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

Coastal erosion reveals a potentially unique Oligocene and possible periglacial sequence at present-day sea level in Port Davey, remote South-West Tasmania

Cut-back of a sea-cliff at Hannant Inlet in remote South-West Tasmania has exposed Oligocene clays buried under Late Pleistocene “colluvium” from which abundant wood fragments protrude. The two units are separated by a transitional interval defined by mixed Oligocene and Pleistocene microfloras. Microfloras preserved in situ in the clay provide a link between floras in Tasmania and other Southern Hemisphere landmasses following onset of major glaciation in East Antarctica during the Eocene-Oligocene transition (c. 34 Ma). The Late Pleistocene “colluvium” preserves abundant fossil pollen of the shrub conifer genus Pherosphaera (al. Microstrobos). Assuming the parent plants had the same upper subalpine-alpine ecology as living Pherosphaera hookeriana, the microflora provides evidence for cold, wet conditions in the Port Davey lowlands during a low sea-level stand. The same data highlight the failure of Pherosphaera to regain its Pleistocene distribution during the Postglacial period. Our data are inconclusive whether Late Pleistocene conditions in Hannant Inlet were periglacial, i.e., the Oligocene sediments were turbated by freeze-thaw processes, or have been reworked by fluvial processes into the Pleistocene “colluvium”. Nevertheless, the inferred cold-climate is consistent with the former hypothesis. The sequence is sealed under cross-bedded coarse quartzite gravels of presumed Last Glacial Stage age

What was the vegetation in northwest Australia during the Paleogene, 66-23 million years ago?

Fossil pollen and spores preserved in drillcore from both the upper South Alligator River (SARV) in the Kakadu National Park, Northern Territory and the North-West Shelf, Western Australia provide the first record of plants and plant communities occupying the coast and adjacent hinterland in north-west Australia during the Paleogene 66 to 23million years ago. The palynologically-dominant woody taxon is Casuarinaceae, a family now comprising four genera of evergreen scleromorphic shrubs and trees native to Australia, New Guinea, South-east Asia and Pacific Islands. Rare taxa include genera now mostly restricted to temperate rainforest in New Guinea, New Caledonia, New Zealand, South-East Asia and/or Tasmania, e.g. Dacrydium, Phyllocladus and the Nothofagus subgenera Brassospora and Fuscospora. These appear to have existed in moist gorges on the Arnhem Land Plateau, Kakadu National Park. No evidence for Laurasian rainforest elements was found. The few taxa that have modern tropical affinities occur in Eocene or older sediments in Australia, e.g. Lygodium, Anacolosa, Elaeagnus, Malpighiaceae and Strasburgeriaceae. We conclude the wind-pollinated Oligocene to possibly Early Miocene vegetation in the upper SARV was Casuarinaceae sclerophyll forest or woodland growing under seasonally dry conditions and related to modern Allocasuarina/Casuarina formations. There are, however, strong floristic links to coastal communities growing under warm to hot, and seasonally to uniformly wet climates in north-west Australia during the Paleocene-Eocene.RSH’s contribution was supported by Australian Research Council Grant Number DP130104314 to J.G. Conran, F.A. McInerney and R.S. Hill

Deletion of human metapneumovirus M2-2 increases mutation frequency and attenuates growth in hamsters

<p>Abstract</p> <p>Background</p> <p>Human metapneumovirus (hMPV) infection can cause acute lower respiratory tract illness in infants, the immunocompromised, and the elderly. Currently there are no licensed preventative measures for hMPV infections. Using a variant of hMPV/NL/1/00 that does not require trypsin supplementation for growth in tissue culture, we deleted the M2-2 gene and evaluated the replication of rhMPV/ΔM2-2 virus <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p><it>In vitro </it>studies showed that the ablation of M2-2 increased the propensity for insertion of U nucleotides in poly-U tracts of the genomic RNA. In addition, viral transcription was up-regulated although the level of genomic RNA remained comparable to rhMPV. Thus, deletion of M2-2 alters the ratio between hMPV genome copies and transcripts. <it>In vivo</it>, rhMPV/ΔM2-2 was attenuated compared to rhMPV in the lungs and nasal turbinates of hamsters. Hamsters immunized with one dose of rhMPV/ΔM2-2 were protected from challenge with 10⁶PFU of wild type (<it>wt) </it>hMPV/NL/1/00.</p> <p>Conclusion</p> <p>Our results suggest that hMPV M2-2 alters regulation of transcription and influences the fidelity of the polymerase complex during viral genome replication. In the hamster model, rhMPVΔM2-2 is attenuated and protective suggesting that deletion of M2-2 may result in a potential live vaccine candidate. A more thorough knowledge of the hMPV polymerase complex and the role of M2-2 during hMPV replication are being studied as we develop a potential live hMPV vaccine candidate that lacks M2-2 expression.</p

Mercury enrichment in anthrosols and adjacent coastal sediments at a Classic Maya site, Marco Gonzalez, Belize

Elevated concentrations of total mercury (THg) are found in the surface soils and flanking wetland sediments at the Classic Maya coastal site of Marco Gonzalez, Belize. Significant concentrations (up to 1.3 µg·g−1 dry mass) of THg occur in leaf litter-rich soils, as well as in the artefact-rich anthrosol spread over the vegetated mound site of structures and occupation debris. The abundance and spatial pattern of major and trace elements measured in the surface soils indicate both site-scale controlling factors of topography, structures and vegetation on soil geochemistry as well as local highs in concentration compared with background, due to human activity. Geochemical stratigraphy of wetland sediment cores shows that a shift from carbonate-reef sediments to mangrove peat in the 13th century AD was attended by an input of allogenic (mineral) elements, including mercury. A THg concentration peak (0.8 μg·g−1) in brackish pool sediment is 210Pb-dated to 1960–1970 AD, but the incorporation of mercury in multiple cores adjacent to the site shows increasing mercury inputs to have occurred before, during Classic-period Maya occupation and following the sites abandonment. Analysis of element values from site-scale soil sampling, combined with results from off-site cores, provides a numerical framework upon which outlier values of THg and other element spatial patterns can be assessed, especially the spatial co-association of elements related to differences in soil–sediment matrices. Geochemical results from active soils developing from occupation deposits (anthrosols) and sediment cores open up questions concerning contemporary and past mercury accumulation at coastal Mayan sites, and the wider interaction of human and natural biogeochemical processes that occur in human-modified soils and coastal wetland sediments

Switching to second-line antiretroviral therapy in resource-limited settings: comparison of programmes with and without viral load monitoring.

In high-income countries, viral load is routinely measured to detect failure of antiretroviral therapy (ART) and guide switching to second-line ART. Viral load monitoring is not generally available in resource-limited settings. We examined switching from nonnucleoside reverse transcriptase inhibitor (NNRTI)-based first-line regimens to protease inhibitor-based regimens in Africa, South America and Asia

Marco Gonzalez, Ambergris Caye, Belize: a Geoarchaeological Record of Ground Raising Associated With Surface Soil Formation and the Presence of a Dark Earth

Marco Gonzalez, on the south-west end of the island of Ambergris Caye, Belize, has well-preserved Maya archaeological stratigraphy dating from Preclassic times (ca. 300 B.C.) to the Late Classic period (ca. A.D. 550/600 to 700/760). Although later occupations are recorded by house platforms and inhumations (Terminal Classic to Early Postclassic), and use of the site continued until the 16th century A.D., intact stratigraphy is rare in these cases owing to a greater degree of disturbance. Nonetheless, understanding site formation entails accounting for all processes, including disturbance. The site’s depositional sequence—as revealed through soil micromorphology and chemistry and detailed here—has yielded critical information in two spheres of research. As regards archaeology and the elucidation of Maya activities on the caye over time, soil micromorphology has contributed beyond measure to what we have been able to distinguish as material remains of cultural activity. Detailed descriptions of the nature of the material remains has in turn helped us to clarify or alter interpretations based on artefacts that have been identified or sediments characterised according to traditional recovery techniques. The other major sphere in which soil micromorphology and chemistry play a critical role is in assessment of the environmental impact of human activity, which enables us to construct and test hypotheses concerning how the site formed over time; what materials and elements contributed to the character of the sediments, especially in the formation of a specific Maya Dark Earth type that is developed from carbonate rich deposits; and how the modern surface soils acquired the appearance of a Dark Earth, but essentially differ from them. In terms of agricultural soil sustainability, the Marco Gonzalez surface soil is neo-formed by a woodland vegetation drawing upon the nutrients and constituents present in both the Dark Earth and underlying better preserved stratified deposits

The Marco Gonzalez Maya site, Ambergris Caye, Belize: assessing the impact of human activities by examining diachronic processes at the local scale

Research at the Maya archaeological site of Marco Gonzalez on Ambergris Caye in Belize is socio-ecological because human activities have been a factor in the formation and fluctuation of the local marine and terrestrial environments over time. The site is one of many on Belize's coast and cayes that exhibit anomalous vegetation and dark-coloured soils. These soils, although sought for cultivation, are not typical 'Amazonian Dark Earths' but instead are distinctive to the weathering of carbonate-rich anthropogenic deposits. We tentatively term these location-specific soils as Maya Dark Earths. Our research seeks to quantify the role of human activities in long-term environmental change and to develop strategies, specifically Life Cycle Assessment (LCA), that can be applied to environmental impact modelling today

Acute administration of dopaminergic drugs has differential effects on locomotion in larval zebrafish

Altered dopaminergic signaling causes behavioral changes in mammals. In general, dopaminergic receptor agonists increase locomotor activity, while antagonists decrease locomotor activity. In order to determine if zebrafish (a model organism becoming popular in pharmacology and toxicology) respond similarly, the acute effects of drugs known to target dopaminergic receptors in mammals were assessed in zebrafish larvae. Larvae were maintained in 96-well microtiter plates (1 larva/well). Non-lethal concentrations (0.2–50 µM) of dopaminergic agonists (apomorphine, SKF-38393, and quinpirole) and antagonists (butaclamol, SCH-23390, and haloperidol) were administered at 6 days post-fertilization (dpf). An initial experiment identified the time of peak effect of each drug (20–260 minutes post-dosing, depending on the drug). Locomotor activity was then assessed for 70 minutes in alternating light and dark at the time of peak effect for each drug to delineate dose-dependent effects. All drugs altered larval locomotion in a dose-dependent manner. Both the D1- and D2-like selective agonists (SKF-38393 and quinpirole, respectively) increased activity, while the selective antagonists (SCH-23390 and haloperidol, respectively) decreased activity. Both selective antagonists also blunted the response of the larvae to changes in lighting conditions at higher doses. The nonselective drugs had biphasic effects on locomotor activity: apomorphine increased activity at the low dose and at high doses, while butaclamol increased activity at low to intermediate doses, and decreased activity at high doses. This study demonstrates that (1) larval zebrafish locomotion can be altered by dopamine receptor agonists and antagonists, (2) receptor agonists and antagonists generally have opposite effects, and (3) drugs that target dopaminergic receptors in mammals appear, in general, to elicit similar locomotor responses in zebrafish larvae