Joining the dots : piloting the work diary as a data collection tool for educational research

This paper demonstrates the affordances of the work diary as a data collection tool for both pilot studies and qualitative research of social interactions. Observation is the cornerstone of many qualitative, ethnographic research projects (Creswell, 2008). However, determining through observation, the activities of busy school teams could be likened to joining dots of a child’s drawing activity to reveal a complex picture of interactions. Teachers, leaders and support personnel are in different locations within a school, performing diverse tasks for a variety of outcomes, which hopefully achieve a common goal. As a researcher, the quest to observe these busy teams and their interactions with each other was daunting and perhaps unrealistic. The decision to use a diary as part of a wider research project was to overcome the physical impossibility of simultaneously observing multiple team members. One reported advantage of the use of the diary in research was its suitability as a substitute for lengthy researcher observation, because multiple data sets could be collected at once (Lewis et al, 2005; Marelli, 2007).

Bi-2212/Ag laminated tapes: Bending and joining effects

Superconducting composite Bi-2212/Ag tapes and their joints are fabricated by a combination of dip-coating and partial melt processing. The heat treated tapes have a critical current (Ic) between 8 and 26A, depending on tape thickness and the number of Bi-2212 layers. Current transmissions between 80% and 100% have been achieved through the joints of tapes. Different types of HTS joints of Bi-2212/Ag laminated tapes are made and their transport properties during winding operations are investigated. Irreversible strain values (ε irrev) for laminated tapes and their joints are determined and it is found that the degradation of Ic during tape bending depends on the type of joint.

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.

Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism

Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.

Joining of dissimilar metals: issues and modelling techniques

Joining of dissimilar metals involves a number of scientific issues, the modelling of which offers unique challenges. This review discusses the complexities in different joining processes and dissimilar combinations, and the corresponding computational techniques that have the potential to address the same. Future directions in modelling at both macroscopic and microscopic scales are also suggested.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development. (C) 2012 Elsevier Ltd. All rights reserved.

An Inhibitor of Nonhomologous End-Joining Abrogates Double-Strand Break Repair and Impedes Cancer Progression

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells, and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens.

Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase

Here, we show that PARP inhibitor-mediated cell death of RAD51C-deficient cells occur by NHEJ-driven illegitimate repair of one-ended double-strand breaks, and the hypomorphic RAD51C pathological mutant cells can be targeted by `synergistic toxicity' induced by low-dose PARP inhibitor and IR.Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function. However, targeting cancer cells that express hypomorphic mutants of RAD51C is highly challenging. Here, we report that RAD51C-deficient cells can be targeted by a `synthetic lethal' approach using PARP inhibitor and this sensitivity was attributed to accumulation of cells in the G(2)/M and chromosomal aberrations. In addition, spontaneous hyperactivation of PARP1 was evident in RAD51C-deficient cells. Interestingly, RAD51C-negative cells exhibited enhanced recruitment of non-homologous end joining (NHEJ) proteins onto chromatin and this accumulation correlated with increased activity of error-prone NHEJ as well as genome instability leading to cell death. Notably, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV rescued this phenotype. Strikingly, stimulation of NHEJ by low dose of ionizing radiation (IR) in the PARP inhibitor-treated RAD51C-deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity `synergistically'. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a `synergistic approach' and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other homologous recombination pathway genes.

Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing

DNA repair, one of the fundamental processes occurring in a cell, safeguards the genome and maintains its integrity. Among various DNA lesions, double-strand breaks are considered to be the most deleterious, as they can lead to potential loss of genetic information, if not repaired. Non-homologous end joining (NHEJ) and homologous recombination are two major double-strand break repair pathways. SCR7, a DNA ligase IV inhibitor, was recently identified and characterized as a potential anticancer compound. Interestingly, SCR7 was shown to have several applications, owing to its unique property as an NHEJ inhibitor. Here, we focus on three main areas of research in which SCR7 is actively being used, and discuss one of the applications, i.e. genome editing via CRISPR/Cas, in detail. In the past year, different studies have shown that SCR7 significantly increases the efficiency of precise genome editing by inhibiting NHEJ, and favouring the error-free homologous recombination pathway, both in vitro and in vivo. Overall, we discuss the current applications of SCR7 to shed light on the unique property of the small molecule of having distinct applications in normal and cancer cells, when used at different cellular concentrations.

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knock-down studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.