Characterization of X-ray Dose in Murine Animals Using microCT,a New Low-Dose Detector and nanoDot Dosimeters

Background

Dose continues to be an area of concern in preclinical imaging studies, especially for those imaging disease progression in longitudinal studies. To our knowledge, this work is the first to characterize and assess dose from the Inveon CT imaging platform using nanoDot dosimeters. This work is also the first to characterize a new low-dose configuration available for this platform.

Methodology/Principal Findings

nanoDot dosimeters from Landauer, Inc. were surgically implanted into 15 wild type mice. Two nanoDots were placed in each animal: one just under the skin behind the spine and the other located centrally within the abdomen. A manufacturer-recommended CT protocol was created with 1 projection per degree of rotation acquired over 360 degrees. For best comparison of the low dose and standard configurations, noise characteristics of the reconstructed images were used to match the acquisition protocol parameters. Results for all dose measurements showed the average dose delivered to the abdomen to be 13.8 cGy±0.74 and 0.97 cGy±0.05 for standard and low dose configurations respectively. Skin measurements of dose averaged 15.99 cGy±0.72 and 1.18 cGy±0.06. For both groups, the standard deviation to mean was less than 5.6%. The maximum dose received for the abdomen was 15.12 cGy and 0.97 cGy while the maximum dose for the skin was 17.3 cGy and 1.32 cGy. Control dosimeters were used for each group to validate that no unwanted additional radiation was present to bias the results.

Conclusions/Significance

This study shows that the Inveon CT platform is suitable for imaging mice both for single and longitudinal studies. Use of low-dose detector hardware results in significant reductions in dose to subjects with a >12x (90%) reduction in delivered dose. Installation of this hardware on another in vivo microCT platform resulted in dose reductions of over 9x (89%).     

Radar Tracking and Motion-Sensitive Cameras on Flowers Reveal the Development of Pollinator Multi-Destination Routes over Large Spatial Scales

Central place foragers, such as pollinating bees, typically develop circuits (traplines) to visit multiple foraging sites in a manner that minimizes overall travel distance. Despite being taxonomically widespread, these routing behaviours remain poorly understood due to the difficulty of tracking the foraging history of animals in the wild. Here we examine how bumblebees (Bombus terrestris) develop and optimise traplines over large spatial scales by setting up an array of five artificial flowers arranged in a regular pentagon (50 m side length) and fitted with motion-sensitive video cameras to determine the sequence of visitation. Stable traplines that linked together all the flowers in an optimal sequence were typically established after a bee made 26 foraging bouts, during which time only about 20 of the 120 possible routes were tried. Radar tracking of selected flights revealed a dramatic decrease by 80% (ca. 1500 m) of the total travel distance between the first and the last foraging bout. When a flower was removed and replaced by a more distant one, bees engaged in localised search flights, a strategy that can facilitate the discovery of a new flower and its integration into a novel optimal trapline. Based on these observations, we developed and tested an iterative improvement heuristic to capture how bees could learn and refine their routes each time a shorter route is found. Our findings suggest that complex dynamic routing problems can be solved by small-brained animals using simple learning heuristics, without the need for a cognitive map.     

Analysis of Mitochondrial Function and Localisation during Human Embryonic Stem Cell Differentiation In Vitro

Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.     

Low temperature effects on leaf physiology and survivorship in the C3 and C4 subspecies of Alloteropsis semialata

The species richness of C(4) grasses is strongly correlated with temperature, with C(4) species dominating subtropical ecosystems and C(3) types predominating in cooler climates. Here, the effects of low temperatures on C(4) and C(3) grasses are compared, controlling for phylogenetic effects by using Alloteropsis semialata, a unique species with C(4) and C(3) subspecies. Controlled environment and common garden experiments tested the hypotheses that: (i) photosynthesis and growth are greater in the C(4) than the C(3) subspecies at high temperatures, but this advantage is reversed below 20 degrees C; and (ii) chilling-induced photoinhibition and light-mediated freezing injury of leaves occur at higher temperature thresholds in the C(4) than the C(3) plants. Measurements of leaf growth and photosynthesis showed the expected advantages of the C(4) pathway over the C(3) type at high temperatures. These declined with temperature, but were not completely lost until 15 degrees C, and there was no evidence of a reversal to give a C(3) advantage. Chronic chilling (5-15 degrees C) or acute freezing events induced a comparable degree of photodamage in illuminated leaves of both subspecies. Similarly, freezing caused high rates of mortality in the unhardened leaves of both subtypes. However, a 2-week chilling treatment prior to these freezing events halved injury in the C(3) but not the C(4) subspecies, suggesting that C(4) leaves lacked the capacity for cold acclimation. These results therefore suggest that C(3) members of this subtropical species may gain an advantage over their C(4) counterparts at low temperatures via protection from freezing injury rather than higher photosynthetic rates.     

Consequences of C4 photosynthesis for the partitioning of growth: a test using C3 and C4 subspecies of Alloteropsis semialata under nitrogen-limitation

C(4) plants dominate the world's subtropical grasslands, but investigations of their ecology typically focus on climatic variation, ignoring correlated changes in soil nutrient concentration. The hypothesis that higher photosynthetic nitrogen use efficiency (PNUE) in C(4) than in C(3) species allows greater flexibility in the partitioning of growth, especially under nutrient-deficient conditions, is tested here. Our experiment applied three levels of N supply to the subtropical grass Alloteropsis semialata, a unique model system with C(3) and C(4) subspecies. Photosynthesis was significantly higher for the same investment of leaf N in the C(4) than C(3) subspecies, and was unaffected by N treatments. The C(4) plants produced more biomass than the C(3) plants at high N levels, diverting a greater fraction of growth into inflorescences and corms, but less into roots and leaves. However, N-limitation of biomass production caused size-dependent shifts in the partitioning of growth. Root production was higher in small than large plants, and associated with decreasing leaf biomass in the C(3), and inflorescence production in the C(4) plants. Higher PNUE in the C(4) than C(3) subspecies was therefore linked with greater investment in sexual reproduction and storage, and the avoidance of N-limitations on leaf growth, suggesting advantages of the C(4) pathway in disturbed and infertile ecosystems.     

Seasonal differences in photosynthesis between the C3 and C4 subspecies of Alloteropsis semialata are offset by frost and drought

The regional abundance of C₄ grasses is strongly controlled by temperature, however, the role of precipitation is less clear. Progress in elucidating the direct effects of photosynthetic pathway on these climate relationships is hindered by the significant genetic divergence between major C₃ and C₄ grass lineages. We addressed this problem by examining seasonal climate responses of photosynthesis in Alloteropsis semialata , a unique grass species with both C₃ and C₄ subspecies. Experimental manipulation of rainfall in a common garden in South Africa tested the hypotheses that: (1) photosynthesis is greater in the C₄ than C₃ subspecies under high summer temperatures, but this pattern is reversed at low winter temperatures; and (2) the photosynthetic advantage of C₄ plants is enhanced during drought events. Measurements of leaf gas exchange over 2 years showed a significant photosynthetic advantage for the C₄ subspecies under irrigated conditions from spring through autumn. However, the C₄ leaves were killed by winter frost, while photosynthesis continued in the C₃ plants. Unexpectedly, the C₄ subspecies also lost its photosynthetic advantage during natural drought events, despite greater water-use efficiency under irrigated conditions. This study highlights previously unrecognized roles for climatic extremes in determining the ecological success of C₃ and C₄ grasses.     

Possible role of pectin-containing mucilage and dew in repairing embryo DNA of seeds adapted to desert conditions

BACKGROUND AND AIMS: Repair of damage to DNA of seed embryos sustained during long periods of quiescence under dry desert conditions is important for subsequent germination. The possibility that repair of embryo DNA can be facilitated by small amounts of water derived from dew temporarily captured at night by pectinaceous surface pellicles was tested. These pellicles are secreted during early seed development and form mucilage when hydrated. METHODS: Seeds of Artemisia sphaerocephala and Artemisia ordosica were collected from a sandy desert. Their embryos were damaged by gamma radiation to induce a standard level of DNA damage. The treated seeds were then exposed to nocturnal dew deposition on the surface of soil in the Negev desert highlands. The pellicles were removed from some seeds and left intact on others to test the ability of mucilage to support repair of the damaged DNA when night-time humidity and temperature favoured dew formation. Repair was assessed from fragmentation patterns of extracted DNA on agarose gels. KEY RESULTS: For A. sphaerocephala, which has thick seed pellicles, DNA repair occurred in seeds with intact pellicles after 50 min of cumulative night dew formation, but not in seeds from which the pellicles had been removed. For A. ordosica, which has thin seed pellicles, DNA repair took at least 510 min of cumulative night dewing to achieve partial recovery of DNA integrity. The mucilage has the ability to rehydrate after daytime dehydration. CONCLUSIONS: The ability of seeds to develop a mucilaginous layer when wetted by night-time dew, and to repair their DNA under these conditions, appear to be mechanisms that help maintain seed viability under harsh desert conditions.     

Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes

Background  

Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining.