in this study that the noncovalently closed configuration of the FD in the infecting Mu donor substrate controls the fate of the strand-transfer joint. Our data show that the RecBCD exonuclease is required in vivo for degradation of the FD. This occurs only after integration of infecting Mu, suggesting that there is a timed mechanism to remove N and allow RecBCD access into the FD. Attempts to recapitulate this reaction in vitro have revealed that the FD is processed in two stages: First, RecBCD degrades the long DNA, and next, a specific endonucleolytic cleavage leaves short 4-nt flanks within the transpososome-protected DNA. Earlier, we had found that a cryptic endonuclease activity of MuA was required for removal of the flanks in vivo, and had interpreted the data to suggest that degradation of this DNA is initiated by endonucleolytic cleavage (15). In light of the results obtained in the present study, we reinterpret our earlier data and propose a new model for repair. We expect that this first biochemical analysis, to our knowledge, of the initial steps of Mu repair will reveal pathways common to the repair of all transposon insertions.     

Dentin biomodification: strategies,renewable resources and clinical applications

Objectives

The biomodification of dentin is a biomimetic approach, mediated by bioactive agents, to enhance and reinforce the dentin by locally altering the biochemistry and biomechanical properties. This review provides an overview of key dentin matrix components, targeting effects of biomodification strategies, the chemistry of renewable natural sources, and current research on their potential clinical applications.

Methods

The PubMed database and collected literature were used as a resource for peer-reviewed articles to highlight the topics of dentin hierarchical structure, biomodification agents, and laboratorial investigations of their clinical applications. In addition, new data is presented on laboratorial methods for the standardization of proanthocyanidin-rich preparations as a renewable source of plant-derived biomodification agents.

Results

Biomodification agents can be categorized as physical methods and chemical agents. Synthetic and naturally occurring chemical strategies present distinctive mechanism of interaction with the tissue. Initially thought to be driven only by inter- or intra-molecular collagen induced non-enzymatic cross-linking, multiple interactions with other dentin components are fundamental for the long-term biomechanics and biostability of the tissue. Oligomeric proanthocyanidins show promising bioactivity, and their chemical complexity requires systematic evaluation of the active compounds to produce a fully standardized intervention material from renewable resource, prior to their detailed clinical evaluation.

Significance

Understanding the hierarchical structure of dentin and the targeting effect of the bioactive compounds will establish their use in both dentin-biomaterials interface and caries management.     

Association between cystatin C and inflammation in patients with essential hypertension

Background

Serum cystatin C is not only a marker of renal function but also acts as an independent risk factor for cardiovascular damage, heart failure, and death. It is known that the initiation and progression of these cardiovascular events contributes to renal dysfunction and chronic inflammation. In this study, we investigated the relationship between cystatin C and proinflammatory cytokines.

Methods

Eighty-eight patients with essential hypertension participated in the study, which involved measuring proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and C reactive protein (CRP).

Results

Positive correlations were detected between cystatin C and estimated glomerular filtration rate (eGFR) (r = ?0.503, p < 0.001), systolic blood pressure (r = ?0.246, p = 0.034), and pulse pressure (r = ?0.295, p = 0.010). In contrast, serum creatinine correlated only with eGFR (r = ?0.755, p < 0.001) and eGFR correlated only with age (r = ?0.339, p = 0.001) and not with the other clinical parameters, whereas cystatin C also correlated with log natural (ln) IL-6 (r = ?0.247, p = 0.033) and ln TNF-α (r = ?0.405, p < 0.001) but not with CRP (r = ?0.188, p = 0.108). In contrast, plasma creatinine and eGFR did not correlate with any of these proinflammatory cytokines. Stepwise regression analysis showed that ln TNF-α, eGFR and pulse pressure were independent determinants of serum cystatin C concentration.

Conclusion

This study showed that cystatin C is a marker of inflammation as well as renal function.     

Mitochondrial respiratory complex I dysfunction promotes tumorigenesis through ROS alteration and AKT activation

Previously, we have shown that a heteroplasmic mutation in mitochondrial DNA-encoded complex I ND5 subunit gene resulted in an enhanced tumorigenesis through increased resistance to apoptosis. Here we report that the tumorigenic phenotype associated with complex I dysfunction could be reversed by introducing a yeast NADH quinone oxidoreductase (NDI1) gene. The NDI1 mediated electron transfer from NADH to Co-Q, bypassed the defective complex I and restored oxidative phosphorylation in the host cells. Alternatively, suppression of complex I activity by a specific inhibitor, rotenone or induction of oxidative stress by paraquat led to an increase in the phosphorylation of v-AKT murine thymoma viral oncogene (AKT) and enhanced the tumorigenesis. On the other hand, antioxidant treatment can ameliorate the reactive oxygen species-mediated AKT activation and reverse the tumorigenicity of complex I-deficient cells. Our results suggest that complex I defects could promote tumorigenesis through induction of oxidative stress and activation of AKT pathway.     

Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression

Genetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10??) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10?2?) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10??). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10??) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype.     

A randomized trial comparing seed displacement of coated seeds to regular loose seeds at 30 days postimplant

PurposeTo compare 30-day seed displacement and seed loss of standard loose seeds to specially engineered coated seeds.Methods and MaterialsForty patients with prostate cancer were randomized and treated with either loose seeds or loose “coated” seeds. Implants were preplanned using transrectal ultrasound and performed using preloaded needles containing either standard or coated iodine-125 seeds according to randomization. Pelvic X-rays and CT were performed on Days 0 and 30 and a pelvic magnetic resonance scan on Day 30. Cranial–caudal displacement relative to the center of mass (COM) of the seed cloud of the six most peripheral basal and apical seeds was determined from Day 0 and 30 CT scans using custom software. Day 30 magnetic resonance–CT fusion was performed using a seed-to-seed match for soft tissue contouring on MRI.ResultsThe mean displacement for the six basal seeds was 0.32 cm (standard deviation [SD], 0.25 cm) and 0.33 cm (SD, 0.27 cm) toward the COM for the regular and coated seeds, respectively (p = 0.35). For the apical seeds, mean displacement was 0.31 cm (SD, 0.35 cm) and 0.43 cm (SD, 0.26 cm) (p = 0.003) toward the COM. More regular seeds (n = 8) were lost from the apical region as compared with one coated seed (p = 0.015). There was a trend to reduction in total seeds lost: 1% for regular seeds as compared with 0.3% for coated seeds.ConclusionsCoated seeds were found to have a significant anchoring effect that was effective in reducing the number of apical seeds lost because of venous migration.