Analysis of radiation-induced bystander effects using high content screening

When cells are exposed to (ionising) radiation there is a rapid phosphorylation of a minor nucleosomal histone protein, H2AX, at the sites where double stranded breaks (DSB) occur. This phosphorylation is one of the earliest events in the repair cascade and extends over several mega base pairs surrounding the break. Nowadays it is generally accepted that the formation of γH2AX functions as a signal enhancer. Using immuno histochemistry we can visualise this phosphorylation as foci in the nucleus, where each foci represents a DSB [1]. For our research we use normal human primary fibroblasts (NHDF’s) to study the so called radiation-induced bystander effects which refer to the responses induced in non-irradiated cells, when neighbouring cells are irradiated. Although the exact pathways of transmission are yet to be determined, studies have shown that gap junction-mediated transport and secretion of soluble extracellular factors play an important role [2]. To exclude variation we first tried synchronisation of the fibroblasts using nocodazole or aphidicolin. Our attempts did not produce the desired synchronisation level. In addition, recent reports doubt the effectiveness of these products in cell synchronisation [3]. To resolve this problem, we used high content screening of cells together with specific cell cycle markers. One of these markers is 5-bromo-2-deoxyuridine (BrdU). BrdU, a synthetic nucleoside, is an analogue of thymidine that can be incorporated in replicating cells and specifically label S-phases [4]. Cells are cultured on membrane inserts, with a pore size of 0,4µm allowing soluble factors to pass but preventing the cells to interchange. These cells are irradiated with different doses and subsequently placed together with NHDF that are grown on cover glasses (see figure 1). Depending on the objectives BrdU is added 20-40 minutes before fixation. We found a differential pattern for γH2AX that we could specifically link to the cell cycle. During the S phase γH2AX is significantly more induced than during other phases of the cell cycle (see figure 2). This is probably due to the increased vulnerability caused by the unwinding of DNA during replication. 1. ¬¬S.H.Macphail, J.P.Banath, T.Y.Yu, E.H.Chu, H.Lambur, P.L.Olive, Int.J.Radiat.Biol. 79 (2003) P. 351-358. 2. H.Yang, N.Asaad, K.D.Held, Oncogene 24 (2005) p. 2096-2103. 3. S.Cooper, G.Iyer, M.Tarquini, P.Bissett, Cell Tissue Res. 324 (2006) p.237-242. 4. R.T.O'Keefe, S.C.Henderson, D.L.Spector, J.Cell Biol. 116 (1992) p.1095-1110

Trade Promotion Authority (TPA): Frequently Asked Questions

Trade Promotion Authority (TPA), formerly called fast track, is the authority Congress has granted to the President for limited periods of time to negotiate reciprocal trade agreements. The authority lays out U.S. trade negotiating objectives, procedures for congressional-executive notification and consultation, and expedited legislative procedures under which bills implementing trade agreements negotiated by the executive branch are to be considered. The most recent authority was enacted in December 2002 and expired as of July 1, 2007. Legislation to reauthorize TPA has been introduced in the 113th Congress. The United States is engaged in several sets of trade agreement negotiations. The issue of TPA reauthorization has raised a number of questions regarding TPA itself and the pending legislation. This report addresses a number of those questions that are frequently asked, including: • What is trade promotion authority? • Is TPA necessary? • What are trade negotiating objectives and how are they reflected in TPA statutes? • What requirements does Congress impose on the President under TPA? • Does TPA affect congressional authority on trade policy

A learning community two years on: reflecting on successes and framing futures

This paper reports the results of a participatory action research (PAR) evaluation conducted with the members of the Granite Belt Learners Group in their rural 'learning community' in South East Queensland, and presents an action research and evaluation framework to guide the community on the next stage of its journey

Parkes-CDSCC telemetry array: Equipment design

A unique combination of Deep Space Network (DSN) and non-DSN facilities in Australia provided enhanced data return from the Voyager spacecraft as it encountered the planet Uranus. Many of the key elements are duplicated from Voyager's encounters with Jupiter and Saturn. Some are unique extensions of that technology