Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PK$_{cs}$ and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). $\textit{In vivo}$ studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. $\textit{In vitro}$ studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.National Institutes of Health, Cancer Research UK Program Grant IDs: C6/A11224, C6946/A14492), Wellcome Trust (Grant IDs: WT092096, WT093167

Overcoming cross-cultural group work tensions: mixed student perspectives on the role of social relationships

As universities worldwide rapidly internationalise, higher education classrooms have become unique spaces for collaboration between students from different countries. One common way to encourage collaboration between diverse peers is through group work. However, previous research has highlighted that cross-cultural group work can be challenging and has hinted at potential social tensions. To understand this notion better, we have used robust quantitative tools in this study to select 20 participants from a larger classroom of 860 students to take part in an in-depth qualitative interview about cross-cultural group work experiences. Participant views on social tensions in cross-cultural group work were elicited using a unique mediating artefact method to encourage reflection and in-depth discussion. In our analysis of emergent interview themes, we compared student perspectives on the role of social relationships in group work by their academic performance level. Our findings indicated that all students interviewed desired the opportunity to form social relationships with their group work members, but their motivations for doing so varied widely by academic performance level

High-sensitivity diamond magnetometer with nanoscale resolution

We present a novel approach to the detection of weak magnetic fields that takes advantage of recently developed techniques for the coherent control of solid-state electron spin quantum bits. Specifically, we investigate a magnetic sensor based on Nitrogen-Vacancy centers in room-temperature diamond. We discuss two important applications of this technique: a nanoscale magnetometer that could potentially detect precession of single nuclear spins and an optical magnetic field imager combining spatial resolution ranging from micrometers to millimeters with a sensitivity approaching few femtotesla/Hz$^{1/2}$.Comment: 29 pages, 4 figure

Does shear wave ultrasound independently predict axillary lymph node metastasis in women with invasive breast cancer?

Shear wave elastography (SWE) shows promise as an adjunct to greyscale ultrasound examination in assessing breast masses. In breast cancer, higher lesion stiffness on SWE has been shown to be associated with features of poor prognosis. The purpose of this study was to assess whether lesion stiffness at SWE is an independent predictor of lymph node involvement. Patients with invasive breast cancer treated by primary surgery, who had undergone SWE examination were eligible. Data were retrospectively analysed from 396 consecutive patients. The mean stiffness values were obtained using the Aixplorer(®) ultrasound machine from SuperSonic Imagine Ltd. Measurements were taken from a region of interest positioned over the stiffest part of the abnormality. The average of the mean stiffness value obtained from each of two orthogonal image planes was used for analysis. Associations between lymph node involvement and mean lesion stiffness, invasive cancer size, histologic grade, tumour type, ER expression, HER-2 status and vascular invasion were assessed using univariate and multivariate logistic regression. At univariate analysis, invasive size, histologic grade, HER-2 status, vascular invasion, tumour type and mean stiffness were significantly associated with nodal involvement. Nodal involvement rates ranged from 7 % for tumours with mean stiffness <50 kPa to 41 % for tumours with a mean stiffness of >150 kPa. At multivariate analysis, invasive size, tumour type, vascular invasion, and mean stiffness maintained independent significance. Mean stiffness at SWE is an independent predictor of lymph node metastasis and thus can confer prognostic information additional to that provided by conventional preoperative tumour assessment and staging

Quercetin elevates p27Kip1 and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1

Our previous work with primary bovine fibroblasts demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 and G2/M, in correlation with p53 activation. The expression of bovine papillomavirus type 4 (BPV-4) E7 overcame this arrest and lead to the development of tumorigenic cells lines (Beniston et al., 2001). Given the possible link between papillomavirus infection, bracken fern in the diet and cancer of the upper gastrointestinal (GI) tract in humans, we investigated whether a similar situation would occur in human cells transformed by human papillomavirus type 16 (HPV-16) oncoproteins. Quercetin arrested primary human foreskin keratinocytes in G1. Arrest was linked to an elevation of the cyclin-dependent kinase inhibitor (cdki) p27Kip1. Expression of the HPV16 E6 and E7 oncoproteins in transformed cells failed to abrogate cell cycle arrest. G1 arrest in the transformed cells was also linked to an increase of p27Kip1 with a concomitant reduction of cyclin E-associated kinase activity. This elevation of p27Kip1 was due not only to increased protein half-life, but also to increased mRNA transcription

Interleukin-1β sequesters hypoxia inducible factor 2α to the primary cilium.

BACKGROUND: The primary cilium coordinates signalling in development, health and disease. Previously we have shown that the cilium is essential for the anabolic response to loading and the inflammatory response to interleukin-1β (IL-1β). We have also shown the primary cilium elongates in response to IL-1β exposure. Both anabolic phenotype and inflammatory pathology are proposed to be dependent on hypoxia-inducible factor 2 alpha (HIF-2α). The present study tests the hypothesis that an association exists between the primary cilium and HIFs in inflammatory signalling. RESULTS: Here we show, in articular chondrocytes, that IL-1β-induces primary cilia elongation with alterations to cilia trafficking of arl13b. This elongation is associated with a transient increase in HIF-2α expression and accumulation in the primary cilium. Prolyl hydroxylase inhibition results in primary cilia elongation also associated with accumulation of HIF-2α in the ciliary base and axoneme. This recruitment and the associated cilia elongation is not inhibited by blockade of HIFα transcription activity or rescue of basal HIF-2α expression. Hypomorphic mutation to intraflagellar transport protein IFT88 results in limited ciliogenesis. This is associated with increased HIF-2α expression and inhibited response to prolyl hydroxylase inhibition. CONCLUSIONS: These findings suggest that ciliary sequestration of HIF-2α provides negative regulation of HIF-2α expression and potentially activity. This study indicates, for the first time, that the primary cilium regulates HIF signalling during inflammation

CP Violation in Supersymmetry with Effective Minimal Flavour Violation

We analyze CP violation in supersymmetry with Effective Minimal Flavour Violation, as recently proposed in arXiv:1011.0730. Unlike the case of standard Minimal Flavour Violation, we show that all the phases allowed by the flavour symmetry can be sizable without violating existing Electric Dipole Moment constraints, thus solving the SUSY CP problem. The EDMs at one and two loops are precisely analyzed as well as their correlations with the expected CP asymmetries in B physics.Comment: 22 pages, 7 figures. v2: Discussion in section 2 extended, conclusions unchanged. Matches published versio

Applications of Direct Injection Soft Chemical Ionisation-Mass Spectrometry for the Detection of Pre-blast Smokeless Powder Organic Additives

Analysis of smokeless powders is of interest from forensics and security perspectives. This article reports the detection of smokeless powder organic additives (in their pre-detonation condition), namely the stabiliser diphenylamine and its derivatives 2-nitrodiphenylamine and 4-nitrodiphenylamine, and the additives (used both as stabilisers and plasticisers) methyl centralite and ethyl centralite, by means of swab sampling followed by thermal desorption and direct injection soft chemical ionisation-mass spectrometry. Investigations on the product ions resulting from the reactions of the reagent ions H3O+ and O2+ with additives as a function of reduced electric field are reported. The method was comprehensively evaluated in terms of linearity, sensitivity and precision. For H3O+, the limits of detection (LoD) are in the range of 41-88 pg of additive, for which the accuracy varied between 1.5 and 3.2%, precision varied between 3.7 and 7.3% and linearity showed R20.9991. For O2+, LoD are in the range of 72 to 1.4 ng, with an accuracy of between 2.8 and 4.9% and a precision between 4.5 and 8.6% and R20.9914. The validated methodology was applied to the analysis of commercial pre-blast gun powders from different manufacturers.(VLID)4826148Accepted versio

Decitabine impact on the endocytosis regulator RhoA, the folate carriers RFC1 and FOLR1, and the glucose transporter GLUT4 in human tumors.

BackgroundIn 31 solid tumor patients treated with the demethylating agent decitabine, we performed tumor biopsies before and after the first cycle of decitabine and used immunohistochemistry (IHC) to assess whether decitabine increased expression of various membrane transporters. Resistance to chemotherapy may arise due to promoter methylation/downregulation of expression of transporters required for drug uptake, and decitabine can reverse resistance in vitro. The endocytosis regulator RhoA, the folate carriers FOLR1 and RFC1, and the glucose transporter GLUT4 were assessed.ResultsPre-decitabine RhoA was higher in patients who had received their last therapy &gt;3&nbsp;months previously than in patients with more recent prior therapy (P = 0.02), and varied inversely with global DNA methylation as assessed by LINE1 methylation (r = -0.58, P = 0.006). Tumor RhoA scores increased with decitabine (P = 0.03), and RFC1 also increased in patients with pre-decitabine scores ≤150 (P = 0.004). Change in LINE1 methylation with decitabine did not correlate significantly with change in IHC scores for any transporter assessed. We also assessed methylation of the RFC1 gene (alias SLC19A1). SLC19A1 methylation correlated with tumor LINE1 methylation (r = 0.45, P = 0.02). There was a small (statistically insignificant) decrease in SLC19A1 methylation with decitabine, and there was a trend towards change in SLC19A1 methylation with decitabine correlating with change in LINE1 methylation (r = 0.47, P &lt;0.15). While SLC19A1 methylation did not correlate with RFC1 scores, there was a trend towards an inverse correlation between change in SLC19A1 methylation and change in RFC1 expression (r = -0.45, P = 0.19).ConclusionsIn conclusion, after decitabine administration, there was increased expression of some (but not other) transporters that may play a role in chemotherapy uptake. Larger patient numbers will be needed to define the extent to which this increased expression is associated with changes in DNA methylation