Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were exactly identical; (3) the power of the Comet assay to detect a low level of genotoxic potential can be elevated to a level higher than that of MN test by using DNA resynthesis inhibitors, such as araC and HU

Age-related mitochondrial DNA depletion and the impact on pancreatic beta cell function

Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes

Magnetic Ordering in V-Layers of the Superconducting System of Sr2VFeAsO3

Results of transport, magnetic, thermal, and 75As-NMR measurements are presented for superconducting Sr2VFeAsO3 with an alternating stack of FeAs and perovskite-like block layers. Although apparent anomalies in magnetic and thermal properties have been observed at ~150 K, no anomaly in transport behaviors has been observed at around the same temperature. These results indicate that V ions in the Sr2VO3-block layers have localized magnetic moments and that V-electrons do not contribute to the Fermi surface. The electronic characteristics of Sr2VFeAsO3 are considered to be common to those of other superconducting systems with Fe-pnictogen layers.Comment: 4 pages, 4 figures, To appear in JPSJ 79 (2010) 12371

Study of Ni-doping Effect of Specific Heat and Transport Properties for LaFe1-yNiyAsO0.89F0.11

Specific heats and transport quantities of the LaFe1-yNiyAsO0.89F0.11 system have been measured, and the results are discussed together with those reported previously by our group mainly for LaFe1-yCoyAsO0.89F0.11 and LaFeAsO0.89-xF0.11+x systems. The y dependence of the electronic specific heat coefficient gamma can basically be understood by using the rigid-band picture, where Ni ions provide 2 electrons to the host conduction bands and behave as nonmagnetic impurities. The superconducting transition temperature Tc of LaFe1-yNiyAsO0.89F0.11 becomes zero, as the carrier density p (=2y+0.11) doped to LaFeAsO reaches its critical value p_c_ ~0.2. This p_c_ value of ~0.2 is commonly observed for LaFe1-yCoyAsO0.89F0.11 and LaFeAsO0.89-xF0.11+x systems, in which the relations p = x+0.11 and p = y+0.11 hold, respectively. As we pointed out previously, the critical value corresponds to the disappearance of the hole-Fermi surface. These results indicate that the carrier number solely determines the Tc value. We have not observed appreciable effects of pair breaking, which originates from the nonmagnetic impurity scattering of conduction electrons and strongly suppresses T_c_ values of systems with sign-reversing of the order parameter over the Fermi surface(s). On the basis of the results, the so-called s_+-_ symmetry of the order parameter with the sign-reversing is excluded.Comment: 4 pages, 7 figures, submitted to J. Phys. Soc. Jpn, (modified version

Exploring the source of TYLCV resistance in Nicotiana benthamiana

Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most devastating pathogens of tomato, worldwide. It is vectored by the globally prevalent whitefly, Bemisia tabaci, and is asymptomatic in a wide range of plant species that act as a virus reservoir. The most successful crop protection for tomato in the field has been from resistance genes, of which five loci have been introgressed fromwild relatives. Of these, the Ty-1/Ty-3 locus, which encodes an RNA-dependent RNA polymerase 3 (RDR3), has been the most effective. Nevertheless, several TYLCV strains that break this resistance are beginning to emerge, increasing the need for new sources of resistance. Here we use segregation analysis and CRISPR-mediated gene dysfunctionalisation to dissect the differential response of two isolates of Nicotiana benthamiana to TYLCV infection. Our study indicates the presence of a novel non-RDR3, but yet to be identified, TYLCV resistance gene in a wild accession of N. benthamiana. This gene has the potential to be incorporated into tomatoes

Benign giant mediastinal schwannoma presenting as cardiac tamponade in a woman: a case report

<p>Abstract</p> <p>Introduction</p> <p>Mediastinal schwannomas are typically benign and asymptomatic, and generally present no immediate risks. We encountered a rare case of a giant benign posterior mediastinal schwannoma, complicated by life-threatening cardiac tamponade.</p> <p>Case presentation</p> <p>We report the case of a 72-year-old Japanese woman, who presented with cardiogenic shock. Computed tomography of the chest revealed a posterior mediastinal mass 150 cm in diameter, with pericardial effusion. The cardiac tamponade was treated with prompt pericardial fluid drainage. A biopsy was taken from the mass, and after histological examination, it was diagnosed as a benign schwannoma, a well-encapsulated non-infiltrating tumor, originating from the intrathoracic vagus nerve. It was successfully excised, restoring normal cardiac function.</p> <p>Conclusion</p> <p>Our case suggests that giant mediastinal schwannomas, although generally benign and asymptomatic, should be excised upon discovery to prevent the development of life-threatening cardiopulmonary complications.</p

Feedback between p21 and reactive oxygen production is necessary for cell senescence

The sustained activation of CDKN1A (p21/Waf1/Cip1) by a DNA damage response induces mitochondrial dysfunction and reactive oxygen species (ROS) production via signalling through CDKN1A-GADD45A-MAPK14- GRB2-TGFBR2-TGFbeta in senescing primary human and mouse cells in vitro and in vivo.Enhanced ROS production in senescing cells generates additional DNA damage. Although this damage is repairable and transient, it elevates the average levels of DNA damage response permanently, thus forming a positive feedback loop.This loop is necessary and sufficient to maintain the stability of growth arrest until a ‘point of no return' is reached during establishment of senescence