Measurement of DNA damage by electrons with energies between 25 and 4000 eV.

All ionizing radiations deposit energy stochastically along their tracks. The resulting distribution of energies deposited in a small target such as the DNA helix leads to a corresponding spectrum in the severity of damage produced. So far, most information about the probable spectra of DNA lesion complexity has come from Monte Carlo studies which endeavour to model the relationship between the energy deposited in DNA and the damage induced. The aim of this paper is to establish methods of determining this relationship by irradiating pBR322 plasmid DNA using low energy electrons with energies comparable with the minimum energy thought to produce critical damage. The technique of agarose gel electrophoresis has been used to ascertain the fraction of DNA single- and double-strand breaks induced by monoenergetic electrons with energies as low as 25 eV. Our data show that the threshold electron energy for induction of single-strand breaks is

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA, Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA, As well as strand breaks, these sites may contain multiple damaged bases, We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH radicals with either Co-60 gamma-rays or alpha-particles from a Pu-238 source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1x10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp, This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute similar to 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.

Quantification of DNA damage by PFGE: development of an analytical approach to correct for the background distribution

Purpose: To analyse the currently existing methods to infer the extent of cellular DNA damage induced by ionizing radiation when the pulsed field gel electrophoresis (PFGE) technique is used.

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms.

An Experimental Assessment of the Effects of Stator Vane Tip Clearance and Back Swept Blading on an Automotive Variable Geometry Turbocharger

Off-design performance is of key importance now in the design of automotive turbocharger turbines. Due to automotive drive cycles, a turbine that can extract more energy at high pressure ratios and lower rotational speeds is desirable. Typically a radial turbine provides peak efficiency at U/C values of 0.7, but at high pressure ratios and low rotational speeds, the U/C value will be low and the rotor will experience high values of positive incidence at the inlet. The positive incidence causes high blade loading resulting in additional tip leakage flow in the rotor as well as flow separation on the suction surface of the blade. An experimental assessment has been performed on a scaled automotive VGS (variable geometry system). Three different stator vane positions have been analyzed: minimum, 25%, and maximum flow position. The first tests were to establish whether positioning the endwall clearance on the hub or shroud side of the stator vanes produced a different impact on turbine efficiency. Following this, a back swept rotor was tested to establish the potential gains to be achieved during off-design operation. A single passage CFD model of the test rig was developed and used to provide information on the flow features affecting performance in both the stator vanes and turbine. It was seen that off-design performance was improved by implementing clearance on the hub side of the stator vanes rather than on the shroud side. Through CFD analysis and tests, it was seen that two leakage vortices form, one at the leading edge and one after the spindle of the stator vane. The vortices affect the flow angle at the inlet to the rotor, in the hub region. The flow angle is shifted to more negative values of incidence, which is beneficial at the off-design conditions but detrimental at the design point. The back swept rotor was tested with the hub side stator vane clearance configuration. The efficiency and MFR were increased at the minimum and 25% stator vane position. At the design point, the efficiency and MFR were decreased. The CFD investigation showed that the incidence angle was improved at the off-design conditions for the back swept rotor. This reduction in the positive incidence angle, along with the improvement caused by the stator vane tip leakage flow, reduced flow separation on the suction surface of the rotor. At the design point, both the tip leakage flow of the stator vanes and the back swept blade angle caused flow separation on the pressure surface of the rotor. This resulted in additional blockage at the throat of the rotor reducing MFR and efficiency.

Nonadiabatic forces in ion-solid interactions:The initial stages of radiation damage

The Born-Oppenheimer approximation is the keystone for molecular dynamics simulations of radiation damage processes; however, actual materials response involves nonadiabatic energy exchange between nuclei and electrons. In this work, time dependent density functional theory is used to calculate the electronic excitations produced by energetic protons in Al. We study the influence of these electronic excitations on the interatomic forces and find that they differ substantially from the adiabatic case, revealing a nontrivial connection between electronic and nuclear stopping that is absent in the adiabatic case. These results unveil new effects in the early stages of radiation damage cascades.

Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

Introduction: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints.

Methods: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography.

Results: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p < 0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p < 0.005).

Conclusion: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.

A wavelet-based damage detection algorithm based on bridge acceleration response to a vehicle

Previous research based on theoretical simulations has shown the potential of the wavelet transform to detect damage in a beam by analysing the time-deflection response due to a constant moving load. However, its application to identify damage from the response of a bridge to a vehicle raises a number of questions. Firstly, it may be difficult to record the difference in the deflection signal between a healthy and a slightly damaged structure to the required level of accuracy and high scanning frequencies in the field. Secondly, the bridge is going to have a road profile and it will be loaded by a sprung vehicle and time-varying forces rather than a constant load. Therefore, an algorithm based on a plot of wavelet coefficients versus time to detect damage (a singularity in the plot) appears to be very sensitive to noise. This paper addresses these questions by: (a) using the acceleration signal, instead of the deflection signal, (b) employing a vehicle-bridge finite element interaction model, and (c) developing a novel wavelet-based approach using wavelet energy content at each bridge section which proves to be more sensitive to damage than a wavelet coefficient line plot at a given scale as employed by others.

Sperm DNA damage has a negative association with live birth rates after IVF.

Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.

Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.

PKB-mediated PHF20 phosphorylation on Ser291 is required for p53 function in DNA damage

PHD finger protein 20 (PHF20) is a transcription factor, which was originally identified in glioma patients. PHF20 appears to be a novel antigen in glioma, and has also termed glioma-expressed antigen 2. PHF20 is thought to contribute to the development of cancers, including glioblastoma, lung cancer, colon cancer and ovarian cancer. However, little is known about the function of PHF20 in various cancers. Here we report that PHF20 contains two consensus sites for protein kinase B (PKB) phosphorylation (RxRxxS/T). PKB can directly phosphorylate PHF20 on Ser291 in vitro and in vivo. It has been shown that PKB participates in the tumor suppressor p53 regulated gene expression program and has a direct effect on p21 regulation after DNA damage. UV-induced DNA damage results in accumulation of p53 and PKB activation. Interestingly, PKB-mediated PHF20 phosphorylation led to an inhibition of p53 induction following UV treatment, leading to the reduction of p21 transcriptional activity. Using anti PHF20 and anti pPKB (S473) antibodies, these events were mapped in various human cancer tissues. Taken together, these data suggest that PHF20 is a novel substrate for PKB and its phosphorylation by PKB plays an important role in tumorigenesis via regulating of p53 mediated signaling. © 2012 Elsevier Inc.

A progressive failure model for composite laminates subjected to low velocity impact damage

This paper presents a 3-D failure model for predicting the dynamic material response of composite laminates under impact loading. The formulation is based on the Continuum Damage Mechanics (CDM) approach and enables the control of the energy dissipation associated with each failure mode regardless of mesh refinement and fracture plane orientation. Internal thermodynamically irreversible damage variables were defined in order to quantify damage concentration associated with each possible failure mode and predict the gradual stiffness reduction during the impact damage process. The material model has been implemented into LS-DYNA explicit finite element code within solid elements and it has proven to be capable of reproducing experimental results with good accuracy in terms of static/dynamic responses, absorbed energy and extent of damage.