Development and validation of 'AutoRIF': Software for the automated analysis of radiation-induced foci

Copyright @ 2012 McVean et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This article has been made available through the Brunel Open Access Publishing Fund.Background: The quantification of radiation-induced foci (RIF) to investigate the induction and subsequent repair of DNA double strands breaks is now commonplace. Over the last decade systems specific for the automatic quantification of RIF have been developed for this purpose, however to ask more mechanistic questions on the spatio-temporal aspects of RIF, an automated RIF analysis platform that also quantifies RIF size/volume and relative three-dimensional (3D) distribution of RIF within individual nuclei, is required. Results: A java-based image analysis system has been developed (AutoRIF) that quantifies the number, size/volume and relative nuclear locations of RIF within 3D nuclear volumes. Our approach identifies nuclei using the dynamic Otsu threshold and RIF by enhanced Laplacian filtering and maximum entropy thresholding steps and, has an application ‘batch optimisation’ process to ensure reproducible quantification of RIF. AutoRIF was validated by comparing output against manual quantification of the same 2D and 3D image stacks with results showing excellent concordance over a whole range of sample time points (and therefore range of total RIF/nucleus) after low-LET radiation exposure. Conclusions: This high-throughput automated RIF analysis system generates data with greater depth of information and reproducibility than that which can be achieved manually and may contribute toward the standardisation of RIF analysis. In particular, AutoRIF is a powerful tool for studying spatio-temporal relationships of RIF using a range of DNA damage response markers and can be run independently of other software, enabling most personal computers to perform image analysis. Future considerations for AutoRIF will likely include more complex algorithms that enable multiplex analysis for increasing combinations of cellular markers.This article is made available through the Brunel Open Access Publishing Fund

An assessment of minimum sequence copy thresholds for identifying and reducing the prevalence of artefacts in dietary metabarcoding data

1. Metabarcoding provides a powerful tool for investigating biodiversity and trophic interactions, but the high sensitivity of this methodology makes it vulnerable to errors, resulting in artefacts in the final data. Metabarcoding studies thus often utilise minimum sequence copy thresholds (MSCTs) to remove artefacts that remain in datasets; however, there is no consensus on best practice for the use of MSCTs. 2. To mitigate erroneous reporting of results and inconsistencies, this study discusses and provides guidance for best-practice filtering of metabarcoding data for the ascertainment of conservative and accurate data. Several of the most commonly used MSCTs were applied to example datasets of Eurasian otter Lutra lutra and cereal crop spider (Araneae: Linyphiidae and Lycosidae) diets. 3. Changes in both the method and threshold value considerably affected the resultant data. Of the MSCTs tested, it was concluded that the optimal method for the examples given combined a sample-based threshold with removal of maximum taxon contamination, providing stringent filtering of artefacts while retaining target data. 4. Choice of threshold value differed between datasets due to variation in artefact abundance and sequencing depth, thus studies should employ controls (mock communities, negative controls with no DNA and unused MID tag combinations) to select threshold values appropriate for each individual study

A vision-based fully automated approach to robust image cropping detection

The definition of valid and robust methodologies for assessing the authenticity of digital information is nowadays critical to contrast social manipulation through the media. A key research topic in multimedia forensics is the development of methods for detecting tampered content in large image collections without any human intervention. This paper introduces AMARCORD (Automatic Manhattan-scene AsymmetRically CrOpped imageRy Detector), a fully automated detector for exposing evidences of asymmetrical image cropping on Manhattan-World scenes. The proposed solution estimates and exploits the camera principal point, i.e., a physical feature extracted directly from the image content that is quite insensitive to image processing operations, such as compression and resizing, typical of social media platforms. Robust computer vision techniques are employed throughout, so as to cope with large sources of noise in the data and improve detection performance. The method leverages a novel metric based on robust statistics, and is also capable to decide autonomously whether the image at hand is tractable or not. The results of an extensive experimental evaluation covering several cropping scenarios demonstrate the effectiveness and robustness of our approac

Subjective Assessment of Image Compression Artefacts on Stereoscopic Display

Image and video quality are important to depict any pictorial information vividly and correctly. With the advancement of technology, we can produce high-quality images and can display those in advanced high-resolution displays. But as high-quality images continue to increase in size, transmitting these exceeds the limited bandwidth of display links. To cope, we need to compress the images but desire that the user cannot perceive any difference between the compressed and uncompressed images. In my thesis, psychophysical experiments with a flicker paradigm were undertaken to do a subjective assessment of the visibility of compression artefacts of two sets of images with two codecs viewed on a stereoscopic display. For one set of images the result shows that artefacts can be silenced in some stereo images relative to 2D while testing with the other set of images was inconclusive. This thesis documented evidence for silencing of artefacts in 3D displays. Other differences between stereoscopic and 2D presentation can be predicted but were not observed here (perhaps due to floor effects). Further large-scale subjective assessment with challenging images may help to get a concrete conclusion

A radar-based rainfall climatology of Great Britain and Ireland

The Met Office 1km radar-derived precipitation-rate composite over 8 years (2006–2013) is examined to evaluate whether it provides an accurate representation of annual-average precipitation over Great Britain and Ireland over long periods of time. The annual-average precipitation from the radar composite is comparable with gauge measurements, with an average error of +23mmyr−1 over Great Britain and Ireland, +29mmyr−1 (3%) over the United Kingdom and –781mmyr−1 (46%) over the Republic of Ireland. The radar-derived precipitation composite is useful over the United Kingdom including Northern Ireland, but not accurate over the Republic of Ireland, particularly in the south

Getting simultaneous red and near-infrared band data from a single digital camera for plant monitoring applications: theoretical and practical study

Multispectral images, including red and near-infrared bands, have proved efficient for vegetation-soil discrimination and agricultural monitoring in remote-sensing applications. However, they remain little used in ground-based and unmanned aerial vehicle (UAV) imagery, due to a limited availability of adequate 2D imaging devices. A methodology is proposed to obtain simultaneously the near-infrared and red bands from a standard single RGB camera, after having removed the near-infrared blocking filter inside. Its ability to provide satisfactory NDVI (normalised difference vegetation index) computation for vegetation and soil has been assessed through spectral simulations. Application in field conditions with Canon 500 D and Canon 350D cameras has then been considered, taking into account signal-noise and demosaicing concerns. The results obtained have proved the practical usability of this approach, opening new technical possibilities for crop monitoring and agricultural robotics

Flow cytometry in plant pathology: a case study on Pseudomonas cichorii

Flow cytometry (FCM) is a powerful and very versatile technique to measure cells in suspension. It is an indispensible method for routine diagnostics in the medical sector, but also for research purposes in very diverse fields of study. Despite its multiple and still increasing use in other sectors, FCM is scarcely used in plant pathology. In this thesis, we explored the possibilities of flow cytometry in plant pathology, focussing on viability and specific detection of the waterborne lettuce pathogen Pseudomonas cichorii. In a first phase we tested different fluorescent dyes and optimal instrument settings to stain, detect and count bacteria with FCM and determine their viability. In a next step, we wanted to develop a specific detection method for P. cichorii in irrigation water. As this pathogen can cause midrib rot on greenhouse-grown lettuce after a single overhead irrigation with water containing only 100 CFU ml-1, very sensitive detection was necessary. Moreover, P. cichorii is most found in rainwater, and this water often contains high bacterial backgrounds, as well as other organic and inorganic pollutants. Therefore we chose to develop a detection system based on immunomagnetic beads, which would allow specific capture and concentration of the target cells out of the water. We wanted to combine specific detection with viability assessment, in order to have a method that is also useful to research in vivo survival of P. cichorii and gain more insight into the epidemiology of the bacteria. The combination of immunomagnetic separation (IMS) and live/dead staining is not easy and has seldom been tried. We tested different bead systems and defined the most important factors influencing IMS and nonspecific staining. We obtained best results with the relatively large (2.6 µm) non-fluorescent Compel beads. After optimizing this bead system, we came to a method in which beads were identified based on scatter properties and bacteria based on fluorescence properties. Bead-bacteria complexes had both the large scatter of the beads and the high fluorescence of the live/dead stained bacteria. Combining those two conditions in a logical combination of gates allowed the exclusion of most noise and resulted in the sensitive enumeration of bead-bacteria complexes. This method was further evaluated on mixed cultures and larger volumes and finally tested on different irrigation waters from commercial lettuce greenhouses. Irrigation water proved to be a difficult and very variable matrix. Despite the extra sample pretreatment steps, we could not reliably detect P. cichorii cells below the infection threshold of 100 cells ml-1, except in one water type. The major problems we encountered were a too low recovery of P. cichorii, combined with a too high background remaining in the final samples. Besides the IMS method itself, of which the binding percentage and binding strength should be improved, both the bacteria and their matrix complicated detection. Their was a significant difference between some of the tested sampling dates and the analysis date had a significant effect on P. cichorii recovery: higher recovery was obtained in the same waters sampled in March, compared to the February samplings. Furthermore, recovery improved when a water sample was spiked and analysed after storage for at least a week. Also PCR recovery may be influenced by sampling date, but here recovery tended to be lower in spring samplings. The combination of low recovery and an unknown influence of water constitution on recovery, made that our IMS method is not (yet) suited as an alternative for the existing PCR detection of P. cichorii. However, when comparing the conventional real-time PCR detection of P. cichorii with our IMS-FCM method, or with IMS pretreatment followed by PCR analysis, conventional RT-PCR is by far the most expensive method. Not the PCR analysis itself, but the sample pretreatment and DNA extraction before PCR is laborious and has, besides very high labour cost, also high material costs. Although PCR will remain the most specific method, IMS and/or FCM could be brought to a comparable sensitivity and have the potential to become a more cost-effective alternative for sample pretreatment and/or PCR analysis. The fact that P. cichorii is a difficult bacterium to detect is not only due to the IMS/FCM methodology, its low infection threshold, or to the complexity of its natural environment. Also the extremely high sensitivity of these bacteria to mechanical stress complicated detection. Mechanical stress seems to induce rapid apoptosis and autolysis, making P. cichorii cells disappear for both FCM or PCR detection. Medium constitution, especially salt concentration and the presence of nutrients, has a big influence on survival. In the absence of H2O2 and presence of 1% LB, recovery percentages of more than 90% could be obtained, while in saline solution, less than 10% was recovered after centrifugation. Although the enigmatic behaviour of P. cichorii complicated our research, such a far-reaching effect of common lab practices on bacterial viability has never been reported before and may be of considerable importance for microbiological practices

Money spider dietary choice in pre‐ and post‐harvest cereal crops using metabarcoding

Money spiders (Linyphiidae) are an important component of conservation biological control in cereal crops, but they rely on alternative prey when pests are not abundant, such as between cropping cycles. To optimally benefit from these generalist predators, prey choice dynamics must first be understood. Money spiders and their locally available prey were collected from cereal crops 2 weeks pre‐ and post‐harvest. Spider gut DNA was amplified with two novel metabarcoding primer pairs designed for spider dietary analysis, and sequenced. The combined general and spider‐exclusion primers successfully identified prey from 15 families in the guts of the 46 linyphiid spiders screened, whilst avoiding amplification of Erigone spp. The primers show promise for application to the diets of other spider families such as Agelenidae and Pholcidae. Distinct invertebrate communities were identified pre‐ and post‐harvest, and changes in spider diet and, to a lesser extent, prey choice reflected this. Spiders were found to consume one another more than expected, indicating their propensity towards intraguild predation, but also consumed common pest families. Changes in spider prey choice may redress prey community changes to maintain a consistent dietary intake. Consistent provision of alternative prey via permanent refugia should be considered to sustain effective conservation biocontrol