DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity

Abstract DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability

Scrapie-Specific Pathology of Sheep Lymphoid Tissues

Transmissible spongiform encephalopathies (TSEs) or prion diseases often result in accumulation of disease-associated PrP (PrPd) in the lymphoreticular system (LRS), specifically in association with follicular dendritic cells (FDCs) and tingible body macrophages (TBMs) of secondary follicles. We studied the effects of sheep scrapie on lymphoid tissue in tonsils and lymph nodes by light and electron microscopy. FDCs of sheep were grouped according to morphology as immature, mature or regressing. Scrapie was associated with FDC dendrite hypertrophy and electron dense deposit or vesicles. PrPd was located using immunogold labelling at the plasmalemma of FDC dendrites and, infrequently, mature B cells. Abnormal electron dense deposits surrounding FDC dendrites were identified as immunoglobulins suggesting that excess immune complexes are retained and are indicative of an FDC dysfunction. Within scrapie-affected lymph nodes, macrophages outside the follicle and a proportion of germinal centre TBMs accumulated PrPd within endosomes and lysosomes. In addition, TBMs showed PrPd in association with the cell membrane, non-coated pits and vesicles, and also with discrete, large and random endoplasmic reticulum networks, which co-localised with ubiquitin. These observations suggest that PrPd is internalised via the caveolin-mediated pathway, and causes an abnormal disease-related alteration in endoplasmic reticulum structure. In contrast to current dogma, this study shows that sheep scrapie is associated with cytopathology of germinal centres, which we attribute to abnormal antigen complex trapping by FDCs and abnormal endocytic events in TBMs. The nature of the sub-cellular changes in FDCs and TBMs differs from those of scrapie infected neurones and glial cells suggesting that different PrPd/cell membrane interactions occur in different cell types

Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy

A comparative study of immature and mature bone marrow-derived dendritic cells (BMDCs) was first performed through an atomic force microscope (AFM) to clarify differences of their nanostructure and adhesion force. AFM images revealed that the immature BMDCs treated by granulocyte macrophage-colony stimulating factor plus IL-4 mainly appeared round with smooth surface, whereas the mature BMDCs induced by lipopolysaccharide displayed an irregular shape with numerous pseudopodia or lamellapodia and ruffles on the cell membrane besides becoming larger, flatter, and longer. AFM quantitative analysis further showed that the surface roughness of the mature BMDCs greatly increased and that the adhesion force of them was fourfold more than that of the immature BMDCs. The nano-features of the mature BMDCs were supported by a high level of IL-12 produced from the mature BMDCs and high expression of MHC-II on the surface of them. These findings provide a new insight into the nanostructure of the immature and mature BMDCs

Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1

Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome

Molecular Dynamics and Quantum Mechanics of RNA: Conformational and Chemical Change We Can Believe In

Structure and dynamics are both critical to RNA’s vital functions in biology. Numerous techniques can elucidate the structural dynamics of RNA, but computational approaches based on experimental data arguably hold the promise of providing the most detail. In this Account, we highlight areas wherein molecular dynamics (MD) and quantum mechanical (QM) techniques are applied to RNA, particularly in relation to complementary experimental studies

Edible bio-based nanostructures: delivery, absorption and potential toxicity

The development of bio-based nanostructures as nanocarriers of bioactive compounds to specific body sites has been presented as a hot topic in food, pharmaceutical and nanotechnology fields. Food and pharmaceutical industries seek to explore the huge potential of these nanostructures, once they can be entirely composed of biocompatible and non-toxic materials. At the same time, they allow the incorporation of lipophilic and hydrophilic bioactive compounds protecting them against degradation, maintaining its active and functional performance. Nevertheless, the physicochemical properties of such structures (e.g., size and charge) could change significantly their behavior in the gastrointestinal (GI) tract. The main challenges in the development of these nanostructures are the proper characterization and understanding of the processes occurring at their surface, when in contact with living systems. This is crucial to understand their delivery and absorption behavior as well as to recognize potential toxicological effects. This review will provide an insight into the recent innovations and challenges in the field of delivery via GI tract using bio-based nanostructures. Also, an overview of the approaches followed to ensure an effective deliver (e.g., avoiding physiological barriers) and to enhance stability and absorptive intestinal uptake of bioactive compounds will be provided. Information about nanostructures potential toxicity and a concise description of the in vitro and in vivo toxicity studies will also be given.Joana T. Martins, Oscar L. Ramos, Ana C. Pinheiro, Ana I. Bourbon, Helder D. Silva and Miguel A. Cerqueira (SFRH/BPD/89992/2012, SFRH/BPD/80766/2011, SFRH/BPD/101181/2014, SFRH/BD/73178/2010, SFRH/BD/81288/2011, and SFRH/BPD/72753/2010, respectively) are the recipients of a fellowship from the Fundacao para a Ciencia e Tecnologia (FCT, POPH-QREN and FSE, Portugal). The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes," REF.NORTE-07-0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. We also thank to the European Commission: BIOCAPS (316265, FP7/REGPOT-2012-2013.1) and Xunta de Galicia: Agrupamento INBIOMED (2012/273) and Grupo con potencial de crecimiento. The support of EU Cost Action FA1001 is gratefully acknowledged

Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.