Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach

Immunoslot blot assays have been used for the analysis of many DNA adducts, but problems are frequently encountered in achieving reproducible results. Each step of the assay was examined systematically, and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimization was performed on both the malondialdehyde–deoxyguanosine (M1dG) adduct and the O6-carboxymethyl–deoxyguanosine (O6CMdG) adduct to demonstrate the applicability to other DNA adducts. Blood samples from the European Prospective Investigation on Cancer(EPIC) study (n = 162) were analyzed for M1dG adducts, and the data showed no correlation with adduct levels in other tissues, indicating that the EPIC blood samples were not useful for studying M1dG adducts. Blood samples from a processed meat versus vegetarian diet intervention (n = 6) were analyzed for O6CMdG, and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 107 nucleotides for the analysis of O6CMdG. Subsequentreanalysis for O6CMdG showed a weakly significant increase in O6CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted. 2010 Elsevier Inc. All rights reserved

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease

Neutrinos

229 pages229 pages229 pagesThe Proceedings of the 2011 workshop on Fundamental Physics at the Intensity Frontier. Science opportunities at the intensity frontier are identified and described in the areas of heavy quarks, charged leptons, neutrinos, proton decay, new light weakly-coupled particles, and nucleons, nuclei, and atoms

Digitise This! A Quick and Easy Remote Sensing Method to Monitor the Daily Extent of Dredge Plumes

Technological advancements in remote sensing and GIS have improved natural resource managers’ abilities to monitor large-scale disturbances. In a time where many processes are heading towards automation, this study has regressed to simple techniques to bridge a gap found in the advancement of technology. The near-daily monitoring of dredge plume extent is common practice using Moderate Resolution Imaging Spectroradiometer (MODIS) imagery and associated algorithms to predict the total suspended solids (TSS) concentration in the surface waters originating from floods and dredge plumes. Unfortunately, these methods cannot determine the difference between dredge plume and benthic features in shallow, clear water. This case study at Barrow Island, Western Australia, uses hand digitising to demonstrate the ability of human interpretation to determine this difference with a level of confidence and compares the method to contemporary TSS methods. Hand digitising was quick, cheap and required very little training of staff to complete. Results of ANOSIM R statistics show remote sensing derived TSS provided similar spatial results if they were thresholded to at least 3 mg L-1. However, remote sensing derived TSS consistently provided false-positive readings of shallow benthic features as Plume with a threshold up to TSS of 6 mg L-1, and began providing false-negatives (excluding actual plume) at a threshold as low as 4 mg L-1. Semi-automated processes that estimate plume concentration and distinguish between plumes and shallow benthic features without the arbitrary nature of human interpretation would be preferred as a plume monitoring method. However, at this stage, the hand digitising method is very useful and is more accurate at determining plume boundaries over shallow benthic features and is accessible to all levels of management with basic training

Opposite polarities of ENSO drive distinct patterns of coral bleaching potentials in the southeast Indian Ocean.

Episodic anomalously warm sea surface temperature (SST) extremes, or marine heatwaves (MHWs), amplify ocean warming effects and may lead to severe impacts on marine ecosystems. MHW-induced coral bleaching events have been observed frequently in recent decades in the southeast Indian Ocean (SEIO), a region traditionally regarded to have resilience to global warming. In this study, we assess the contribution of El Niño-Southern Oscillation (ENSO) to MHWs across the mostly understudied reefs in the SEIO. We find that in extended summer months, the MHWs at tropical and subtropical reefs (divided at ~20°S) are driven by opposite ENSO polarities: MHWs are more likely to occur at the tropical reefs during eastern Pacific El Niño, driven by enhanced solar radiation and weaker Australian Monsoon, some likely alleviated by positive Indian Ocean Dipole events, and at the subtropical reefs during central Pacific La Niña, mainly caused by increased horizontal heat transport, and in some cases reinforced by local air-sea interactions. Madden-Julian Oscillations (MJO) also modulate the MHW occurrences. Projected future increases in ENSO and MJO intensity with greenhouse warming will enhance thermal stress across the SEIO. Implementing forecasting systems of MHWs can be used to anticipate future coral bleaching patterns and prepare management responses

The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms

The natural exclusion of red deer from large boulder grazing refugia and the consequences for saxicolous bryophyte and lichen ecology

Large boulder grazing refugia permitted comparison of saxicolous bryophyte and lichen assemblages with those boulder tops accessible to red deer (Cervus elaphus) on a sporting estate in northwest Scotland. Plant succession was predicted to occur unchecked by grazing on the tops of these large boulders with cascading effects on bryophytes and lichens—assuming boulders had been in place over the same time period. Fifty pairs of boulders (one ≥2 m and the other accessible to red deer) were selected at random from various locations below north-facing crags. Percentage cover of each bryophyte and lichen species was estimated from three randomly placed quadrats on each boulder top. Due consideration was given to the influence of island biogeography theory in subsequent model simplification. Mean shrub cover and height, leaf-litter, bryophyte cover and bryophyte species richness were significantly higher within quadrats on large boulder tops that naturally excluded red deer. Lichen cover and lichen species richness were significantly higher on boulder tops accessible to red deer. Lichen cover was in a significant negative relationship with bryophyte cover, shrub cover and litter cover. Bryophyte cover showed a significant positive relationship with shrub height but there was an optimum shrub cover. Natural exclusion of red deer from the tops of large boulders has facilitated plant succession. The results suggest that grazing arrests the lithosere on boulder tops accessible to red deer at an early plagioclimax favouring saxicolous lichens. The results are relevant to situations where red deer might be excluded from boulder fields that hold lichen assemblages of conservation value