DNA fiber assay for the analysis of DNA replication progression in human pluripotent stem cells

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell‐based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication‐fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside‐enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single‐molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters

A Note on Fluxes and Superpotentials in M-theory Compactifications on Manifolds of G_2 Holonomy

We consider the breaking of N=1 supersymmetry by non-zero G-flux when M-theory is compactified on a smooth manifold X of G_2 holonomy. Gukov has proposed a superpotential W to describe this breaking in the low-energy effective theory. We check this proposal by comparing the bosonic potential implied by W with the corresponding potential deduced from the eleven-dimensional supergravity action. One interesting aspect of this check is that, though W depends explicitly only on G-flux supported on X, W also describes the breaking of supersymmetry by G-flux transverse to X.Comment: 15 pages, harvmac, v2: reference adde

Pyridazine-bridged cationic diiridium complexes as potential dual-mode bioimaging probes

A novel diiridium complex [(N^C^N)2Ir(bis-N^C)Ir(N^C^N)2Cl]PF6 (N^C^N = 2-[3-tert-butyl-5-(pyridin-2-yl)phenyl]pyridine; bis-N^C = 3,6-bis(4-tert-butylphenyl)pyridazine) was designed, synthesised and characterised. The key feature of the complex is the bridging pyridazine ligand which brings two cyclometallated Ir(III) metal centres close together so that Cl also acts as a bridging ligand leading to a cationic complex. The ionic nature of the complex offers a possibility of improving solubility in water. The complex displays broad emission in the red region (λem = 520–720 nm, τ = 1.89 μs, Φem = 62% in degassed acetonitrile). Cellular assays by multiphoton (λex = 800 nm) and confocal (λex = 405 nm) microscopy demonstrate that the complex enters cells and localises to the mitochondria, demonstrating cell permeability. Further, an appreciable yield of singlet oxygen generation (ΦΔ = 0.45, direct method, by 1O2 NIR emission in air equilibrated acetonitrile) suggests a possible future use in photodynamic therapy. However, the complex has relatively high dark toxicity (LD50 = 4.46 μM), which will likely hinder its clinical application. Despite this toxicity, the broad emission spectrum of the complex and high emission yield observed suggest a possible future use of this class of compound in emission bioimaging. The presence of two heavy atoms also increases the scattering of electrons, supporting potential future applications as a dual fluorescence and electron microscopy probe

Transition metal complexes as photosensitisers in one- and two-photon photodynamic therapy

Photodynamic therapy (PDT) exploits light-activated compounds for therapeutic use. It relies on a photosensitiser (PS) that is inactive in the absence of light. When irradiated, the PS absorbs light and is promoted to a higher energy, “excited” state (PS ∗ ), which is either toxic to cells in itself or triggers formation of other species which are toxic to cells, and hence particular wavelengths of light can be used to induce light-dependent cell killing. In PDT occurring via the so-called type I and type II mechanisms, the PS ∗ engages in energy transfer to dioxygen present in cells and tissues. This process generates highly reactive singlet oxygen ( 1 O 2 ) and/or other reactive oxygen species (ROS), which in turn cause damage in the immediate vicinity of the irradiation and ultimately can lead to cell death. Whilst the main focus of research for the last 50 years has been on organic molecules or porphyrins as sensitisers, there is now emerging interest in extending the use to transition metal (TM) complexes, which can display intense absorptions in the visible region, and many also possess high two-photon absorption cross-sections, enabling two-photon excitation with NIR light. As with any other type of photosensitiser, the issues to consider whilst designing a TM complex as a photosensitiser include cell permeability, efficient absorption of NIR light for deeper penetration, preferential affinity to cancer cells over healthy cells, targeted intracellular localisation and lack of side effects. This review summarises recent developments involving photosensitisers containing Ru(II), Os(II), Pt, Ir(III), and Re(I) ions, and the approaches used to address the above requirements. Several remarkable recent advances made in this area, including the first clinical trial of a metal complex as a photosensitiser, indicate the bright future of this class of compounds in PDT

Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand

Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1–1 μM) by comparatively low dose of 405 nm light (3.6 J cm−2) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin

Together, yet still not equal? Sex integration in equestrian sport

Sex segregation is a core organising principle of most modern sports and is a key element in the marginalisation and subordination of girls and women in sport and beyond. In this article I explore the only Olympic-level sport which is not organised around sex segregation – equestrian sport – in order to consider the implications of sex integration for female participants. I draw on a study conducted on elite riders that found that although sex integration in equestrian sport does not lead to female participants being excluded from high-level competition, men continue to perform disproportionately well. This suggests that although sex integration may be an important step towards breaking down gender hierarchies in sport, without accompanying wider changes in gender norms and expectations, sex integration alone will not be enough to achieve greater gender equality in equestrian sport

Inclusive One Jet Production With Multiple Interactions in the Regge Limit of pQCD

DIS on a two nucleon system in the regge limit is considered. In this framework a review is given of a pQCD approach for the computation of the corrections to the inclusive one jet production cross section at finite number of colors and discuss the general results.Comment: 4 pages, latex, aicproc format, Contribution to the proceedings of "Diffraction 2008", 9-14 Sep. 2008, La Londe-les-Maures, Franc

A solution of a problem of Sophus Lie: Normal forms of 2-dim metrics admitting two projective vector fields

We give a complete list of normal forms for the 2-dimensional metrics that admit a transitive Lie pseudogroup of geodesic-preserving transformations and we show that these normal forms are mutually non-isometric. This solves a problem posed by Sophus Lie.Comment: This is an extended version of the paper that will appear in Math. Annalen. Some typos were corrected, references were updated, title was changed (as in the journal version). 31 page

Spin and energy transfer in nanocrystals without transport of charge

We describe a mechanism of spin transfer between individual quantum dots that does not require tunneling. Incident circularly-polarized photons create inter-band excitons with non-zero electron spin in the first quantum dot. When the quantum-dot pair is properly designed, this excitation can be transferred to the neighboring dot via the Coulomb interaction with either {\it conservation} or {\it flipping} of the electron spin. The second dot can radiate circularly-polarized photons at lower energy. Selection rules for spin transfer are determined by the resonant conditions and by the strong spin-orbit interaction in the valence band of nanocrystals. Coulomb-induced energy and spin transfer in pairs and chains of dots can become very efficient under resonant conditions. The electron can preserve its spin orientation even in randomly-oriented nanocrystals.Comment: 13 pages, 3 figure