Biological Evaluation of DNA Biomarkers in a Chemically Defined and Site-Specific Manner

As described elsewhere in this Special Issue on biomarkers, much progress has been made in the detection of modified DNA within organisms at endogenous and exogenous levels of exposure to chemical species, including putative carcinogens and chemotherapeutic agents. Advances in the detection of damaged or unnatural bases have been able to provide correlations to support or refute hypotheses between the level of exposure to oxidative, alkylative, and other stresses, and the resulting DNA damage (lesion formation). However, such stresses can form a plethora of modified nucleobases, and it is therefore difficult to determine the individual contribution of a particular modification to alter a cell’s genetic fate, as measured in the form of toxicity by stalled replication past the damage, by subsequent mutation, and by lesion repair. Chemical incorporation of a modification at a specific site within a vector (site-specific mutagenesis) has been a useful tool to deconvolute what types of damage quantified in biologically relevant systems may lead to toxicity and/or mutagenicity, thereby allowing researchers to focus on the most relevant biomarkers that may impact human health. Here, we will review a sampling of the DNA modifications that have been studied by shuttle vector technique

Triggering necroptosis in cisplatin and IAP antagonist-resistant ovarian carcinoma.

Ovarian cancer patients are typically treated with carboplatin and paclitaxel, but suffer a high rate of relapse with recalcitrant disease. This challenge has fostered the development of novel approaches to treatment, including antagonists of the 'inhibitor of apoptosis proteins' (IAPs), also called SMAC mimetics, as apoptosis-inducing agents whose action is opposed by caspase inhibitors. Surprisingly, IAP antagonist plus caspase inhibitor (IZ) treatment selectively induced a tumor necrosis factor-α (TNFα)-dependent death among several apoptosis-resistant cell lines and patient xenografts. The induction of necroptosis was common in ovarian cancer, with expression of catalytically active receptor-interacting protein kinase-3 (RIPK3) necessary for death, and in fact sufficient to compromise survival of RIPK3-negative, necroptosis-resistant ovarian cancer cells. The formation of a necrosome-like complex with a second critical effector, receptor-interacting serine-threonine kinase-1 (RIPK1), was observed. RIPK1, RIPK3 and TNFα were required for the induction of death, as agents that inhibit the function of any of these targets prevented cell death. Abundant RIPK3 transcript is common in serous ovarian cancers, suggesting that further evaluation and targeting of this RIPK3-dependent pathway may be of clinical benefit

Amine-Gold Linked Single-Molecule Junctions: Experiment and Theory

The measured conductance distribution for single molecule benzenediamine-gold junctions, based on 59,000 individual conductance traces recorded while breaking a gold point contact in solution, has a clear peak at 0.0064 G$_{0}$ with a width of $\pm$ 40%. Conductance calculations based on density functional theory (DFT) for 15 distinct junction geometries show a similar spread. Differences in local structure have a limited influence on conductance because the amine-Au bonding motif is well-defined and flexible. The average calculated conductance (0.046 G$_{0}$) is seven times larger than experiment, suggesting the importance of many-electron corrections beyond DFT

Correlation of interfacial bonding mechanism and equilibrium conductance of molecular junctions

We report theoretical investigations on the role of interfacial bonding mechanism and its resulting structures to quantum transport in molecular wires. Two bonding mechanisms for the Au-S bond in an Au(111)/1,4-benzenedithiol(BDT)/Au(111) junction were identified by ab initio calculation, confirmed by a recent experiment, which, we showed, critically control charge conduction. It was found, for Au/ BDT/Au junctions, the hydrogen atom, bound by a dative bond to the Sulfur, is energetically non-dissociative after the interface formation. The calculated conductance and junction breakdown forces of H-non-dissociative Au/BDT/Au devices are consistent with the experimental values, while the H-dissociated devices, with the interface governed by typical covalent bonding, give conductance more than an order of magnitude larger. By examining the scattering states that traverse the junctions, we have revealed that mechanical and electric properties of a junction have strong correlation with the bonding configuration. This work clearly demonstrates that the interfacial details, rather than previously believed many-body effects, is of vital importance for correctly predicting equilibrium conductance of molecular junctions; and manifests that the interfacial contact must be carefully understood for investigating quantum transport properties of molecular nanoelectronics.Comment: 18 pages, 6 figures, 2 tables, to be appeared in Frontiers of Physics 9(6), 780 (2014

Green function techniques in the treatment of quantum transport at the molecular scale

The theoretical investigation of charge (and spin) transport at nanometer length scales requires the use of advanced and powerful techniques able to deal with the dynamical properties of the relevant physical systems, to explicitly include out-of-equilibrium situations typical for electrical/heat transport as well as to take into account interaction effects in a systematic way. Equilibrium Green function techniques and their extension to non-equilibrium situations via the Keldysh formalism build one of the pillars of current state-of-the-art approaches to quantum transport which have been implemented in both model Hamiltonian formulations and first-principle methodologies. We offer a tutorial overview of the applications of Green functions to deal with some fundamental aspects of charge transport at the nanoscale, mainly focusing on applications to model Hamiltonian formulations.Comment: Tutorial review, LaTeX, 129 pages, 41 figures, 300 references, submitted to Springer series "Lecture Notes in Physics

On the stability of peptide nucleic acid duplexes in the presence of organic solvents

Nucleic acid double helices are stabilized by hydrogen bonding and stacking forces (a combination of hydrophobic, dispersive and electrostatic forces) of the base pairs in the helix. One would predict the hydrogen bonding contributions to increase and the stacking contributions to decrease as the water activity in the medium decreases. Study of nucleobase paired duplexes in the absence of water and ultimately in pure aprotic, non-polar organic solvents is not possible with natural phosphodiester nucleic acids due to the ionic phosphate groups and the associated cations, but could be possible with non-ionic nucleic acid analogues or mimics such as peptide nucleic acids. We now report that peptide nucleic acid (PNA) (in contrast to DNA) duplexes show almost unaffected stability in up to 70% dimethylformamide (DMF) or dioxane, and extrapolation of the data to conditions of 100% organic solvents indicates only minor (or no) destabilization of the PNA duplexes. Our data indicate that stacking forces contribute little if at all to the duplex stability under these conditions. The differences in behaviour between the PNA and the DNA duplexes are attributed to the differences in hydration and counter ion release rather than to the differences in nucleobase interaction. These results support the possibility of having stable nucleobase paired double helices in organic solvents

Early inflammation precedes cardiac fibrosis and heart failure in desmoglein 2 murine model of arrhythmogenic cardiomyopathy.

The study of a desmoglein 2 murine model of arrhythmogenic cardiomyopathy revealed cardiac inflammation as a key early event leading to fibrosis. Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure due to abnormalities in the cardiac desmosome. We examined how loss of desmoglein 2 (Dsg2) in the young murine heart leads to development of AC. Apoptosis was an early cellular phenotype, and RNA sequencing analysis revealed early activation of inflammatory-associated pathways in Dsg2-null (Dsg2-/-) hearts at postnatal day 14 (2 weeks) that were absent in the fibrotic heart of adult mice (10 weeks). This included upregulation of iRhom2/ADAM17 and its associated pro-inflammatory cytokines and receptors such as TNFα, IL6R and IL-6. Furthermore, genes linked to specific macrophage populations were also upregulated. This suggests cardiomyocyte stress triggers an early immune response to clear apoptotic cells allowing tissue remodelling later on in the fibrotic heart. Our analysis at the early disease stage suggests cardiac inflammation is an important response and may be one of the mechanisms responsible for AC disease progression

SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity

Plant defense responses need to be tightly regulated to prevent auto-immunity, which is detrimental to growth and development. To identify negative regulators of Resistance (R) protein-mediated resistance, we screened for mutants with constitutive defense responses in the npr1-1 background. Map-based cloning revealed that one of the mutant genes encodes a conserved TPR domain-containing protein previously known as SRFR1 (SUPPRESSOR OF rps4-RLD). The constitutive defense responses in the srfr1 mutants in Col-0 background are suppressed by mutations in SNC1, which encodes a TIR-NB-LRR (Toll Interleukin1 Receptor-Nucleotide Binding-Leu-Rich Repeat) R protein. Yeast two-hybrid screens identified SGT1a and SGT1b as interacting proteins of SRFR1. The interactions between SGT1 and SRFR1 were further confirmed by co-immunoprecipitation analysis. In srfr1 mutants, levels of multiple NB-LRR R proteins including SNC1, RPS2 and RPS4 are increased. Increased accumulation of SNC1 is also observed in the sgt1b mutant. Our data suggest that SRFR1 functions together with SGT1 to negatively regulate R protein accumulation, which is required for preventing auto-activation of plant immunity