Two-photon imaging using adaptive phase compensated ultrashort laser pulses.

An adaptive pulse shaper controlled by multiphoton intrapulse interference phase scanning (MIIPS) was used, together with a prism-pair, to measure and cancel high-order phase distortions introduced by a high-numerical-aperture objective and other dispersive elements of a two-photon laser-scanning microscope. The delivery of broad-bandwidth (approximately 100 nm), sub-12-fs pulses was confirmed by interferometric autocorrelation measurements at the focal plane. A comparison of two-photon imaging with transform-limited and second-order-dispersion compensated laser pulses of the same energy showed a 6-to-11-fold improvement in the two-photon excitation fluorescence signal when applied to cells and tissue, and up to a 19-fold improvement in the second harmonic generation signal from a rat tendon specimen.

In search of drug treatment for genetic defects in the DNA damage response: the example of ataxia-telangiectasia.

Most genetic disorders are severe diseases that cannot be treated. In the majority of them, enzyme and gene therapy can be significantly curtailed by technical difficulties and the nature of the physiological defects and affected tissues. A rational search for drug treatment for such diseases must be based on understanding the corresponding molecular defects. For example, in a disease stemming from a defective signaling pathway, a drug that can activate redundant pathways could be useful. Screening for such a drug would then depend on the availability of a laboratory assay that faithfully reflects the molecular defect in the corresponding disease, and a technology for applying the assay in a high throughput setup. Deficiencies in various components of the DNA damage response lead to genomic instability syndromes characterized by tissue degeneration, sensitivity to DNA damaging agents, and cancer predisposition. A typical example is ataxia-telangiectasia (A-T), caused by deficiency of the nuclear protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. ATM phosphorylates a multitude of substrates, each of which in turn modulates a branch of the damage response network. A certain redundancy among ATM and related proteins gives hope that activation of ATM-redundant activities might form a basis for drug treatment of A-T. This article describes a high throughput strategy for drug screening for A-T that is based on the above principles. A similar strategy can potentially be applied to drug screening for other genetic disorders.

Requirement of the MRN complex for ATM activation by DNA damage.

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.