In recent years, a number of approaches have been applied to the problem of deformable registration validation. However, the challenge of assessing a commercial deformable registration system – in particular, an automatic registration system in which the deformable transformation is not readily accessible – has not been addressed. Using a collection of novel and established methods, we have developed a comprehensive, four‐component protocol for the validation of automatic deformable image registration systems over a range of IGRT applications. The protocol, which was applied to the Reveal‐MVS system, initially consists of a phantom study for determination of the system's general tendencies, while relative comparison of different registration settings is achieved through postregistration similarity measure evaluation. Synthetic transformations and contour‐based metrics are used for absolute verification of the system's intra‐modality and inter‐modality capabilities, respectively. Results suggest that the commercial system is more apt to account for global deformations than local variations when performing deformable image registration. Although the protocol was used to assess the capabilities of the Reveal‐MVS system, it can readily be applied to other commercial systems. The protocol is by no means static or definitive, and can be further expanded to investigate other potential deformable registration applications.PACS numbers: 87.19.xj, 87.56.Da, 87.57.nj Varied objectives in these and other potential applications, compounded by the fact that often the lack of a gold standard makes true assessment impossible, make validation of deformable image registration algorithms very difficult.In recent years, deformable image registration has become a very important component in a number of image‐guided radiation therapy (IGRT) and adaptive radiation therapy (ART) protocols. It has been applied to problems such as autosegmentation,,2 fo It has become common practice to compare algorithms by evaluating similarity measures such as the sum of square intensity differences (SSD), correlation coefficient (CC), and mutual information (MI) upon completion of image registration. It is assumed that there is a direct correlation between enhanced similarity values and registration accuracy. Although these methods may be sufficient for relative comparison, they provide little information on the absolute accuracy of registration.A number of approaches have been applied to the problem of deformable registration validation. Although inherently limited, visual assessment has been used in combination with other methods to qualitatively validate registration results.,,, It and pelvic phantoms with varying levels of design complexity have been manufacturing and applied to deformable registration validation. Xiong et al. used inflatable balloons with radio‐opaque markers to assess their algorithm for bladder deformation in CT images acquired prior to high dose rate (HDR) vaginal cuff brachytherapy. A gel‐balloon phantom containing plastic beads molded to the gel that propagate based on levels of balloon inflation was used by Lu et al. to validate their free‐form deformable registration program. Although practical, authors have argued that phantoms have limited value in validating deformable registration algorithms because they cannot fully assess the impact of anatomical variations on the algorithm's performance.Various deformable thoracic,,20 applied a harmonic function to a 2D pelvic CT image, while Lau et al. warped 2D T1‐weighted MR brain images with five types of synthetic mathematical transformations. Intuitively, the closer the simulated transformation resembles clinical anatomical variations, the more relevant this method is for validation. As such, this procedure is enhanced by using a second independent deformable registration algorithm to generate the known transformation for validation of the algorithm in question. Fiducial based thin‐plate spline registration has been used to warp thoracic CT and abdominal CT images for synthetic transformation based deformable image registration assessment. The major downside of using simulated warped images for registration validation is that the method is not applicable for registration of images of differing modalities.A useful quantitative method of assessing registration is to apply a known simulated mathematical transformation to a patient image, generating a synthetic warped image. Registration of the original and warped images allows for direct comparison of the true displacement field with that generated by registration. Lu et al.15 app Brock et al. also identified landmarks on thoracic and abdominal MR images when validating a finite element model (FEM)‐based deformable registration system. Various measures of volume overlap evaluated using radiation oncologist delineated prostate contours have been used for validation of deformable registration of T2‐weighted pelvic images with and without inflated endorectal coils. Volumetric‐based methods have also been used when validating deformable registration of pelvic CT and head and neck CT images.The most commonly employed and perhaps the most accurate method of quantitatively validating deformable registration is to compare the position of anatomical landmarks or the overlap of regions of interest (ROI) in registered images. Measuring distances between manually identified points such as vascular and bronchial bifurcations is a common approach to validating thoracic CT deformable registration.,,,, The majority of the aforementioned references share a commonality in that the authors develop a novel algorithm that requires some procedure to assess its capabilities. However, the problem of assessing a commercial black box deformable registration system prior to clinical implementation has not been addressed – in particular, an automatic registration system in which the deformable transformation is not readily accessible. Proper quality assurance and acceptance testing of such systems is necessary to identify subtle limitations for various clinical applications. Using a collection of novel and established methods, we have developed a comprehensive protocol for the validation of automatic deformable image registration systems over a range of IGRT applications. This work predominantly focuses on anatomical images in which one‐to‐one correspondence is desired between registered images. Deformable registration involving functional images will be addressed briefly. The protocol has been applied to the Reveal‐MVS Fusion Workstation .As is the case for other technological advancements implemented in radiation therapy, the extent of the capabilities and limitations of a commercial system must be understood prior to its use in any IGRT deformable registration application. Given the wide range of anatomical variations across the population, different imaging modalities and scanning protocols and the number of potential applications of deformable registration in IGRT, a single standardized algorithm is probably insufficient. As such, an automatic commercial deformable registration system would require multiple algorithms or, at the very least, multiple registration settings in order for it to be relevant over a spectrum of applications. With this in mind, our protocol consists of four separate components: a phantom study to determine the system's general tendencies, relative validation of different registration settings, absolute verification of the system's intra‐modality and inter‐modality deformable registration tools. All patient images were retrospectively incorporated into this study with local research ethics board consent. The terms source and target will be used to describe registrations throughout this paper, using the standard notation that the source image is registered to the target image. In all deformable registrations on the Reveal‐MVS system, images were initially aligned using the system's automatic rigid registration tools. All analysis was performed using 3D image sets. The automatic deformable registration and PET/CT motion correction tools have user defined settings for stiffness , speed , and refinement , giving rise to 36 permutations of setting combinations. Although the methods are not open source, some information about the settings is provided with the software's documentation. If stiffness is set to ‘None’, no constraints are set to the registration, whereas a stiffness of ‘Stiff’ typically limits displacements to less than 0.5 cm. ‘Soft’ and ‘Medium’ settings typically limit displacements to 5 cm and 2 cm, respectively. The speed setting sets the approximate registration time to less than a minute, 3–4 minutes or in excess of 10 minutes, for ‘Fast’, ‘Medium’ and ‘Slow’ settings, respectively. The refinement setting determines the level of localization at which the deformations are applied to images. For example, the ‘Coarse’ setting results in deformations applied at a resolution of approximately 5 cm. Resolutions of 2 cm and 0.5 cm are typical of the ‘Medium’ and ‘Fine’ settings, respectively. The deformation function generated after registration of the source and target images is not accessible, although it can be saved and subsequently loaded and reapplied to the original source or any other image. The system allows for export of deformed images, features built‐in contouring tools with exporting capabilities, but is unable to import ROI contours delineated on other systems. All import and export functions are based on the DICOM standard.The Reveal‐MVS system has automatic rigid and deformable registration capabilities, as well as a separate PET/CT scanner automatic motion correction option specifically designed to account for the typically minor anatomical variations present between PET and CT scans on a combined or hybrid PET/CT system. The deformable registration algorithm is proprietary, thus severely limiting the authors' knowledge of its actions. However, the roots of the company Mirada Solutions Ltd.  can be traced to the Wolfson Medical Vision Laboratory at the University of Oxford.,33 T In principle, we agree with this assertion. However, it was believed that a simple, site nonspecific, multi‐modality phantom may yield pertinent information about an unknown deformable registration algorithm. As such, we included a phantom study in our protocol not for quantitative evaluation of Reveal‐MVS, but as a qualitative assessment tool that was designed and implemented with two objectives. First, to determine if the system has any general tendencies that might influence the registration of patient images; and second, to determine whether or not the system's actions when registering images were modality dependent.A number of authors have argued that phantoms have limited value in quantitatively evaluating deformable registration software because they cannot fully assess the impact of anatomical variations on the algorithm's performance.,25 I12 × 12 grid of quarter‐inch threaded holes 8 mm apart and attached the slab to the inside base of the phantom. Seven solid plastic spheres of varying diameters attached to quarter‐inch diameter rods were inserted in the base so as to be fairly evenly distributed throughout the phantom. Reference target CT , T1‐weighted MRI  and PET  images  images  were acq images  patients. Combining functional PET and anatomical CT information presents many challenges, but a number of them have been addressed with the advent of combined PET/CT scanners. In a single imaging study, these systems consecutively acquire CT and PET images with the patient in a fixed position on a single imaging couch in a timeframe on the order of minutes. The design does not eliminate, but significantly reduces the level of anatomical variations between patient PET and CT images acquired on independent systems. PET (144×144×178 (median) voxels; 4.0×4.0×4.0 mm3) and CT (512×512×151 (median) voxels; 1.2×1.2×5.0 mm3) images were acquired on a Gemini PET/CT unit . System resolutions are approximately 0.7 mm and 6.5 mm for the CT and PET components of the Gemini unit, respectively.Longitudinal PET: Longitudinal or temporal imaging studies where a patient is successively imaged to monitor change in a disease state, or to assess the effectiveness of treatment is a common practice in radiation therapy. We address this particular application by registering post‐treatment follow‐up images to pre‐treatment baseline images (144×144×213 (median) voxels; 4.0×4.0×4.0 mm3) acquired on an Allegro PET scanner, which has a measured system resolution of 6.5 mm.Longitudinal thoracic CT: The utilization of deformable image registration in 4D radiotherapy of the lung has been well documented. Registration of thoracic CT images acquired during different respiratory phases allows for modeling of respiratory motion and improved delineation of target margins. For simplicity we register longitudinal thoracic CT (512×512×43 (median) voxels; 0.8×0.8×7.5 mm3) studies imaged on a diagnostic quad‐slice Mx8000 scanner , which has a measured resolution of approximately 0.7 mm.Pelvic MRI to planning CT: It has been cited that the positions of both the prostate apex and base are often misidentified on pelvic CT images, and that rigid registration of MRI and CT can improve target delineation during prostate treatment planning. However, if the prostate has translated or has become slightly deformed with respect to surrounding anatomy between imaging studies, rigid registration may not result in accurate overlap of the MRI and CT prostate volumes. Deformable registration has the potential to further improve target delineation by accounting for internal prostate motion. T1‐weighted MRI (512 × 512×35 (median) voxels; 0.8×0.8×6.0 mm3) and planning CT (256×256×91 (median) voxels; 1.9×1.9×3.0 mm3) images were acquired on an Intera 3T  magnet and a PQ 5000 CT scanner, respectively. The system resolution for the protocol used for MRI image acquisition is approximately 2.2 mm, while the corresponding value for the CT images is 0.7 mm.Pelvic megavoltage CT (MVCT) to planning CT: The protocol for patients treated on the Hi·Art II  helical tomotherapy unit at our clinic calls for the acquisition of daily megavoltage CT (MVCT) (512×512×29 (median) voxels; 0.8×0.8×6.0 mm3) images with the patient in treatment position. Pre‐treatment MVCT images are beneficial in that they provide daily image guidance and allow for calculation of the daily delivered dose. It has been demonstrated that accurate deformable registration of daily MVCT and planning CT images permits the evaluation of accumulated dose distributions delivered over the coarse of treatment. Planning CT (256×256×91 (median) voxels; 1.9×1.9×3.0 mm3) images were acquired on a PQ 5000 system. Approximate resolutions for the MVCT and planning CT acquisition systems are 1.7 mm and 0.7 mm, respectively.The objective of the second component of the protocol is to determine the optimal deformable registration settings for a collection of IGRT applications. It should be re‐iterated that patient images were acquired retrospectively and, unlike in the phantom study, the authors could not dictate which imaging systems were used. As such, different scanners were used for different applications; however, there were no variations amongst each individual application. The following five image combinations, selected based on availability and the desire to include multiple anatomical sites and imaging modalities, were investigated.Five image pairs for each individual application were imported into the Reveal‐MVS system for this study. After initial automatic normalized mutual information based rigid alignment, every source image was automatically registered to its respective target image using each of the possible 36 automatic deformable registration settings. The automatic motion correction tool was used for PET/CT registration in place of automatic deformable registration. Deformed images were exported and postregistration similarity measures were evaluated using in‐house software developed in C++. Symmetric correlation ratio, mutual information, and correlation coefficient values were calculated for each intra‐modality image pair, with the latter excluded from inter‐modality analysis. A simple qualitative visual assessment of fused images was used to evaluate the generally accepted relationship that similarity measure optimization corresponds to improved registration accuracy.T0 were registered using Reveal‐MVS. Absolute comparison of the true transformation T0 and that produced by the commercial system T was achieved by calculation of the displacement error, which is defined asTwo of the registration applications evaluated in the previous section, one intra‐modality and the other inter‐modality, were selected for analysis of absolute registration accuracy. For the intra‐modality application, mathematically simulated deformations were used for absolute validation of longitudinal thoracic CT deformable registration. Unedited and synthetically deformed patient images that differed by a known transformation nt error,26 whiN is the number of image voxels and for each voxel i, Ei is the voxel error, the displacement between the true position of each deformed voxel and the position obtained by registration. In addition to full 3D analysis, axial components of DE values were also evaluated.where In order to best represent clinical reality, transformations were selected by registering baseline to follow‐up patient images with a standard B‐spline parameterized free form deformable (FFD) registration algorithm implemented in the Insight Segmentation and Registration Toolkit (ITK). ITK is an open‐source, object‐oriented software package that consists of a collection of C++ classes designed for image processing, segmentation and registration that can be implemented in user‐developed software. The registration need not be perfect so long as the generated transformation and the synthetic deformed baseline image were clinically relevant and plausible. Synthetic images were then registered to the original baseline images on the Reveal‐MVS system using the optimal settings determined by relative evaluation.Upon conducting this procedure on each of the five image pairs analyzed in the previous section, we recognized that additional information could be extracted from the process in order to further enhance analysis of the commercial system's absolute capabilities. FFD registration is an iterative process and not only can the final iteration be considered a clinically relevant transformation, each preceding iteration can be viewed as representing clinical plausibility. As such, B‐spline transformation parameters for nine additional iterations from each FFD registration were used to produce synthetic images. In all five FFD registrations, convergence required greater than ten iterations, so additional iterations were selected to obtain B‐spline transformations representing varying degrees of deformation.T0 applied to the original baseline image and deformed images were subsequently imported into Reveal‐MVS. The Reveal‐MVS registration derived transformation T was applied to the deformed grid image, which was then exported for analysis. Perfect registration on Reveal‐MVS would result in the exported image being equivalent to the original nondeformed nonuniform grid image and a displacement error value of nil. Both grid images were imported into analysis software developed in C++ and the approximate displacement error was calculated using the locations of each of the grid points. A flowchart is used to summarize the intra‐modality registration validation procedure in Calculation of the displacement error for Reveal‐MVS registrations was hindered by the fact that transformations in Reveal‐MVS cannot be accessed by the user. To overcome this problem, we developed a grid‐based system in which baseline patient images were modified by initially setting the intensity of each to zero. A set of randomly selected individual image voxels was then modified to have non‐zero intensity values creating a nonuniform grid image. All image modification was performed with a MATLAB  script. Grid images were then deformed using the same known B‐spline transformation Recognizing that the accuracy of our proposed DE evaluation method was limited by a number of factors including statistics, and the potential merging or crossover of grid points, we felt that validation of its efficacy was required. A collection of known B‐spline transformations defining varying magnitudes of deformation was applied to a randomly selected grid image. B‐spline functions were obtained for all iterations during the registration of the selected longitudinal thoracic CT images with our FFD registration program based on ITK. After setting the intensity of all image voxels in the baseline image to zero, either 500, 1000, 3000, or 5000 randomly selected voxels were set to non‐zero values. The deformed and original grids were imported into our evaluation software for calculation of approximate DE values, which were compared with true displacement error values extracted from the known B‐spline functions. The process was repeated using differing quantities of random grid points to determine the optimal number of non‐zero intensity voxels to employ. Kagawa et al. observed that CT contours often erroneously included sections of seminal vesicles, the base of the bladder, adjacent structures such as venous plexus and fibromuscular stroma, neurovascular bundles, and the anterior rectal wall. In fact, inter‐modality variation has been demonstrated to exceed inter‐observer variation and its consequences should influence the procedural design of this study.Finally, we evaluated the Reveal‐MVS system's ability to deform pelvic MRI images to planning CT images through analysis of radiation oncologist delineated prostate contours. However, an important factor must be considered when performing volumetric analysis of prostate contours drawn and CT and MRI images. It has been demonstrated through independent studies that contoured prostate volumes are greater in CT than in MRI images.,40 KEach patient MRI image was registered to its respective planning CT using the optimal deformable registration setting determined through relative validation. Prostate volumes were contoured on each of the five CT and five unregistered MRI pelvic images by a single radiation oncologist on Reveal‐MVS. Each patient's unregistered MRI image was displayed on a monitor adjacent to the registration system in an attempt to improve prostate CT delineation. Contours were exported and a C++ program was used to convert patient images into binary images in which voxels corresponding to prostate had intensity of one, while all other voxel intensities were set to zero. Binary MRI images were imported into Reveal‐MVS and appropriate transformations were applied, generating deformed binary MRI images for comparison with each patient's binary CT image. the Tannimoto Coefficient (TC) and Dice Similarity Coefficient (DSC). Given a CT contour volume VCT and a MRI contour volume VMRI, TC and DSC are defined byVolumetric analysis of binary CT and deformed binary MRI images consisted of evaluation of the mean surface distance between contours and the two most common measures of region overlap,25 theBoth measures have possible values ranging from 0 for no overlap to 1 for perfect agreement between volumes. A flowchart is used to summarize the inter‐modality registration validation procedure in A very consistent trend was observed in all of the phantom image deformable registrations on the Reveal‐MVS system regardless of phantom modification or image modality. Whether one of the plastic spheres was translated or replaced with a different sized sphere, the registration software performs little or no image warping in the vicinity of the modified sphere. This is illustrated in Mean and individual longitudinal thoracic CT post‐deformable registration normalized mutual information values for all 36 setting combinations are plotted in An important observation from Our method for determining approximate displacement error values for images registered on systems that do not provide access to the transformation function was tested by applying known B‐spline functions to a set of non‐uniform grid images. True displacement error and true axial displacement error values are plotted against the approximate values calculated by our in‐house software for each of the deformed grid images in After generating synthetic images by applying mathematically known deformations to patient images followed by the registration of synthetic to original thoracic CT images on Reveal‐MVS, postregistration axial and 3D displacement errors were evaluated. Resultant values are plotted against known predeformable registration values for all five patients in Based on mean NMI and SCR values, the optimal registration settings for prostate MR to CT deformable registration are None‐Fast‐Medium and None‐Slow‐Medium, respectively. Pre‐ and postregistration prostate DSC, TC, and mean contour separation values for both settings are given in The use of phantoms in deformable registration validation is an ongoing area of discussion. Researchers continue to utilize them even though any phantom, no matter how complex the design, will not be able to simulate the range of anatomical variations that may occur in clinical imaging studies. We included a phantom study in our validation protocol not for quantified assessment, but with the intent that it may provide important insight on the tendencies of a black box commercial deformable image registration system. Based on phantom images, we established that the deformable registration algorithm on Reveal‐MVS must have strict constraints on the amount of local deformation that may occur. In addition, various intra‐ and inter‐modality phantom registrations revealed little or no evidence that the commercial system's apparent preference for global over local deformation is modality dependent. Although not quantifiable, these observations provide useful information that may be extrapolated to predict the system's ability to perform clinical deformable image registration applications. For example, if many local image deformations are required, this system may not be up for the task. but has had limited use in applications directly relevant to radiation therapy. By registering multiple B‐spline warped images to original thoracic CT images, we quantified the commercial system's performance over a range of potential initial clinical deformations. The resultant plot of postregistration versus preregistration displacement errors in After establishing the optimal registration settings for five deformable registration applications through relative measures, absolute quantitative evaluation of an intra‐ and inter‐modality registration was performed. Intra‐modality deformable registration validation using known transformations is common in inter‐subject brain studies, Acquisition of anatomical MRI images with surface or other noninvasive coils significantly reduces problems related to prostate deformation but daily prostate motion remains a major concern. As a result, any translational offset between prostate positions in the MRI and CT with respect to surrounding anatomy may lead to inaccurate overlap of the MRI and CT prostate volumes after rigid registration. Based on our analysis in The use of deformable registration instead of conventional rigid registration to improve structure delineation in the planning of prostate radiation therapy is an interesting concept. The predominant application of deformable registration of prostate MRI to CT images in the literature is to account for the deformation of the prostate caused by the insertion of an endorectal coil during magnetic resonance spectroscopic imaging (MRSI).,30 AAlthough contour analysis was used for inter‐modality registration validation in this study, point landmark‐based evaluation is also an option. Both methods have the potential to provide useful quantitative information on the capabilities of a deformable registration system but, ultimately, we selected volumetric analysis simply because radiation therapy planning and delivery is based on doses delivered to volumetric regions of interest (ROI). However, for larger regions of interest that are susceptible to internal local deformations or ROI whose borders cannot be visually delineated with adequate accuracy, point‐based validation may be required. Ultimately, there may be some application dependence involvement in absolute inter‐modality deformable registration validation. For the most part, consistency methods have been used for rigid registration validation, but have recently been applied to deformable models by Malsch et al. A novel registration assessment tool recently introduced is the concept of unbalanced energy whereby, instead of using gold standards, the physical fidelity of the deformation field is quantified through finite element models (FEMs). It has been applied with success to deformable registration of truncated pelvic CT images that include only a small region surrounding the prostate gland. Although promising, its relevancy for full 3D images of sites prone to significant anatomical deformations must be established. Like consistency testing, unbalanced energy certainly requires further research. Both validation methods were excluded from our protocol mainly because they rely heavily on the extensive knowledge of the deformation field which, as demonstrated by Reveal‐MVS, may not necessarily be the case for commercial systems.In additional to landmark‐based validation, two other validation methods excluded from our protocol have been the focus of recent research. The first is the consistency approach: in three given images , a comparison of transformations produced by registering A to B, B to C, and C to A provides a measure of registration error, assuming that errors are random and distributed evenly between each transformation.47 ForThe primary intent of this work was the development of a protocol for the assessment of commercial deformable registration systems, but while applying our protocol, two additional matters were also investigated. First, we developed and validated a method to compare true displacement fields with those produced by registration when the registration software does not allow access to the deformable transformation. We also tested the generally accepted notion that the quality of different deformable registration procedures can be ranked by comparing postregistration similarity measures. Whether the results of both investigations are specific to the deformable registration applications analyzed or are relevant to other applications requires further investigation.In the known transformation study, we showed that by applying known B‐splines to a blank image with one thousand randomly positioned grid points followed by locating the discernible deformed grid points, the calculated displacement error from the sample points is highly accurate for a range of displacement error values. However, an upper limit is eventually reached due to our analysis software's reduced ability to correctly identify corresponding grid points in heavily deformed images. When registering images on the commercial system that differ by displacement error values greater than these upper limits, the accuracy of measured postregistration values may be questionable. Although an acceptable value for postregistration displacement errors has yet to be discussed, these upper limits certainly exceed any ideal acceptable quantity.In using our random grid point method for DE evaluation of deformable registration on the Reveal‐MVS system, we make two fundamental assumptions. First, we assume that the commercial system improves or, at the very least, does not significantly reduce correspondence between the synthetically deformed and original thoracic CT images during the deformable registration procedure. The concern is that deformable registration may reduce correspondence to the point that postregistration displacement error values exceed the aforementioned threshold in which results can no longer be considered accurate and measured DE values may provide an erroneous picture of the system's capabilities. However, a problem such as this would be observable through visual inspection of registrations and this was certainly not the case. The second assumption is based on the accuracy of using a sample of voxels instead of the entire image to calculate displacement errors. We have demonstrated it to be acceptable for B‐splines, but whether or not the same is true for deformable transformations on the Reveal‐MVS system cannot be verified. Here the concern is that the commercial system performs significant local deformations whose contributions to the postregistration displacement error would not be picked up by sampling a limited number of voxels. Based on the observed global tendencies of Reveal‐MVS in comparison to B‐spline deformation, once again, we feel this is not a significant problem. In fact, part of the reason we chose to model synthetic transformations with B‐splines is that they are well‐known for allowing tremendous local control of image warping in comparison to other deformable models.50The second investigation beyond the scope of the validation protocol was the establishment of a relationship between absolute measures of registration accuracy and post‐registration similarity measure comparison. We have presented a protocol for the validation of automatic commercial deformable image registration systems. Through a series of tests, the protocol provides a well‐balanced general assessment of a commercial system's capabilities. It initially consists of a qualitative phantom study to determine the system's general tendencies, followed by a relative comparison of the system's registration settings through similarity measure evaluation. The protocol also includes an assessment of the system's absolute intra‐modality registration accuracy using synthetic transformations and, finally, a contour‐based evaluation of the system's inter‐modality registration capabilities. Developed software and acquired images are readily available for assessment of other commercial systems that may be purchased for clinical or research objectives by our center. Access to the registration displacement field is not required as long as it can be stored and later re‐applied, which would probably be considered mandatory in a system approved for clinical use. The protocol is by no means static or definitive and can readily be expanded to investigate other potential deformable registration applications. Future work will include applying the protocol to additional deformable registration systems and the establishment of standard minimum benchmarks for the evaluated absolute measures.


A scores, was poorer in the very preterm compared with the control group. Analysis of fMRI data focused on differences in neural activity between the recognition and encoding trials. Very preterm born adults showed decreased activation in the right middle frontal gyrus and posterior cingulate cortex/precuneus and increased activation in the left inferior frontal gyrus and bilateral lateral occipital cortex (LOC) compared with controls. Hippocampi, fornix and cingulum volume was significantly smaller and fornix HMOA was lower in very preterm adults. Among all the structural and functional brain metrics that showed statistically significant group differences, LOC activation was the best predictor of online task performance (P = 0.020). In terms of association between brain function and structure, LOC activation was predicted by fornix HMOA in the preterm group only (P = 0.020). These results suggest that neuroanatomical alterations in very preterm born individuals may be underlying their poorer recognition memory performance. Hum Brain Mapp 38:644–655, 2017. © 2016 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.Very preterm (<32 weeks of gestation) birth is associated with structural brain alterations and memory impairments throughout childhood and adolescence. Here, we used functional MRI (fMRI) to study the neuroanatomy of recognition memory in 49 very preterm‐born adults and 50 controls (mean age: 30 years) during completion of a task involving visual encoding and recognition of abstract pictures. T1‐weighted and diffusion‐weighted images were also collected. Bilateral hippocampal volumes were calculated and tractography of the fornix and cingulum was performed and assessed in terms of volume and hindrance modulated orientational anisotropy (HMOA). Online recognition memory task performance, assessed with  Very preterm birth (<32 weeks of gestation) is associated with structural and functional brain alterations that persist into early adulthood and often correlate with cognitive performance . Differential activation patterns have been found in very preterm samples compared to controls during visual , prolonged gestation (greater than 42 weeks), history of psychiatric illness, severe hearing and motor impairments, and mental retardation indicated by IQ < 70. Participants gave full informed consent and the experiment was approved by the appropriate local ethics committees and in compliance with national legislation and the code of ethical principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki).The aim of this experiment was to investigate the neural networks that are recruited during a recognition memory task by adults who were born very preterm. We adapted a task designed by Dove et al.  which el2. These were co‐registered with T1‐weighted anatomical image , allowing for 196 slices with no gap and an isotropic resolution of 1.1 × 1.1 × 1.1 mm3. Diffusion weighted images were acquired using a multislice spin echo EPI sequence, obtaining 60 contiguous near‐axial slice locations with isotropic (2.4 × 2.4 × 2.4 mm3) voxels . Maximum b‐value was 1300 s/mm2, with 32 diffusion‐weighted directions and 4 non‐diffusion weighted volumes. Peripheral cardiac gating was applied with an effective TR of 20/30 RR interval.Data were collected using a GE 3 tesla Signa MR scanner  at the Institute of Psychiatry/Maudsley Hospital, Kings College London. A gradient‐echo EPI sequence was used to collect data from 39 non‐contiguous slices of 3.5 mm thickness separated by a distance of 0.5 mm, and with in‐plane voxel resolution of 3.75 × 3.75 mmhttp://www.fmrib.ox.ac.uk/fsl). The three initial volumes were removed to minimize the effects of magnetic saturation. Motion correction was followed by spatial smoothing  and temporal high‐pass filtering (sigma = 50 s). Regressors for each condition in the general linear model were convolved with a gamma hemodynamic response function. For the encoding trials, only the trials that were subsequently successfully recognised in the recognition trials were modelled. For the recognition trials, only those in which participants gave a correct response were modelled separately from incorrect trials. Only correct recognition trials were modelled in the analyses. Individual participant data were then entered into a higher‐level analysis using a mixed effects design  whole‐brain analysis. Neural responses from each individual were registered to a study specific T1‐weighted template, which is an average of 78 brain images from term born and preterm individuals  and from the resulting cluster maps clusters of activity that significantly differed between groups were identified. No statistically significant results were found when comparing very preterm adults with brain injury, very preterm adults with normal ultrasound classification  and false alarms (F)  and a relative threshold  at each voxel to remove the general noise floor and surviving noise local maxima, respectively. Each FOD that survived the threshold were used as seeds to perform whole‐brain tractography. Fibre orientation streamlines were propagated using Euler integration with a step‐size of 1 mm. Propagation stopped if the angle between two successive steps exceeded 60°. Tractography reconstruction was performed using software written in Matlab  using a two‐region method . Each multiple linear regression model was built first across the whole sample, then subsequently for separate groups. Multicollinearity was tested for all independent variables and all variables had a variance inflation factor < 3. Partial correlation was performed to explore the relationship between multiple correlated variables. All models and tests were corrected for multiple comparison using the Benjamini‐Hochberg procedure  were used as independent variables in a multiple linear regression model to find the best predictors of episodic memory performance (A scores (P = 0.021). There were no very preterm participants who had severe hearing and/or motor impairments or mental retardation indicated by IQ < 70. Four very preterm participants had a current psychiatric diagnosis.Only participants who achieved an accuracy higher than 50% on the fMRI task were included in the analysis to ensure they were actively performing the task. Three very preterm born participants were thus excluded. The final sample studied included 49 very preterm participants and 50 term born controls Table . The verAge was added as a covariate in the fMRI analysis as it was close to being significantly different between groups. In terms of neural response, regions differentially activated between the groups in the encoding > low level baseline and the recognition > low level baseline contrasts are reported in Table When contrasting the recognition trials to the encoding trials, the very preterm group showed less activation in the PCC/precuneus and the right middle frontal gyrus (MFG) compared with controls. However, the very preterm group showed more activation in the left inferior frontal gyrus (IFG) and bilateral lateral occipital cortex (LOC) compared with controls Table , Fig. 2.n = 2; control, n = 1) and unsuccessful Freesurfer parcellation . The volume of the dorsal, ventral cingulum, and fornix were significantly different between the very preterm and control groups after controlling for intracranial volume. The HMOA of the white matter tracts was only significantly different between the groups in the fornix, with the very preterm group having lower HMOA than controls. Very preterm participants had smaller volumes of the hippocampi than controls, after adjusting for intracranial volume 2 to meet the assumptions of multiple linear regression. Regions with bilateral activation were averaged and used as one variable to avoid multicollinearity issues. Three multiple linear regression models were built using activation differences in encoding > low level baseline, recognition > low level baseline, and recognition > encoding contrasts. Structural brain variables were included in all models. Only the model built using activation differences in recognition > encoding was significant and LOC activation emerged as the best predictor of A scores . However, only LOC recognition, inferior division was significant in explaining on‐line task performance, not LOC encoding in the same cluster. This result shows that while accounting for LOC activation during encoding, it is activation of the recognition > encoding contrast that significantly explains on‐line task performance.As recognition performance relies to a degree on the effectiveness of the previous encoding stage, we extracted LOC activation in the same location of the significant recognition > encoding contrast  in the encoding > low level baseline  contrast. We then built a multiple linear regression model including LOC recognition, inferior division, LOC encoding, inferior division, white matter structures, and volume of the hippocampi. In the whole sample, this model was significant (P = 0.077). However, when investigating the model separately for the very preterm and control groups, LOC activation was predicted by fornix HMOA in the very preterm group .To further explore the relationship between neural activation and local structure, subsequent multiple linear regression models were built to find variables that explained LOC activation. In the whole sample, this model was not significant . The same result was found in the control group , but not in the very preterm group .Both on‐line performance and fornix HMOA were found to be associated with LOC activation; therefore, we used a partial correlation to assess if fornix HMOA influenced the association between on‐line performance and LOC activation. The correlation between on‐line performance and LOC activation persisted after partialling out fornix HMOA in the whole sample  compared to controls. However, significant differences in white matter microstructure in the very preterm group was only found in the fornix . Contrary to the present findings, in our recent study using the same cohort, Froudist‐Walsh et al.  found thOur findings of increased neural activation in right MFG and PCC in the control compared with the very preterm group are in support of the predictive coding hypothesis, a stimulus recognition scheme using an empirical Bayesian approach Friston, . This thStrengths of this study include the use of multiple MRI modalities to assess both functional and structural aspects of recognition memory. The HMOA measure we used to assess white matter structure can provide a tract‐specific diffusion measure, even in the presence of multiple fibres crossing in a single voxel, unlike conventional measures such as fractional anisotropy. This gives us higher sensitivity to detect white matter alterations and a more accurate representation of its microstructure.This study complements and extends our previous work, which focused on working memory (Froudist‐Walsh et al., A scores, the very preterm group performed significantly worse than controls, suggesting they were less able to correctly reject novel images. Furthermore, we did not assess visual acuity, which can be affected in very preterm born adults (Birch and O'Connor, A limitation of this study is we did not have a large enough number of foil trials to test the differences between recognition and foil trials. Every participant achieved a higher than chance accuracy in recognising previous images, but when also considering correct rejections of new images in A further limitation of the study refers to the fact that in our analyses we considered bilateral hippocampal volume, despite evidence of hemispheric specialization of the hippocampi, whereby the left hippocampus is involved in the processing of verbal material and the right hippocampus in the elaboration of nonverbal stimuli (Kennepohl et al., This study was the first to investigate multimodal imaging correlates of visual recognition memory in very preterm adults, providing a unique opportunity to investigate neural plasticity in a neurodevelopmental framework. Very preterm born adults displayed different neuronal activation patterns during completion of a visual recognition memory task compared to controls. Specifically, increased activation in the LOC in the very preterm group was associated with worse task performance, which may reflect weaker sensory prediction, and with structural integrity of memory‐related white matter tracts. These findings are inconsistent with our previous study, which indicated that increased activation in a perisylvian region was associated with functional adaptation to successfully perform a working memory task (Froudist‐Walsh et al.,


Functional genomics has transformed from futuristic concept to well‐established scientific discipline during the last decade. Cotton functional genomics promise to enhance the understanding of fundamental plant biology to systematically exploit genetic resources for the improvement of cotton fibre quality and yield, as well as utilization of genetic information for germplasm improvement. However, determining the cotton gene functions is a much more challenging task, which has not progressed at a rapid pace. This article presents a comprehensive overview of the recent tools and resources available with the major advances in cotton functional genomics to develop elite cotton genotypes. This effort ultimately helps to filter a subset of genes that can be used to assemble a final list of candidate genes that could be employed in future novel cotton breeding programme. We argue that next stage of cotton functional genomics requires the draft genomes refinement, re‐sequencing broad diversity panels with the development of high‐throughput functional genomics tools and integrating multidisciplinary approaches in upcoming cotton improvement programmes. Gossypium hirsutum) is a foundation of the global economy, prized for its important renewable fibre resource. It serves as an ideal plant for different biological studies such as genome evolution, polyploidization and single‐celled biological processes  in its nucleus. About 5–10 million years ago (MYA), the African‐derived ‘A’ diploid genome diverged from the eudicot progenitor simultaneously with the diploid genome ‘D’ which was native to Mexican  to the New World and hybridized with a D‐genome ancestor (Gossypium raimondii) followed by chromosome doubling, which produced the allotetraploid cotton  have been sequenced; the precise species that directs the development of the tetraploid cotton species about 1–2 MYA currently not exists  is prized due to its superior quality and extra‐long fibre for the fabrication of high‐quality textiles. Considering its importance, genome of the G. barbadense was sequenced  at the whole‐genome level, even for the 2.5‐Gb genome of allotetraploid cotton. In cotton, SNP63K has been developed that contains assays for 45 104 and 17 954 putative intraspecific and interspecific SNP markers  , Cotton Functional Genomic Database , Cotton Genome Resource Database , Cotton Genome Database , Cotton Genome Project , Platform of Functional Genomics Analysis in Gossypium raimondii , Comparative Evolutionary Genomics of Cotton (http://cottonevolution.info/), Join Genome Institute  and Database for Co‐expression Networks with Function Modules .A comprehensive study of any genome depends on the availability of information regarding genome sequence, map position, mRNA and protein expression, metabolism and allelic variation. Hence, with the development of enormous omics data sets, it is important to have functional genomics database that permits users to easily obtain and visualize genomic information. Currently, many functional genomics databases are available for the cotton research community: the CottonGen ‐associated protein 9 (Cas9) from et al., et al., et al., et al., GhARG), discosoma red fluorescent protein2 (DsRed2) and chloroplast development (GhCLA1) genes proves that it is highly reliable and effective for cotton genome editing . The distributions of these genes  is preferentially expressed during fibre initiation by the HDZIP2ATATHB2 core cis element  gene highly expressed in developing fibres encodes a cell wall protein and regulates cell wall loosening  gene disturbed the fibre elongation, signifying the contribution of SuSy in osmosis regulation. In contrast, proline‐rich protein‐coding gene (GhPRP5) worked as a negative regulator during cotton fibre development , which was up‐regulated during secondary wall cellulose synthesis in cotton fibre. Besides secondary wall cellulose synthesis, maturation stage of fibre development begins. During fibre maturation, the majority of the expressed genes belong to cellular respiration  family showed strong expression during early fibre elongation  and self‐pruning (SP) genes regulate the balance between monopodial and sympodial growth habits in woody perennial plants  gene from G. hirsutum was expressed in the shoot apex  gene positively regulates defence response but mediates reduced tolerance to biotic and abiotic stresses in transgenic Nicotiana benthamiana  gene showed higher chlorophyll content, increased peroxidase activities and lower lipid peroxidation levels, which ultimately increase the salt and drought stress tolerance in transgenic cotton  and sucrose nonfermenting 1‐related protein kinase 2 (SnRK2) genes are also involved in abiotic stress tolerance in cotton  and highly divergent C‐terminal regions  and ethylene response factors (ERF) are among the largest and most diverse transcription factor families involved in stress tolerance in many plant species. However, in cotton, few members of these families, that are GhABF2  gene could function as positive regulators to enhance abiotic stress tolerance in cotton  cascades have also been shown to regulate a number of stress responses. In cotton, GhMPK16 from D‐MAPK group has been characterized, which is involved in disease resistance  gene showed the significant resistance to disease and mycotoxin causing fungal pathogens  is also one of the important rising threats to cotton production in different countries. It has been reported that resistance against CLCuV is conferred by two dominant and one suppressor gene  and truncated AC1 gene were targeted for restricting viral replication and movement in transgenic cotton  is correlated with larval growth and its expression was induced by gossypol  (Nida et al., CP4 EPSPS gene has played an important role in vegetative tolerance to glyphosate; however, its expression is critical for the development of male reproductive organ in response to high glyphosate application during late developmental stage (Chen et al., et al., Insect herbivores and cotton plant have waged war from millions of years. In cotton plant, transgenic technology has been mainly used to induce the resistance against insect herbivores. Among the different transgenic approaches against insects, et al., et al., et al., et al., et al., et al., et al., et al., For determining the entire set of genes with their functions, genome sequencing of an organism is an important prerequisite resource. At present, the sequenced and re‐sequenced genomes of diploid and allotetraploid (Fang et al., Additionally, the development of a well‐organized system for molecular breeding by various functional components is necessary. Previously, few efforts have been made to develop a multiscale crop system for high‐throughput association studies of composite traits, that is the ePlant model (Zhu Harnessing the full potential of functional genomics requires a multidisciplinary approach and integrated knowledge of the molecular and other biological processes underlying different traits because gene functions cannot be inferred by only one approach. The addition of information obtained from genomics, transcriptomics, proteomics and epigenomics studies of cotton will help us to critically explore and investigate the different regulatory pathways underlying different traits. Also, new user‐friendly bioinformatics tools and software with better resolving power and technological improvements need to be developed to increase the potential offered by functional genomics. The resulting huge amount of data from different high‐throughput techniques should, in turn, be further organized, stored and interconnected into fundamental timely updated databases in order to let easy extraction and comparison that will increase the understanding and opportunities for future functional genomics advancements in cotton.Arabidopsis and rice. Therefore, next stage of cotton genomics requires draft genome refinement, re‐sequencing broad diversity panels and diverse wild relatives to better understand its genome. However, for taking the full benefits of the available genomic information on cotton genes, only the multidisciplinary integrated approach allows their functional characterization. So, advances in functional genomics of cotton will depend on developing high‐throughput technologies and integrating multidisciplinary approaches including genomics, transcriptomics, proteomics, epigenomics and bioinformatics in upcoming cotton improvement programmes.The strong background of cotton genetics and the great efforts of the cotton genome consortium led to the start of cotton genome sequencing in 2007. With the wealth of cotton genome sequence information, cotton genomics research has entered the phase of fast functional characterization of all genes. However, despite great efforts in whole‐genome sequencing and re‐sequencing of cotton, large knowledge gaps still persist as compared to model plants The authors declare that there is no conflict of interests regarding the publication of this manuscript.JA and GS conceived and designed the experiments; all authors performed data analysis and interpretation; JA, WM, MAA and GS wrote the manuscript.


This five‐year plan was developed in 2011 with the aim of reducing new paediatric HIV infections by 90% and AIDS‐related maternal and paediatric mortality by 50% As more fragile countries have graduated to middle income status, development assistance for HIV will likely reduce, as for instance, domestic contribution thresholds by eligible middle income countries for grants from the GFATM is 20% to 60% – a significant rise from 5% for developing economies 3acute or protracted, could have different needs requiring different programme designs. This risk analysis could form the basis of risk‐informed joint planning, allowing synergies between HIV and humanitarian interventions.The humanitarian‐development nexus provides an important framework that could bridge a divide between these two fields that are guided by two separate, but complementary global processes – Agenda 2030 for sustainable development Joined‐up programming could be facilitated with sustainable financing by optimizing development and humanitarian funding mechanisms Start Free, Stay Free, AIDS Freenew way of working arising from the global humanitarian summit and a major theme in the Sendai framework for Disaster Risk Reduction 2015 to 2030 Collective outcomes should be defined from the onset of the emergencies, and a robust monitoring and evaluation system put in place to track progress towards SDGs. Sustaining peace resolutions, is critical for maintaining this progress. The renewed momentum in HIV workstreams spearheaded by the new post‐Global Plan programming framework, Authors declare no competing interests DC, CL and PI have not received grants or speakers fees from any commercial body in the preparation or submission of this manuscriptDC conceptualized the article, established links between emergencies and HIV, and drafted the first draft. CL and PI reviewed the draft, elaborated links with SDGs and contributed to the writing of the manuscript


This study aimed to examine whether fear of cancer recurrence (FCR) was related to two important health behaviours  in a large sample of colorectal cancer patients.I have fear about my cancer coming back’), demographics, treatment and health variables. Physical activity (PA) was recorded as number of days per week doing at least 30 min of brisk activity, and smoking status was reported.Ten thousand nine hundred sixty nine patients, diagnosed in 2010–11, and in remission in 2013, completed the ‘Living with and Beyond Colorectal Cancer’ survey. The survey included purpose‐designed questions on fear of recurrence  1.58; 95% confidence interval (CI) 1.46, 1.71)) more younger than older patients  and slightly more patients from deprived areas  reported FCR. Independently of demographics and treatment, compared with those meeting the PA guidelines, those who were doing only ‘some’  or ‘no’ PA  reported higher FCR. Compared with non‐smokers, more current smokers reported fear  and slightly more ex‐smokers .This cross‐sectional study provided novel data showing that colorectal cancer survivors with poorer health behaviours (those with lower activity levels and those who smoked) were more likely to experience FCR. Future research should replicate findings using detailed measures of fear, objective measures of health behaviours and identify directions of associations. © 2016 The Authors. Psycho‐Oncology Published by John Wiley & Sons Ltd. Colorectal cancer is the fourth most common cancer in the UK, with around 40 000 new cases each year There is emerging evidence that practising healthful behaviours  following a diagnosis of colorectal cancer may reduce the risk of recurrence. In a meta‐analysis of six prospective cohort studies examining post‐diagnosis physical activity, those who reported higher levels of physical activity following a diagnosis of colorectal cancer had significantly reduced risk of cancer‐specific mortality Most literature has focused on demographic factors relating to FCR , generally demonstrating that fear is higher in women than men and higher in younger than older patients (reviewed in 14). Treatment types have been related to FRC, but findings are less consistent, probably reflecting the varying cancers studied In a repeat measures study of 73 patients with oral or oropharyngeal cancer, past and current tobacco use were associated with higher levels of psychological distress, and smokers reported higher FCR Therefore, the aims of this study were to use the largest existing relevant dataset of colorectal cancer patients to examine associations between FCR two important health behaviours. Associations between fear and socio‐demographic and treatment factors were also explored.Living with and Beyond Colorectal Cancer survey, commissioned by the UK Department of Health in 2013 as part of a programme of work designed to ensure that UK cancer patients' needs are met across a range of health, psychosocial and lifestyle domains, through recording patient reported outcomes (PROMS) n = 34 467 were eligible). Permission to approach patients was granted to NCRS by the National Information Governance Board [ref ECC 5‐02(FT8)/12] and patients consented by returning completed questionnaires. Authors of the current study applied formally to NCRS for permission to perform secondary analyses on health and lifestyle data from the PROMS. Data were provided fully anonymised and authors had no access to any patient‐identifiable data. The current study was reviewed by NCRS and University College London, and in line with the University College London Ethics Committee, additional ethical approval was not required for secondary analyses of anonymous survey data. Content and face validity for the PROMS survey were identified through expert review and consultation with patients, experts and charity advisory groups Data were from the I have fear about my cancer coming back’ (‘fear of recurrence’) with responses on a 5 point Likert scale (strongly disagree/disagree/neither agree or disagree/agree/strongly agree: coded 1–5) or ‘does not apply to me’ (coded as missing for the current study). The Living with and Beyond CRC survey was developed to audit multi‐dimensional aspects of care with a focus on quality of life and function. The current study involved secondary analyses of existing data, and a validated scale of FCR was not included in the survey because FCR and lifestyle were not a primary focus. However, the data gathered provide a unique opportunity to capture the views and behaviours of an extremely large and representative sample of CRC survivors. Additionally, much of the literature on FCR has used single‐item assessments of FCR, and findings are in accordance with more detailed validated scales ’. Responses were number of days from 0–7. Activity categories were ‘meeting guidelines’ (≥5 days per week), ‘some’ (1–4 days) and none (0 days). Smoking status was assessed using the question ‘Do you consider yourself to be a smoker/ ex‐smoker / non‐smoker?’.Current physical activity was assessed by asking ‘How has your CRC responded to treatment’ . Length of time since treatment was also assessed. Comorbidities were assessed with ‘Do you have a long‐standing health condition other than cancer’ . Virtually all patients reported having had surgery; however, in addition, they were asked to record whether they had received chemotherapy or radiotherapy.Potential socio‐demographic and treatment confounders were selected a priori based on the existing literature on factors associated with FCR For ease of interpretation, for main analyses, FCR data scores were categorised to those reporting no fear (categories 1–3) or reporting fear (categories 4–5). These analyses were repeated excluding category 3 (‘neither agree nor disagree’). However, because results did not change, these patients were included, giving approximately equal‐sized groups. Binary logistic regressions (fear/no fear as the outcome) were carried out, and simple associations were presented between demographics, health behaviours and treatment and health variables and fear presented. Adjusted models were also run for all demographics, both health behaviours adjusting for demographics, treatment and health and treatment and health adjusting for demographics. To illustrate the magnitude of the effect for health behaviours, graphs were also presented with FCR as a continuous outcome, using data analyses of covariance with polynomial contrasts.et al. p < 0.001). Ethnicity of non‐responders was not available for comparison, as this was gathered from the survey. Two hundred three participants who reported having Alzheimer's or dementia were excluded  and only those patients who reported being in remission were included . Of those, 602 (4.9%) recorded that FCR ‘did not apply to them’ (reasons for reporting this were not reported), leaving 11 686 patients. Ten thousand nine hundred sixty nine patients had complete data on all key outcomes for the current study.The overall response rate to the survey was 63.3%, reported by Downing Sample characteristics are presented in Table p < 0.001). Associations with ethnicity could not be assessed because only 338 patients were from a non‐white ethnic group and of those, 182 classified themselves only as ‘other’. All significant associations between demographics and FCR remained in adjusted models. Treatment factors were related to FCR; patients who had more recently completed treatment were more likely to report fear (p < 0.001). In simple models, patients who received chemotherapy or radiotherapy were more likely to report fear (p < 0.001). However in adjusted models, only chemotherapy was significant.Factors associated with FCR are shown in Table Associations between FCR and health behaviours are also shown in Table This was the first study to explore whether health behaviours related to FCR in colorectal cancer patients. We found that those with lower levels of PA, and current smokers, were more likely to experience FCR although the magnitude of those associations was relatively small. Additionally, this study adds to the literature demonstrating that demographic and treatment factors are related to fear.In our study, current smokers (compared with never or ex‐smokers) were more likely to report fear. Although no studies in colorectal cancer survivors have previously examined associations between FCR and smoking status, comparable literature comes from head and neck cancer patients, where it has been observed that smokers report significantly higher levels of fear We found that patients who reported doing more physical activity reported slightly lower levels of FCR. The reasons for these findings are unclear. However, there is good evidence that physical activity reduces levels of general anxiety and depression and improves well‐being in cancer survivors In line with theories of health behaviour change, it is also feasible that those who experience higher levels of fear or perceive themselves to be at higher risk of recurrence are more motivated to change their behaviour To our knowledge, only one other study has reported the associations between physical activity levels and FCR. In a study of 1336 long‐term testicular cancer survivors, there was no association between reported physical activity level and FCR Fifty per cent of patients in our sample expressed some FCR, which adds to the large body of literature showing that FCR is common and pervasive in cancer survivors The finding that more women than men, and younger than older participants, reported fear is consistent with a number of other studies, and the associations between fear and socioeconomic status that were observed in our study have been demonstrated in prostate, testicular and breast cancer survivors A clear and unique strength of the current study is the large population‐based sample of CRC survivors. However, there are limitations that must be acknowledged. The majority of the sample was from a white ethnic group, therefore results may not be generalisable to other ethnicities. The Living with and Beyond Colorectal Cancer PROMS were developed to gather data on a large number of aspects of life after diagnosis. The questionnaire was around 30 pages long, and therefore, it was not feasible to use validated scales to assess every item. As previously mentioned, in future research, where FCR is a key outcome, validated scales should be used, and there are now validated brief scales that could be used when questionnaire length is a consideration This study provided novel data showing that colorectal cancer survivors with poorer health behaviours (those with lower activity levels and those who smoked) were more likely to experience FCR. Further research should replicate findings using more detailed measures of fear, objective measures of health behaviours and aim to identify the direction of the associations.The authors have declared no conflicts of interest.


The unified neutral theory of biodiversity and biogeography has gained the status of a quantitative null model for explaining patterns in ecological (meta)communities. The theory assumes that individuals of trophically similar species are functionally equivalent. We empirically evaluate the relative contribution of neutral and deterministic processes in shaping fruit‐feeding butterfly assemblages in three tropical forests in Africa, using both direct  and indirect approaches . Abundance data were obtained by sampling butterflies using banana baited traps set at the forest canopy and understorey strata. Our results indicate a clear consistency in the kind of species or species groups observed at either the canopy or understorey in the three studied communities. Furthermore, we found significant correlation between some flight‐related morphological traits and species abundance at the forest canopy, but not at the understorey. Neutral theory's contribution to explaining our data lies largely in identifying dispersal limitation as a key process regulating fruit‐feeding butterfly community structure. Our study illustrates that using species abundance data alone in evaluating neutral theory can be informative, but is insufficient. Species‐level information such as habitat preference, host plants, geographical distribution, and phylogeny is essential in elucidating the processes that regulate biodiversity community structures and patterns. This issue has long fascinated ecologists and remains open even today Chesson, . Two maiThe alternative is that communities are unstructured collections of species that have happened to be adapted to the same biome. This neutral theory of biodiversity Caswell,  is a genJM) and the rate (v) at which new species arise at random when an individual mutates to become a new species, a process assumed to be similar to mutation of alleles in genetics. The other fundamental parameter is the migration parameter m : a local community and a metacommunity. The local community consists of an assemblage of trophically similar species that  compete for the same or similar resources in a localized area and is connected to the larger regional pool of species (metacommunity) through dispersal. The metacommunity is maintained by the balance between speciation and extinction. Stochastic ecological processes of birth, death, and immigration are assumed to operate at the local community level. The neutral model requires just two parameters to characterize an ecological community. One parameter is the fundamental biodiversity number θ, which summarizes the speciation process in the metacommunity and is a function of both the metacommunity size  species  from samples of the species abundances. Then, they used the estimated parameters values to generate artificial communities. Finally, the actual test of neutral theory involved the comparison of the predicted ecological patterns or communities with those of the real biological surveys. However, this approach should be regarded as a preliminary step of evaluating a model  fitted the standard neutral model simultaneously to multiple samples of butterfly abundances at local and regional scales, (2) tested the within‐species consistency of vertical stratification across the three forests, and (3) assessed the extent to which species‐specific morphological traits and geographical range size  predicts its occurrence or relative abundance in particular sites or strata.22.12, and lies in a transitional zone between moist semi‐deciduous and moist evergreen zone and forms part of the upper Guinea rainforest—one of the Conservation International global biodiversity hotspots  and one in Uganda . Bia National Park (BIA) is found in the southwestern part of Ghana and borders the forests of Côte d'Ivoire to the west. BIA (06°20′N 06°39′W) covers a total area of 304 km2.2We sampled butterflies using fruit‐baited traps between August 2006 and June 2007 in BIA and December 2006 to November 2007 in BOB. The sampled butterflies were almost exclusively Nymphalidae, which is a well‐defined clade recovered by molecular phylogenetics  for each of the four samples using maximum‐likelihood estimation neutral sampling formulae for multiple samples with varying dispersal limitation  one “combined” but not lumped sample  and (2) three samples of pairs of “combined” local communities; that is, BIA & BOB, BIA & KIB, BOB & KIB. We estimated the neutral model parameters  of observing a particular species abundance dataset D, given the neutral model parameters  and the number of individuals in the sample (J). We estimated the neutral model parameters using the code provided in Etienne  and sample size vector of the observed data (J). We then computed for the real data and each of the 100 simulated communities the maximum log‐likelihood and the dissimilarity (Bray‐Curtis) between local community pairs.The second stage of the direct model evaluation employed Etienne's , 2009a “To assess the extent to which our neutral model generated artificial communities resembling the observed data, we compared the maximum log‐likelihood value of the real dataset to the frequency distribution of the values of the simulated communities. We performed a similar test with the Bray‐Curtis values to assess the extent to which the observed species turnover departs from those expected under neutrality. We would conclude that the observed community is highly unlikely to be structured by neutral processes if the probability of the real data is significantly smaller than most of the artificial datasets Etienne, . If, how2.5Z‐score  for each species.To evaluate the plausibility of the dispersal tendencies impartially, suggested by the neutral model, we obtained species‐specific distributional range information of our sampled species using literature Larsen, . Based o2.6I) of the neutral model inform us about the migration tendencies in the different local communities. Differences in I between local communities for instance suggest either that the local communities differ in the ease with which they are reached by dispersal  or that they differ in the success of establishment of new arrivals in the local community  is simple to calculate and interpret, and based on presence–absence rather than abundance data. All biodiversity indices were computed using the EstimateS software , (2) Wing width WW (distance between the leading and trailing edge of the forewing), (3) Thoracic length LT (section between the head and abdomen), (4) Thoracic width WT (distance between forewing bases), and (5) Abdomen length LA. Due to high colinearity in morphological traits, we condensed some variables into single factors. For instance, thoracic stoutness (WT/LT) was used as a combined effect of thorax length and thorax width. Likewise, wing aspect ratio (4LW2/WW × LW) indexed the forewing parameters. To correct for sexual size dimorphism, morphological data were taken from male specimens only.For each sampled individual, using vernier calipers five morphological parameters were measured: (1) Wing length Using a phylogenetic tree reconstructed from a five‐gene matrix of sequences used in previous species‐level phylogenetic studies  for different samples from the three local fruit‐feeding butterfly communities (Table I) hinted at two kinds of ecological communities, depending on the scale of metacommunity considered. On a “Ghana” metacommunity scale , the θ and I estimates suggested a closed ecological system with low regional diversity (low θ) and low dispersal and/or recruitment limitation (high I). In contrast, on the “Africa” metacommunity scale , the parameter estimates depicted a system of high regional diversity (high θ) and strong dispersal and/or recruitment limitation (low I)  Table . This reIBIA) were consistently the highest, regardless of the metacommunity scale looked at. IKIB‐values on the other hand were always the lowest in all its “combined” samples  suggested low dispersal limitation in BIA compared to BOB. The high IBIA values suggest that either BIA is relatively easier to reach by dispersal  or that it is easier for dispersers to establish themselves in BIA. Within the “Ghana metacommunity,” our neutral model parameters (I) also suggested less dispersal and/or recruitment limitation in the canopy compared to the understorey. In other words, it is relatively easier for immigrants to establish in the canopy than in the understorey. To evaluate the plausibility of these model predictions, we compared the abundance of individuals with their different distribution ranges in the canopy and understorey stratum communities. The results of our distributional range analyses were consistent with those expected under neutrality. There were relatively more individuals (~78%) of taxa with higher dispersal abilities  at the canopy compared to the understorey . When we analyzed the species abundance distributions of canopy and understorey samples separately, we found no significant difference in abundance distribution between the two local communities in Ghana . When either BIA or BOB was contrasted with KIB in the separate stratum analysis, we observed significant differences in the fruit‐feeding butterfly community structures in understorey but not in the canopy (ESM3). Although there were no substantial differences in abundance distribution at the canopy between the three study sites, the two communities in Ghana (BOB and BIA) were more similar to each other in abundance distribution than when either was contrasted with KIB.We observed considerable differences in both the structure and compositions of fruit‐feeding butterfly assemblages found at the two strata. On average, there was ~52% overlap in species presence (measured as Sørensen index) and ~11% overlap when relative abundances are taken into account (measured by Morisita‐Horn) between the understorey and canopy communities. The similarity values were relatively lower  in Ghana; Sørensen 36%, Morisita‐Horn 9%, compared with KIB Sørensen 84% and Morisita‐Horn 15%. The species abundance distribution patterns in the forest understorey were significantly different from those observed at the canopy. This was true for all the three sampled locations  were captured at the forest canopy. Not even a single of the nearly 2,000 individuals (comprising 56 species) of these four genera was captured at the canopy during the entire sampling period in Ghana. An even more entrenched pattern was exhibited by members of the Satyrinae subfamily. This species‐group contributed the largest  to the overall understorey species abundance pool and only 4% of the total Satyrinae individuals trapped were recorded from the forest canopy.The compositions of the butterfly communities were strikingly different between the canopy and understorey at all taxonomic levels: subfamily, genus, and species, and this was consistent across sites Figure , ESM1. GCharaxes and Palla), Apaturinae, Libytheinae, and Biblidinae subfamilies  is an understory specialist. The Apaturinae subfamily in continental Africa is represented by a single species, Apaturopsis cleochares. We recorded 64 individuals of this species in Ghana and all were from the canopy. In KIB, 111 of 138 (84%) individuals of this species were captured in the canopy.In contrast, the canopy was preferred largely by the Charaxinae  and species abundance at the canopy in BIA and when BIA and BOB dataset were pooled, but not when BOB dataset was considered alone Table . Aside t4We investigated the relative importance of neutral and deterministic processes in determining fruit‐feeding butterfly communities in Africa. This is one of the first studies testing neutral theory on mobile animals  are usually locally mono‐ or oligophagous tree foliage feeders that would rarely compete with each other , but there may be some specializations based on proboscis morphology , it is indeed likely that dispersal limitation due to geographic distance plays an important role in determining this general biogeographic pattern.However, our analyses also provide evidence for dispersal limitation that is intrinsically linked to the life history traits of the fruit‐feeding butterfly species found in the two vertical strata. The measured species‐specific thoracic traits appear to predict species abundance in the canopy community in BOB but not in BIA Table . The lacI and θ) estimates for the different local communities were frequently not significantly different from each other at the “Africa” metacommunity level  in one simultaneous analysis led to conflicting conclusions. For instance, at the “Africa” metacommunity scale, the neutral model parameters estimates suggested less dispersal limitation  at the understorey, compared to the forest canopy. This apparently contradicts the inference at the Ghana level and also the results of the species distributional range analysis which indicates a rather opposite trend; more individuals (of species) with wider distributional ranges at the canopy than at the forest understorey Figure . More soel Table .We may explain the weakening in the neutral model parameters estimates’ information as the metacommunity extends from “Ghana” to “Africa” as follows: First, KIB and the two local communities in Ghana (BIA and BOB) may not belong to the same metacommunity. The essence of including KIB in the study was to provide, at least, an approximate answer to a rather unanswered fundamental question; at what distance apart, can two samples or local communities of butterflies (in the current case) be said to belong to the same metacommunity? Etienne  offers aI, either a combination of low θ and high I, or vice versa, and this seems to be the case regardless of whether a single global optimum or several local optima exist for the two parameters. Subsequently, when trying to fit two local communities, the parameter fitting flexibility can be stretched sufficiently to allow a combination of a single θ and two I values that are still compatible with the data. However, when three communities  are fitted simultaneously, the inherent flexibility of the classical  neutral theory may no longer suffice.Secondly, it could be that fitting the three communities simultaneously simply results in a poorer fit than for two communities at a time. The log‐likelihood values of the three local communities are approximately double those for two local communities, irrespective of the stratum considered (canopy, understory, or pooled, Table We have shown that using species abundance data alone in investigating the factors or processes regulating biodiversity community structures and patterns can be informative, but is not sufficient. Neutral theory performed well in identifying dispersal limitation as one key factor structuring fruit‐feeding butterfly communities. By including other useful ecological and evolutionary information, we also identify the vertical dimension of fruit‐feeding butterfly assemblages as an important aspect of community structure. Clearly vertical differences at the scale of meters in fruit‐feeding assemblages are much larger than horizontal ones at the scale of 100s of kilometers. Deterministic dispersal limitation is probably playing a role as some flight‐related morphological traits were found in some cases predictor of species abundance, at least in the canopy stratum. We have shown that among species, differences in the traits of fruit‐feeding butterflies do matter in determining their presence and abundance in ecological communities. These results show that neutral models can be a good starting point to test for the relative importance of deterministic processes and to compare different habitats such as understory and canopy.None declared.KAP and RSE conceived the idea. KAP, WO, and SKO designed the study. KAP and FM collected the field data. KAP and RSE performed the analysis. KAP wrote the manuscript with active participation of WO, SKO, FM, DJL, and RSE. Click here for additional data file. Click here for additional data file.


CO2 uptake. Rapid development of modern crop genetics research now makes it possible to link such canopy models with genetic variations of crops to develop genetics‐based dynamic systems models of canopy photosynthesis. Such models can guide marker‐assisted breeding or genomic selection or engineering of crops to enhance light and nitrogen use efficiencies for different regions under future climate change scenarios.Improving canopy photosynthetic light use efficiency instead of leaf photosynthesis holds great potential to catalyze the next “green revolution”. However, leaves in a canopy experience different biochemical limitations due to the heterogeneities of microclimates and also physiological parameters. Mechanistic dynamic systems models of canopy photosynthesis are now available which can be used to design the optimal canopy architectural and physiological parameters to maximize  Photosynthesis is the primary determinant of plant biomass. Canopy photosynthesis is the sum of photosynthetic rates for all photosynthetic tissues  within the canopy. Since the room to further increase crop harvest index is small, improving canopy photosynthesis and hence biomass production are now widely recognized as a major avenue to increase crop yields. A number of options to increase canopy photosynthesis has been proposed, such as optimizing Rubisco kinetic parameters, increasing the speed of recovery from photo‐protective states, decreasing the antenna size of photosystems, etc.  and Realizing Improved Photosynthetic Efficiency (http://www.ripe.uiuc.edu) projects, the Center of Excellence for Photosynthesis Research (http://photosynthesis.org.au/). Though it is recognized that there are several targets and approaches to improve canopy photosynthesis, most of the current research focuses on photosynthetic efficiency at the leaf level, more efforts should be taken to explore the impacts of different engineering options on the canopy photosynthesis. In fact, there has been many reports regarding the lack of correlation between instantaneous measurements of leaf photosynthesis and crop yields   and maximal rate of photosynthetic electron transfer (Jmax), can be effectively represented in such a model. Furthermore, the sunlit‐shaded model is relatively simple and easy to use  the canopy architectural parameters, for example leaf length, leaf angle, leaf curvature, shape, leaf number, tiller number, planting density etc.; 2) physiological and biochemical parameters, which include leaf chlorophyll content, nitrogen content, content and activation state of key enzymes related to photosynthesis and parameters related to the recovery from the photoprotective state, parameters related to engagement of cyclic electron transfer, parameters related to stomatal responses, etc. The ideo‐type design is to identify optimal combination of these different parameters for a crop grown under a defined geological location (Fig.  physioloAs discussed earlier, the Song et al.  model caHow far are we from realizing such a genetics‐based canopy photosynthesis model for one plant species, such as rice? Rice possibly represents the best studied crop species so far. Many functional relationships between allele variations and canopy architectural parameters have been established already for rice (Zuo and Li 2 uptake for a particular crop species. This optimal allelic combination can then be used to guide parental line selection and marker‐assisted breeding of new cultivars. On the other side, the genetics‐based model can be used to explore the best breeding or engineering strategy for a particular rice accession. In other words, by parameterizing such a genetics‐based model for a particular accession, we can use the model to identify the step exerting the highest control over canopy photosynthesis and further define the optimal allele to use to improve canopy photosynthetic efficiency in this particular rice line.There are two potential applications of using a genomics‐based model to guide the ideo‐type design. On one side, the genetics‐based model can be used to identify the most limiting step or parameter for light or nitrogen use efficiency for a particular crop grown at a particular region Fig. . It can The heterogeneity of microclimate inside a canopy requires using a mechanistic model of canopy photosynthesis to identify the optimal architectural and physiological parameters to support modern crop breeding or breeding. A mechanistic model of canopy photosynthesis is now available which enables one to evaluate impacts of manipulating canopy architectural and physiological parameters on canopy photosynthesis. The model can be used to define region‐specific optimal crop parameters and management practices. The challenge now is to develop a genetics‐based model of canopy photosynthesis by incorporating functional relationship between allelic variations and canopy parameters. Such a genetics‐based model holds great potential in guiding marker‐assisted breeding or genomic selection in the post‐genomics era.None declared.