The segregation of image parts into foreground and background is an important aspect of the neural computation of 3D scene perception. To achieve such segregation, the brain needs information about border ownership; that is, the belongingness of a contour to a specific surface represented in the image. This article presents psychophysical data derived from 3D percepts of figure and ground that were generated by presenting 2D images composed of spatially disjoint shapes that pointed inward or outward relative to the continuous boundaries that they induced along their collinear edges. The shapes in some images had the same contrast (black or white) with respect to the background gray. Other images included opposite contrasts along each induced continuous boundary. Psychophysical results demonstrate conditions under which figure-ground judgment probabilities in response to these ambiguous displays are determined by the orientation of contrasts only, not by their relative contrasts, despite the fact that many border ownership cells in cortical area V2 respond to a preferred relative contrast. Studies are also reviewed in which both polarity-specific and polarity-invariant properties obtain. The FACADE and 3D LAMINART models are used to explain these data. Because occluding objects occur closer in depth than the objects they occlude, border ownership in response to a 3D scene typically coexists with a percept of being closer in depth. The importance of surface border ownership to what may seem nearer to us was already noticed by Galileo  representation from a two-dimensional (2D) stimulus input. During viewing of a natural 3D scene, objects that are closer to the viewer may block or occlude the view of objects that are further away. Boundaries of these occluding objects are perceived as belonging to them, a property called Figure 1). Such spontaneous changes in figure-ground perception occur only under particular circumstances due to competition between multiple, approximately balanced, 3D interpretations of the 2D image.An important problem in visual perception concerns how border ownership assignment occurs in response to 2D pictures, and what role it may play in determining 3D percepts of such pictures. In response to 2D pictures, there are famous examples where the perceptual assignment of surface borders to 3D percepts of foreground and background may be reversible, leading to totally different interpretations of the objects in each representation  model of 3D vision and figure-ground perception, and its further development and extension by the 3D LAMINART laminar cortical model, have explained and predicted many data about how the brain consciously sees 3D surface percepts in response to 2D pictures and 3D scenes, including, but not restricted to, percepts that involve figure-ground perception . Along tNeurophysiological experiments have also been done to record properties of neurons those activities contribute to figure-ground percepts. In this series, Von der Heydt et al. have published important data in a series of neurophysiological experiments about the border ownership properties of neurons in cortical area V2 of monkeys. In particular, absolute binocular disparity are transformed into V2 cells that are sensitivity to relative binocular disparity—namely, the difference in absolute disparity of two visible features  as an example of long-range grouping across opposite contrast polarities in response to polarity-specific inputs from spatially disjoint, oriented detectors. In addition to this boundary-grouping property, the percepts of filled-in surface brightness caused by different inducer configurations had also to be explained.The great pioneering work of Both the boundary grouping and surface brightness properties were simulated in a series of neural modeling articles from Grossberg and his colleagues; e.g., Figure 2, part 2). These studies hark back to earlier observations on the Ehrenstein illusion , considerably weaker groupings are found. Figure 2, part 2, lower left display), which is only perceived in fragmented radial lines of one and the same polarity. These findings suggest that the ways in which contrast polarity variations are locally distributed, and the exposure duration of the stimuli, matter critically in the perceptual genesis of shape illusions. At identical physical luminance, opposite contrast signs within one and the same local inducing element may largely cancel each other out and become less effective in perceptual grouping when viewing durations are not long enough. Analogous effects of local contrast changes on long-range perceptual groupings may be observed in percepts of Glass patterns and reverse-contrast Glass patterns . Key classical data and neural explanations of how mixtures of contrast-dependent and contrast-invariant mechanisms influence percepts ranging from spatial location and hyperacuity ,b to briFigure 5 from Figure 2, part 3. The display in the first row, left panel, generates a percept of unimodal transparency, with an emergent square surface lying in front of four partially occluded pac man figures. The two white pac men are more luminous than the gray background, whereas the two black pac men are less luminous than the background. The illusory Kanizsa square that emerges in this percept thus bridges between opposite-polarity inducers. Opposite polarity inducers also occur in the display in the first row, right panel, but no percept of transparency obtains.Figure 2  illustrates two of these possibilities. A key factor in determining whether such a display looks transparent or not is whether the curved pac man boundary segments at each side of a Kanizsa square boundasry segment have the same contrast, or opposite contrasts, relative to the background. In Figure 3 , the answer is “same” from pac man to background, and transparency is perceived. In Figure 2 , the answer is “opposite” and no transparency is perceived.Many researchers have noted how contrast relations within an image can cause or eliminate a percept of transparency e.g., . GrossbeFigure 2  are derived by removing the outer pac man shapes from the displays in Figure 2 . The resulting identical displays generate a percept of an inner background rectangle that is further away than the open rectangular figure that surrounds it. This kind of display is explained in the Discussion section in the same way that the percepts that are generated by Figure 3 (top row) in the new experiments are explained.The displays in Figure 3) do not change polarity on a spatial scale within the size of individual simple cell receptive fields. Moreover, these displays are conceptually and mechanistically more challenging to explain than previously tested configurations, and the percepts that they generate are quantified in the Experimental Results. The displays used here specifically tested for figure-ground assignment in terms of what is seen as standing out “in front” and what is seen as as “lying behind” by creating configurations in which inducers of varying sign were displayed on either of two sides of a perceptual boundary while the contrast sign within one and the same inducing element was always homogenous. In these configurations, the orientation, direction and polarity of contrast are locally controlled, and may be mixed or switched from one direction and/or polarity to the opposite across the stimulus elements that produce the resulting figure-ground percept. The duration of presentation was not limited in time, as in natural free viewng conditions. An alternative forced choice task similar to that from earlier studies was employed  as well as inducers with inward-oriented edges . The former case induces percepts with the inducers seen “lying behind” within a closed rectangular background region. This situation requires a subtle analysis of the intercortical mechanisms that are responsible for the depth, surface, and persistence properties of such a background region.A key variable of the FACADE theory relative to the orientation of surface-inducing contrast edges was tested by presenting inducing elements with outward-oriented contrast edges  and with the full approval of the corresponding author’s institutional (CNRS) ethics committee. Informed written consent was obtained from each of the participants of the psychophysical experiments. Experimental sessions were run under laboratory conditions of randomized free trial-by-trial image viewing using a Dell PC computer equipped with a mouse device and a high resolution color monitor (EIZO LCD ‘Color Edge CG275W’). This screen has an in-built calibration device which uses the Color Navigator 5.4.5 interface for Windows. The images were generated in Photoshop using selective combinations of Adobe RGB increments to generate contrast inputs see also . The lumTen unpracticed observers, mostly students in computational engineering who were unaware of the hypotheses of the study, participated in the experiments. All subjects had normal or corrected-to-normal visual acuity.Figure 3) consisted of six images with different edge contrast inputs. The luminance of the background was 50.5 cd/m2  in all eight images. The luminance of the black contrast fragments was 1.5 cd/m2  and the luminance of the white contrast fragments was 99.5 cd/m2 , yielding perfectly balanced Weber contrasts (Lfeature–Lbackground/Lbackground) of –0.97 and 0.97 for negative and positive polarities in the six images with the fragmented edge contrasts. The height of the central surfaces was 10 cm on the screen, whereas the width was 12 cm. In the six images with the ambiguous fragmented edge contours, about 50% of the inner surface contour was void of a contrast, so that 50% of the boundary contour had to be completed perceptually . Observers were asked to indicate whether the central surface appeared to “stand in front” of, to “lie behind”, or to be in the “same plane” as the surrounding surface. It was made sure that all observers understood the instructions correctly before an experimental session was initiated.Subjects were seated at a distance of 1 m from the screen and asked to look at the center of the screen. The experiments were run in a dimmed room (mesopic conditions), with blinds closed on all windows. The six images were presented in random order for about one second each, and each image was presented four times in a session. Inter-stimulation intervals were measured. They typically varied from one to three seconds, depending on the observer, who initiated the next image presentation by striking a key on the computer keyboard. The experiment produced a total of 300 observations from 30 trials per subject in an individual session.Systat 11  occurred within a given experimental condition. These frequency distributions, permit conclusions relative to event saliency, and allow plotting probabilities e.g., , based osee also , 2014. TFigure 4) show that the configurations generate a higher event probability for the central surface to be perceived as figure (“in front”) when the local contrast edges of the fragmented contour elements are inward directed, as indicated by the distribution of the response frequencies RF, with the following average values: RF(infront) = 0.83 (SEM = 0.05), RF(behind) = 0.07 (SEM = 0.03), and RF(same) = 0.10 (SEM = 0.04). The configurations generate a higher event probability for the central surface to be perceived as ground (“behind”) when the local edges are outward directed: . These perceptual decisions do not depend on the contrast signs of the local edges. Configurations with negative like-contrasts, positive like-contrasts and mixed contrast polarities produced similar response frequency distributions, with average values as follows: RF(infront) = 0.51 , RF(behind) = 0.48 , and RF(same) = 0.10  for negative like-contrasts; RF(infront) = 0.42 , RF(behind) = 0.43 , and RF(same) = 0.15  for positive like-contrasts; RF(infront) = 0.43 , RF(behind) = 0.42 , RF(same) = 0.15  for mixed polarities.The results  = 228.30, p < 0.001] and “behind” . As expected  = 2.58, NS on response frequencies for “in front” and F = 0.25, NS on response frequencies for “behind”] was observed.ANOVA on the response frequencies for “in front” and “behind” for the two levels of the factor “contrast edge direction” and the three levels of the factor “contrast sign” returned statistically significant effects of “contrast edge direction” on perceptual decisions for “in front” [ed e.g., , no effeFigure 3 images using FACADE and 3D LAMINART model mechanisms . In particular, model mechanisms are summarized with enough detail to achieve a self-contained explanation of the new data using boundary and surface stream interactions within and between cortical areas V1, V2, and V4, while also clarifying the insufficiency of V2 neurophysiological data about border ownership to explain the resulting conscious percepts.A unified mechanistic explanation is here provide of the percepts induced by the Figure 3, boundaries can be completed inwardly between pairs of adjacent colinear inducers. The completion process uses the oriented long-range horizontal cooperation of bipole grouping cells in layer 2/3 of cortical area V2, balanced by shorter-range disynaptic inhibition . Bipole cells can complete boundaries in response to colinear inducers with the same relative contrasts with respect to the background, as in the leftmost two columns of Figure 3, as well as between inducers with opposite relative contrasts with respect to the background, as shown repeatedly in psychophysical experiments . Complex cells, in turn, pool inputs from simple cells in layer 4 of V1 that have the same preferences for position and orientation, but opposite contrast polarities. As a result, bipole cells can complete boundaries around objects that lie in front of textured backgrounds whose relative contrasts reverse along the perimeter of the object. In the present cases, bipole cells complete rectangular boundaries that abut all their inducers.In response to all of the images in ts e.g., . This isFigure 7A). In the images with incomplete boundaries, there are no explicit T-junctions in the image. However, when a rectangular boundary is completed, T-junctions are created at the corners of the colinear inducing contrasts. The bipole cells that lie along the orientation of a completed boundary (the “head” of the T) get more excitatory input than do the bipole cells that lie near the head of the T, but whose orientational preference is along the perpendicular or oblique orientation of the inducing contrast (the “stem” of the T). This is true because the bipole cells that are activated along the head of the T receive strong excitatory inputs from both sides of their receptive fields, whereas the bipole cells that are activated along the stem of the T receive excitatory inputs from just one side of their receptive fields . The more strongly activated bipole cells inhibit surrounding bipole cells more than conversely through a spatially short-range competitive network. As a result, the bipole cells near the head that are along the stem get inhibited. An end gap hereby forms in each boundary near where the stem of a T touches its head .The long-range cooperation and short-range competition processes whereby bipoles complete boundaries are sensitive to any T-junctions that lie along the boundaries that they complete  can be explained in this way  displays appear in front of their surrounding regions, but the percepts of the completed rectangular surfaces in response to the Figure 3 (top row) displays look further away than their surrounding regions.As originally explained in  way see , as can d darker . These cIn order to motivate these theoretical explanations, it is useful to ask the following question: If it is indeed the case that these figure-ground relationships do not depend on having inducers with the same contrast polarity, then why do so many cortical area V2 cells that are sensitive to border ownership also exhibit a particular contrast preference; e.g., Figures 5 and 6) detail how the figure-ground perception process utilizes feedback between the boundary completion process in the interblob cortical stream and the surface filling-in process in the blob cortical stream within V1, V2, and V4 of visual cortex, This feedback enables boundaries and surfaces to generate a consistent percept, despite the fact that they obey computationally complementary laws. This property is called complementary consistency. As will be noted shortly, the mechanisms that ensure complementary consistency also contribute to 3D figure-ground separation. The FACADE and 3D LAMINART models . Only if the boundary of the rectangle is closed, with no significant gaps, can it fully contain its surface-filling in. In the percepts that are generated by the displays in Figure 3, the inducers that are inside or outside these rectangles are surrounded by closed boundaries, since the frame of the image provides another closed boundary that can contain filling-in between it and the bipole-generated rectangular boundary that lies within it. The significance of this fact will be discussed below.In particular, the completed boundaries with their end gaps are projected topographically from the interstripes, or pale stripes, of V2, at which boundaries are completed, to the thin stripes of V2, at which one stage of surface filling-in occurs. When surface filling-in occurs within these boundary inducers, brightness and color can flow out of the end gaps, thereby equalizing the filled-in brightness and color on both sides of the remaining boundaries near these gaps . These feedback signals occur from each active Filling-In DOmain, or FIDO. They are surface contours that are generated by contrast-sensitive on-center off-surround networks that act across position and within the depth represented by each FIDO. These contrast-sensitive networks sense sufficiently large and steep spatial discontinuities in the filled-in brightnesses or colors within their FIDO. They hereby generate surface contour output signals only at the surface positions that are surrounded by closed boundaries. In response to the incomplete inducers in the top row of Figure 3, these regions lie on both sides of the completed boundaries. However, due to the end gaps, surface contour signals are not generated at the boundary positions of the inducers themselves.As filling-in occurs, feedback can occur from the surfaces in the thin stripes to the boundaries in the pale stripes . These feedback signals are delivered to the boundary representations via an on-center off-surround network whose inhibitory off-surround signals act within position and across depth . The on-center signals strengthen the boundaries that generated the successfully filled-in surfaces at the same depth, whereas the off-surround signals inhibit spurious boundaries at the same positions but further depths. This inhibitory process is called boundary pruning. Surface contour signals hereby strengthen consistent boundaries and prune, or inhibit, redundant boundaries.The surface contour output signals generate topographic feedback signals to a subset of the boundary representations that induced them  is inhibited, thereby enabling the horizontal boundaries corresponding to the smaller rectangles to be colinearly completed within depth D2. In response to the picture in Figure 10B, the redundant vertical rectangular boundary is inhibited at depth D2, thereby restoring the boundary fragments at depth D2 that previously were inhibited by the D2 vertical boundaries at end gaps. For this reason, end gaps are not seen in the final depthful percept.Remarkably, by eliminating the spurious boundaries, the off-surround signals that are activated by surface contours also enable figure-ground separation to proceed. They do so by separating occluding and partially occluded surfaces onto different depth planes, after which partially occluded boundaries and surfaces can be amodally completed behind their occluders without interference from the now-inhibited spurious boundaries. For example, the three rectangles in Figure 3, the inducers always appear to lie on a surface behind an occluding surface. Whether the end gaps form inside an illusory rectangle, as in response to the images in Figure 3 (top row) or outside an illusory rectangle, as in response to the images in Figure 3 (bottom row), they will be seen as further away than the surface that contains no end gap boundaries.The above interactions help to explain how, in response to the images in Further analysis is, however, needed to explain how any surfaces are consciously seen—since, as further explained below, V2 boundaries and surfaces are predicted to support recognition, but not conscious seeing, of completed occluders and their partially occluded objects—and also to explain how spurious end cut boundary fragments at both depths do not interfere with the recognition process.Figure 10A are eliminated by surface contours at the further depth D2, they are not eliminated in this way from depth D1. These near-depth boundary fragments are eliminated by the disparity filter , an inhibitory circuit in layer 2/3 of V2 that operates along the line of sight and across depth to help solve the correspondence problem ; see Figure 10A could generate visible surface qualia. How these V2 and V4 mechanisms may cooperate to achieve both effective recognition and seeing were first described in In order to explain how these spurious boundaries are also eliminated, it needs to be explained how additional mechanisms generate the Before summarizing these V2-to-V4 mechanisms for conscious seeing, it is worth noting here that surface contour signals also help to control where the eyes look and to thereby help to regulate how the brain learns invariant object categories. The first role arises because surface contour signals are strongest at the distinctive features of an attended object, such as at high curvature positions along a boundary. In addition to the (thin stripe)-to- feedback that enhances some boundaries while pruning others, a parallel pathway, that is predicted to occur through cortical area V3A, clarifies how these enhanced surface contour positions can also determine target positions of eye movements that explore an attended object’s surface. These signals are proposed to determine where the eyes will look next on an attended surface, and thereby enable inferotemporal cortex to learn view-, size-, and positionally-invariant object categories as the eye movements explore this surface. Thus, the 3D LAMINART model is part of a more comprehensive 3D ARTSCAN Search architecture for active vision wherein 3D boundary and surface representations help to control eye movements for attending, seeing, searching, learning, and recognizing invariant object categories .Figure 10A. Moreover, when the redundant occluding boundaries collapse, the redundant surfaces that they momentarily supported collapse as well. Occluding surfaces are hereby seen to lie in front of occluded surfaces.To set the stage for explaining these V2-to-V4 processes, keep in mind that the boundary pruning process spares the closest surface representation that successfully fills-in at a given set of positions, while removing redundant copies of the boundaries of occluding objects that would otherwise form at further depths. This process illustrates “the asymmetry between near and far”. When the competition from redundant occluding boundaries is removed, the boundaries of partially occluded objects can be amodally completed behind them on boundary copies that represent further depths, as in the percept induced by the display in Figure 5). They are said to be computed within monocular Filling-In-DOmains, or FIDOs. The computation of binocular surfaces that combine brightness and color signals from both eyes is proposed to take place in V4 . These networks are called binocular FIDOs. Here monocular surface signals from both eyes are binocularly matched . The successfully matched binocular signals are pruned by inhibitory signals from the monocular FIDOs . These surface pruning inhibitory signals eliminate redundant feature contour signals at at their own positions and further depths. As a result, occluding objects cannot redundantly fill-in surface representations at multiple depths. This surface pruning process is a second example of the “the asymmetry between near and far”.These surface representations in V2 are depth-selective due to their depth-selective capture by binocular boundaries, but they do not combine brightness and color signals from both eyes  survive within-depth binocular feature contour matching (via pathways 8) and across-depth feature contour inhibition (via pathways 9); (b) are spatially coincident and orientationally aligned with the binocular boundaries (pathways 10); and (c) are surrounded by a connected boundary, or fine web of such boundaries.As in the case of the monocular FIDOs, the feature contour signals to the binocular FIDOs can initiate filling-in only where they are spatially coincident and orientationally aligned with binocular boundaries. Boundary pathways 10 in Figure 11B). This third instance of the asymmetry between near and far is called boundary enrichment. When the vertical right boundary of the vertical rectangle at depth D1 in V4 enriches the boundaries at depth D2, a closed horizontal rectangular boundary is completed at D2, as shown in Figure 11C. This closed boundary can then modally fill-in its surface brightness at D2. These enriched boundaries prevent opaque occluding objects, such as the D1 vertical rectangle in Figure 11C, from looking transparent by duplicating its boundaries at further depths, and thereby blocking filling-in of occluded objects behind them, much as the horizontal rectangle at D2 is prevented from filling-in behind the vertical rectangle at D1 in Figure 11C.In addition, at the binocular FIDOs, the binocular boundaries of nearer depths are added topographically to those that represent further depths . These top–down signals are contour-sensitive and obey the ART Matching Rule . The filled-in surface representations within V4 are predicted to support conscious percepts of the qualia of the unoccluded parts of opaque surfaces, while their boundaries also enhance the strength of the boundary fragments at corresponding positions in V2.The 3D boundary and surface representations that are depicted in Although the present exposition focuses on the perception of opaque surfaces in V4, both unique and bistable transparent percepts have also been explained by these FACADE and 3D LAMINART mechanisms .This article presents additional experimental evidence to complement the fact that many cells in cortical area V2 that are sensitive to border ownership, and thus implicated in the process of figure-ground perception, also exhibit a preferred contrast polarity. The experimental results here, with configurations that match previously established criteria for sign-invariant boundary grouping, show that contrast polarity is often unimportant in determining what part of a 2D picture generates a 3D percept of a closer figure, and what part generates a 3D percept of a further background. Both same-polarity and mixed-polarity sets of figural inducers, with either darker or lighter contrasts compared to the background, can generate the same percepts of relative depth.The results support the hypothesis that V2 is just one stage in a cortical hierarchy that also includes V1 and V4 in the generation of surface percepts with figure-ground properties. Using model interactions among all of these cortical areas, FACADE theory and the 3D LAMINART model explain the psychophysical experimental data here, as well as many other psychophysical data about 3D vision and figure-ground perception in previously published articles. These models also explain many data about identified cells and circuits in these cortical areas, notably, in All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The corpus callosum is the largest white matter structure in the brain, and it is the most consistently reported to be atypical in diffusion tensor imaging studies of autism spectrum disorder. In individuals with typical development, the corpus callosum is known to undergo a protracted development from childhood through young adulthood. However, no study has longitudinally examined the developmental trajectory of corpus callosum in autism past early childhood.The present study used a cohort sequential design over 9 years to examine age-related changes of the corpus callosum in 100 males with autism and 56 age-matched males with typical development from early childhood (when autism can first be reliably diagnosed) to mid-adulthood  (3 to 41 years of age).The group with autism demonstrated a different developmental trajectory of white matter microstructure in the anterior corpus callosum’s (genu and body) fractional anisotropy, which suggests atypical brain maturation in these regions in autism. When analyses were broken down by age group, atypical developmental trajectories were present only in the youngest participants (10 years of age and younger). Significant main effects for group were found in terms of decreased fractional anisotropy across all three subregions of the corpus callosum  and increased mean diffusivity, radial diffusivity, and axial diffusivity in the posterior corpus callosum.These longitudinal results suggest atypical early childhood development of the corpus callosum microstructure in autism that transitions into sustained group differences in adolescence and adulthood. This pattern of results provides longitudinal evidence consistent with a growing number of published studies and hypotheses regarding abnormal brain connectivity across the life span in autism.The online version of this article (doi:10.1186/s13229-015-0001-8) contains supplementary material, which is available to authorized users. Autism spectrum disorder (ASD) is a genetically complex neurodevelopmental disorder that may be marked by atypical functional connectivity within and between particular neural networks and regions in the brain -4. Atypiin vivo neuroimaging studies of autism [Multiple sources of evidence suggest corpus callosum atypicalities in persons with ASD. The corpus callosum was one of the first brain structures observed to be abnormal in f autism -14. Decrf autism , is one f autism ). Functif autism ,17-19. Bf autism , it is wf autism  to examif autism ,23. AD if autism . FA is aFA has been the most common measure of white matter microstructure used in DTI studies of autism , but it A number of recent studies have used DTI methods in cross-sectional examinations of white matter in ASD, and one of the most consistent findings is decreased FA and increased MD of the corpus callosum in ASD . In addDespite the consistency of these results, there are a handful of studies that have found either increased FA or no group differences in corpus callosum microstructure ,35-37. TOnly longitudinal studies can satisfactorily examine developmental trajectories, and to date, only one longitudinal DTI study in ASD has been conducted in very young children. Wolff and colleagues  found thThe present study extends the cross-sectional study of Alexander and colleagues  to longiThe study employed a cohort sequential design  , which sThe study protocol was approved by both the University of Utah and University of Wisconsin-Madison Institutional Review Boards. Consent was obtained for all participants, and for participants under the age of 18 years, both parental consent and participant assent were obtained. All TDC and the majority of the ASD participants were community ascertained. Participants for this study included 100 males with ASD and 56 males with typical development between the ages of 3 and 41. These participants were selected from participants in the broader longitudinal neuroimaging study (110 ASD and 78 TDC). Participants who met criteria for ASD or TDC at enrollment but who were found to be atypical at future time points , participants who did not have an age match in the other group, or participants whose scans did not meet quality control checks (see ‘Imaging protocol’ below) were excluded. Time 1 data from a subset of these participants were reported in the study by Alexander and colleagues . The parStandard scores in verbal, nonverbal, and overall ability  were assessed in the participants with ASD and the participants with typical development at each of the four time points. At time 1, participants completed the Differential Abilities Scales , Wechsleb = 1,000 s/mm2 and a single b = 0 image. Sixty contiguous axial slices 2.5 mm thick were acquired over the cerebrum and cerebellum . The acquisition was repeated four times  in 6.5 min. Thirty-two young participants with ASD received sedation for scanning at time 1, and fifteen of these same participants received sedation again at time 2. No sedation was used at times 3 or 4. Sedation, using a combination of remifentanil and propofol, followed a strict clinical protocol approved by the institutional review board by the University of Utah and performed by an onsite faculty anesthesiologist. No complications occurred. In several cases, rehearsal was used to practice lying in the scanner.Participants were recruited and scanned at the University of Utah. Image processing and analyses were conducted at the University of Wisconsin-Madison. A total of 346 scans were obtained from the participants (221 ASD scans and 125 TDC scans). Magnetic resonance images were acquired on a Siemens Trio 3.0 Tesla Scanner  at the University of Utah. At all four time points, the DTI acquisition used a product single-shot, spin-echo, echo planar imaging (EPI) pulse sequence with diffusion weighting, performed with bipolar gradients with dual-echo refocusing to reduce eddy currents . ParalleBetween times 1 and 2, the scanner hardware  and software were upgraded (which accounted for the TE change described above). To account for systematic differences in the imaging measurements at time 1 versus times 2, 3, and 4 due to this upgrade, a linear regression ‘head coil’ variable  was created and included in all statistical models. To further ascertain that the present longitudinal results were not solely due to noise from this upgrade, all patterns of results were tested by running time 1 analyses only and then by dropping time 1 and using only times 2, 3, and 4 data  were corrected for distortion, translation, and rotation from eddy currents and bulk head motion using an affine registration tools implemented in the fMRIB FSL software library . To excl1, λ2, λ3) were computed from the estimated diffusion tensors. Maps of the MD , FA , AD , and RD (λ3 diffusion perpendicular to the major eigenvector) were calculated. The units for MD, RD, and AD were square millimeter per second, scaled 10−3. Quality control checks were manually performed on the DTI images, looking for instances of slice intensity banding, FA hyperintensities, frontal lobe distortions, or blurring that could affect the analyses. Additionally, the white matter masks were checked to make sure that they were adequately covering the white matter in each brain scan. After quality control checks on the data, all but six ASD scans (98.6%) met the quality control standards.The eigenvalue maps (λP = 0.84) nor across ages (P = 0.48), but TMI was used as a covariate in all analyses to ascertain that group differences were not due to head motion. Additionally, to account for possible physiological noise, we computed the signal-to-noise ratio (SNR) for each region of the corpus callosum in each scan by taking the mean signal from all the volumes of the scan (b0 and the diffusion weighted volumes), dividing the signal by the noise in a voxel  for each participant as described in ,63. The endix of ), and thA population-specific template was estimated iteratively, aligning all the subjects using affine and diffeomorphic diffusion tensor registration implemented in DTI-TK . To segmTo assess the reliability of the longitudinal DTI measurements, an intraclass correlation coefficient (ICC) was calculated for each statistical model by taking the within-individual variability in the mixed-effects model and dividing it by the sum of within and between variability . BecauseTo examine the reliability of the present DTI data, we calculated ICCs for each DTI measurement of each subregion  and their longitudinal trajectories. FA measurements obtained from each of the three subregions of the corpus callosum  can be viewed in Figure A generalized additive mixed-effects model analysis suggested decreased mean FA values in individuals with ASD in each of the corpus callosum subregions  time 1 only (using 8-channel head coil), 2) times 2 to 4 (using the 12-channel head coil), 3) without participants who were on centrally active medications, and 4) without participants who were sedated during scanning. The figures and tables of results for these differential analyses can be seen in the supplementary materials . This atypical developmental trajectory in ASD was characterized by atypically high FA values in early childhood that decreased with age, crossed the TDC curve, and plateaued below the FA values of the typically developing group. Follow-up analyses conducted within particular age brackets reiterated that the developmental slope of FA was atypical only in the youngest ASD cohort (less than 10 years of age). This result is consistent with the only previous longitudinal investigation of corpus callosum microstructure in ASD , which el. study , but ourl. study ,35,75,76l. study ,78.We also found atypical longitudinal development of corpus callosum microstructure during adolescence and young adulthood. FA of the genu and body was persistently decreased; rate of change was similar to the TDC group. Cross-sectional studies of ASD have consistently found decreased FA in adolescents and adults with ASD -31,79-85in vivo DTI with more specific imaging measures of myelination, including multicomponent relaxometry and magnetization transfer [In addition to differences in FA, the only corpus callosum region that the ASD group exhibited significantly increased MD, RD, and AD was in the splenium, although the effect size of the splenium AD was much smaller than the other effects. Further, there was a significant group difference in the developmental trajectory of splenium MD, but this should be interpreted with caution because our follow-up analyses did not converge to show that this was a significant effect in any of our age brackets. Although DTI measures do not specify the type of white matter microstructural changes that may be present , the present results  are consistent with abnormal myelination in ASD . Speculatransfer . Althouget al. noted that group differences in the corpus callosum DTI metrics appeared to be driven by a minority of ASD participants [It is further important to note that we observed great variability in the corpus callosum DTI values of the ASD group. As can be seen in the individual data points presented in Figure icipants . The preCorpus callosum group differences have been the most consistently reported finding in the DTI in ASD literature . AdditioAlthough there is converging evidence across multiple lines of research that suggest that the corpus callosum is commonly affected in ASD, the critical question of how corpus callosum atypicalities relate to the core behavioral symptoms of ASD remains. The similarity in some of the cognitive and symptom profiles between individuals with callosal agenesis and individuals with ASD is intriguing -93. HoweThere are several potential limitations associated with this present study. First, a limitation of the cohort sequential design is the use of a multiple cohorts across ages, which may make this design less sensitive to age-specific changes during narrow windows of development. However, the cohort sequential design allowed us to more rapidly answer this key longitudinal question in autism than would have been possible with a longitudinal panel design, and the accelerated design also minimized potential cohort-related confounds . A seconThe present study used DTI methods to longitudinally examine the white matter microstructure of the corpus callosum in individuals with ASD compared to typically developing controls (ages 3 to 41 years). The results suggest that corpus callosum microstructure  develops atypically in autism before the age of 10 years and that this early atypical development becomes a persistent group difference in later childhood, adolescence, and adulthood. Because the corpus callosum is the largest white matter tract in the brain, corpus callosum atypicalities likely have lasting effects on many aspects of cognition and brain synchronization in ASD.Additional file 1:Supplementary materials for differential analyses. This file contains five tables (Tables S1-S5) and eight figures  that present the results of our differential analyses.Additional file 2:Projections of the genu, body, and splenium subsections of the corpus callosum. This picture file demonstrates the cortical projections of the genu (red), body (green), and splenium (blue) subregions of the corpus callosum in our study’s template space.


GSTM1, GSTT1 and GSTP1, which influence antioxidant defences.Increased bronchial responsiveness is characteristic of asthma. Gas cooking, which is a major indoor source of the highly oxidant nitrogen dioxide, has been associated with respiratory symptoms and reduced lung function. However, little is known about the effect of gas cooking on bronchial responsiveness and on how this relationship may be modified by variants in the genes GSTM1, GSTT1 and GSTP1-rs1695. Information on the use of gas for cooking was obtained from interviewer-led questionnaires. Effect modification by genotype on the association between the use of gas for cooking and bronchial responsiveness was assessed within each participating country, and estimates combined using meta-analysis.The study was performed in subjects with forced expiratory volume in one second at least 70% of predicted who took part in the multicentre European Community Respiratory Health Survey, had bronchial responsiveness assessed by methacholine challenge and had been genotyped for GSTM1 significantly modified this effect , with GSTM1 null subjects showing more responsiveness if they cooked with gas. No effect modification by GSTT1 or GSTP1-rs1695 genotypes was observed.Overall, gas cooking, as compared with cooking with electricity, was not associated with bronchial responsiveness . However, GSTM1 null genotype. This may reflect the oxidant effects on the bronchi of exposure to nitrogen dioxide.Increased bronchial responsiveness was associated with gas cooking among subjects with the  GSTM1, GSTT1, GSTP1)?Is the relationship of bronchial responsiveness with use of gas for cooking modified by variants in oxidative stress-related genes , GSTT1 (null genotype) and GSTP1 (rs1695[G]) modify the association between gas cooking and BR.Nitrogen dioxide is an oxidant species that induces upregulation of the expression of T helper type 2 cell cytokines and ICAM1 as well as neutrophilic inflammation in bronchial epithelium.Data included in the following analysis were collected from subjects participating in the ECRHS, an international multicentre cohort study designed to identify risk factors for asthma.Ethical approval for the study from local research ethics committees and written consent from subjects were obtained.GSTM1, GSTP1 and GSTT1. Genotyped subjects were slightly older than those who were not genotyped, and as there were some between-country differences in response to genotyping, the distribution by country was not the same between the two groups (see online supplementary T1). DNA was extracted from blood samples using a commercial kit . At the Centre for Genomic Regulation , GSTM1 and GSTT1 null genotypes were determined by PCRs, and GSTP1 polymorphism  was genotyped using a specific pyrosequencing assay.GSTP1-rs1695 deviated from Hardy–Weinberg equilibrium (HWE) in France and Germany. HWE could not be calculated for GSTM1 and GSTT1 because our data did not distinguish between one and two copies of the variant allele. Population stratification was assessed with 26 unlinked markers using the genomic control approach and the EIGENSTRAT software. There was no evidence of relevant population stratification.20In total, 19 of 29 centres (8109 out of 10 933 subjects) in ECRHS II collected blood samples for genotyping . HoweverParticipants were asked, “What kind of stove do you mostly use for cooking?” Subjects who answered ‘gas (gas from the mains)’ or ‘gas (gas from bottles or other non-mains source)’ were classified as being exposed to gas cooking. Those who answered ‘electric’ or ‘microwave’ were classified as the reference group. Subjects in four centres in Norway (N=436) and Sweden (N=643) were not included in the analysis because the use of gas for cooking in these countries is extremely low (<1%). Subjects who used other types of stove, such as ‘coal, coke or wood (solid fuel)’ or ‘paraffin (kerosene)’, were also excluded .BR was assessed by methacholine bronchial challenge test as previously described.GSTM1, GSTT1 and GSTP1 modify the association between gas cooking and BR, linear regression models with the slope as the continuous dependent variable were fitted for each of the three genes and a gene–gas cooking interaction term was entered in the models. GSTM1 and GSTT1 were modelled as dichotomous variables (‘null’ vs ‘present’), whereas GSTP1-rs1695[G] was modelled per number of minor alleles  under an additive mode of inheritance. The coefficient (β) for the interaction term between each of the genes and gas cooking was estimated for the whole sample (‘random’ and ‘enriched’) in each country and then combined using a random effects meta-analysis.To assess whether genetic variants in Cladosporium herbarum), total IgE, baseline FEV1 expressed as a standardised difference from an internally derived sex-specific predicted value and baseline FEV1 as a percentage of FVC. An age–sex interactionGSTM1 or GSTT1, and 2444 for models assessing the interaction with GSTP1 , most were from Spain, France and Germany, and cooking with gas was more prevalent than cooking with electricity . GSTM1 aGSTM1, with GSTM1 null subjects showing a strong significant association of increased BR with use of gas for cooking  . The interaction was present among males  and females . Adjustment of these models for the type of sample, a priori confounders and for the other genes did not materially alter the estimates for the interaction (see online supplementary T3). The association of BR with use of gas for cooking was not modified by GSTT1 or GSTP1-rs1695 genotypes (None of the three genetic variants was significantly associated with BR (see online supplementary T2). Overall, gas cooking, versus cooking with electricity, was also not significantly associated with BR . However, this association was different depending on p=0.017) . There wGSTM1 was present and of the same magnitude in the ‘random’ sample and the smaller sample that was enriched in symptomatic subjects .GSTM1 was not present , and there was some evidence of minor heterogeneity between countries in this relationship . As this post hoc analysis included participants from Belgium (who did not conduct BR measures at the follow-up), the analysis was repeated excluding participants from that country but still no interaction was observed . Exclusion of Spain (country contributing the most to heterogeneity of results at ECRHS I) did not alter results . Of note, Spain showed the largest change in the use of gas cooking between the two surveys . Some subjects changed cooking fuel between surveys . Restriction of the analysis to those who used the same fuel at baseline and follow-up still did not show an interaction between gas cooking and GSTM1 in the ECRHS I. As in the second survey, interactions between gas cooking and GSTT1 and GSTP1-rs1695 were not observed in the baseline survey .Some analyses were repeated using all available data obtained at ECRHS I . The interaction between gas cooking and GSTM1 null genotype and who cooked with gas, compared with those who cooked with electricity, throughout the period of follow-up showed some sign of greater increase in BR . There was no evidence that GSTT1 or GSTP1-rs1695 genotypes modified the change in BR due to the use of gas .Overall, there was no evidence that use of gas over the approximately 8 years of follow-up was associated with greater increases in BR . However, participants with the GSTM1, that is, gas cooking was associated with increased BR among subjects with the null genotype for GSTM1, but not among carriers of that gene. Furthermore, the interaction was stronger among those who cooked in conditions where higher exposure levels are expected . To our knowledge, this is the first study to assess and report this interaction.In this population-based study of adults of European ancestry and FEV1 at least 70% of predicted, the association of BR with gas cooking was different depending on One of the strengths of the present study is its large population sample derived from several areas of Europe. There was some loss to follow-up between the two surveys but little reason to believe that this would result in the detection of false gene–environment interactions as loss to follow-up in subjects with respiratory symptoms and cooking with gas, or electricity, are unlikely to be related to genetic characteristics. The general decrease in prevalence of cooking with gas across countries, from the first to the second survey, probably reflects a change in house building trends and is also unlikely due to genetic makeup. A strength of the study was the use of a standardised protocol across participating centres to perform methacholine bronchial challenge tests.26GSTT1 and GSTP1, we had enough power (>90%) to detect an interaction with GSTM1 in both surveys. However, we only observed it in ECRHS II. This may be due to heterogeneity between countries being lower in ECRHS II or to lack of information on some environmental exposure that might have confounded the effect of gas cooking in the first survey. It may also be due to better insulated houses and consequently increased concentration of indoor gases in the second survey, but we do not have data to confirm or reject this argument. We could not detect within-survey differences between age groups, although there was some evidence of increasing BR due to gas cooking among older participants with GSTM1 null genotype, when comparing the second with the first survey (data not shown). While the change in cooking fuel between the two surveys may, in part, explain that the interaction was statistically significant only in the most recent survey, we should not exclude the potential role of other genes, epigenetic mechanisms and cellular phenomena during normal ageing. Adjustment for GSTT1 and GSTP1-rs1695 did not make a difference on the estimates for the interaction between GSTM1 and gas cooking, nor did adjusting for NQO1-rs1800566, which has been proposed to interact with GSTM1 and air pollutants on lung function (data not shown).Despite the fact we were underpowered to detect interactions between gas cooking and GSTM1 and some environmental pollutants on asthma and airway obstruction. In a study of school children, prevalence of asthma was associated with maternal smoking during pregnancy, but only among children with GSTM1 deletion.GSTM1, but not among carriers of at least a copy of that gene.GSTM1, but not among those with at least a copy of GSTM1.33There is evidence of the influence of interactions between GSTM1 is located on chromosome 1 where it encodes a phase II enzyme involved in the detoxification of electrophilic xenobiotics, by conjugation with glutathione, and the protection against oxidative and nitrosative stress.GSTM1 in normal human bronchial epithelial cells has shown that this gene may regulate diesel exhaust particle-induced expression of IL-8 and IL-1β by modulation of reactive oxygen species.gstm1 have low ability to conjugate 1,2-dichloro-4-nitrobenzene with glutathione.GSTM1, common in approximately half of the population of European ancestry, may lead to an increased susceptibility to the effects of gas cooking, which is the main source of indoor nitrogen dioxide and particulate matter.2GSTM1. This may be an indication of the oxidant effects on the bronchi of exposure to nitrogen dioxide originating from cooking with gas. Further larger studies are recommended to confirm this finding and better understand its mechanism of action.In summary, increased BR was associated with gas for cooking among subjects with the null genotype for


UMOD), renin (REN) and hepatocyte nuclear factor-1β (HNF-1β) genes as well as linkage to chromosome 2p22.1-21 have previously been identified. Knowledge of the genetics of this phenotype has provided important clues to developmental pathways in the kidney.Familial juvenile hyperuricaemic nephropathy is a rare inherited nephropathy with genetic heterogeneity. Categorised by genetic defect, mutations in uromodulin (We report a novel phenotype, with the typical features of hyperuricemia and renal deterioration, but with the additional unexpected feature of unilateral renal hypoplasia. Mutation analyses of the existing known genes and genetic loci were negative indicating a new monogenic cause. Interestingly two cousins of the index case did not share the latter feature, suggesting a modifier gene effect.Unilateral renal hypo/aplasia is usually sporadic and relatively common, with no genetic cause to date identified. This reported pedigree reveals the possibility that a new, unknown renal developmental gene may be implicated in the FJHN phenotype. Firstly, there is the penetrant feature of a single hypoplastic kidney alongside a normal sized kidney in three out of the five affected family members. A unilateral hypoplastic kidney in a single case of FJHN has previously been described . To our ogenesis ,13-15. NRenal hypoplasia is defined as abnormally small kidneys (greater than two standard deviations below the expected mean when correlated with age or other growth parameters) with normal morphology but reduced nephron number . Whilst PAX2, SIX2, BNP4, SALL1, UMOD and TCF2. Unilateral renal hypoplasia has been reported as a single feature of low penetrance in a small number of studies; however extrarenal features suggestive of FJHN are lacking [TCF2 and UMOD have been linked to HNFJ through mutation screening [Nephrourogenesis commences at four weeks of gestation, when signals from metanephric mesenchyme (MM) initiate the formation of the ureteric bud (UB) from the nearby Wolffian duct. Contact and interactions between the cell lineages subsequently lead to proliferation and epithelial transformation of the MM, which goes on to form a functional nephron. At the same time, branching of the distal UB occurs to form the mature collecting system ,16. The  lacking ,20. Of tcreening ,6,8,9,12TCF2 is the gene encoding for HNF1β, an embryonic transcription factor expressed in the liver, kidney, and pancreas [HNF1β have been identified in families with FJHN particularly those with atypical features such as renal cysts or other anomalies of renal development. Although the mechanism for the phenotype in these patients is unclear, it has been speculated that HNF1β which is expressed at an early stage in the proximal tubule may reduce transcription of human urate transporters, although this is yet to be proven [pancreas . Mutatioe proven .HNF1β is known to induce UMOD, a gene encoding the protein uromodulin. Several uromodulin mutations have been discovered in patients with the FJHN phenotype, now categorised as FJHN type 1 [N type 1 ,6-9. AlsN type 1 ,21. DefeREN gene in the juxtaglomerular cells over time results in renin-angiotensin dysregulation with reduced renin synthesis, apoptosis and nephron loss leading to progressive renal failure [Mutations in the renin gene have been identified in three families with a FJHN phenotype. It is proposed that high expression of the mutant  failure . While n failure .et al. identified a genetic region of up to 28 candidate genes on 2p22.1-21 linked to five families with FJHN which has since been termed FJHN3. It is speculated that mutations within this region may account for the majority of FJHN cases that prove negative for UMOD, REN or HNF-1β mutations [Piret utations . While iURAT1 and hUAT has been identified [Returning to our index case and his family, the penetrant feature of a renal developmental anomaly in three out of five affected members may implicate an as yet unidentified developmental gene in the pathogenesis of FJHN. The genetic heterogeneity of the condition is well described, and our pedigree supports the possibility of a new, unidentified gene implicated in both the FJHN phenotype as well as the development of renal hypoplasia ,4,9. Othentified .What remains to be identified is the causal gene for the phenotype described in our index case and his affected family members. The overwhelming likelihood here is of a dominantly inherited single gene defect, rather than multiple genetic defects or multifactorial elements at play due to the precise inheritance seen and highly penetrant feature of a unilateral hypoplastic kidney. Interestingly, the lack of a unilateral small kidney in the two cousins of our index case, who both have exactly the same renal impairment phenotype otherwise, would imply the effect of a modifier gene to cause this feature. The genetic identification may have major implications for the commonly seen isolated hypoplastic/absent kidney phenotype in children and adults, for which no candidate gene has to date been identified. It is important to report this pedigree, as the collection of further pedigrees worldwide will help simplify identification of the responsible gene, particularly given the possibility now of complete exome sequencing for affected patients. This will also provide insight into the disease processes present in FJHN, enabling a greater understanding of how a phenotype that often presents later in life is associated with a renal developmental anomaly. Discovery of the mutation will provide an explanation to family members; will allow appropriate counselling for future generations as well as enable clinicians to implement management strategies to prevent disease progression.Written informed consent was obtained from the patient for publication of this Case report. A copy of the written consent is available for review by the Editor of this journal.UMOD: Uromodulin gene; HNF1β: Hepatocyte nuclear factor-1β gene; FJHN: Familial juvenile hyperuricaemic nephropathy; GFR: Glomerular filtration rate; MM: Metanephric mesenchyme; UB: Ureteric bud; RDGs: Renal developmental genes.None declared. Genetic analysis performed by HM was supported by the Cambridge Biomedical Research Centre, and the National Institute of Health Research.LAP (corresponding author) helped to design the manuscript. She researched, drafted and revised the final manuscript for submission. MM was involved in the design of the manuscript, performed data collection on the pedigree and helped to revise the manuscript for submission. AB interpreted the data from the exome sequencing analysis and helped to revise the manuscript. HM, KB and SA performed exome sequencing on DNA from the index case, and helped to redesign and revise the manuscript for re-submission. MAS created the original concept for the manuscript and critically revised the manuscript for important intellectual content. All authors have given their approval for publication of the manuscript. MAS has given overall approval for this version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/15/76/prepub


Escherichia coli lipopolysaccharide (LPS) as innate immunity agonist, ovalbumin as adaptive immunity agonist, or both agonists in three different udder quarters of lactating cows. There was a significant amplification of the initial milk leukocytosis in the quarters challenged with the two agonists compared to leukocytosis in quarters challenged with LPS or ovalbumin alone. This synergistic response occurred only with the cows that developed the ovalbumin-specific inflammatory response, and there were significant correlations between milk leukocytosis and production of IL-17A and IFN-γ in a whole-blood ovalbumin stimulation assay. The antigen-specific response induced substantial concentrations of IL-17A and IFN-γ in milk contrary to the response to LPS. Such a synergy at the onset of the reaction of the mammary gland suggests that induction of antigen-specific immune response with bacterial antigens could improve the initial immune response to infection, hence reducing the bacterial load and contributing to protection.The mammary gland is able to detect and react to bacterial intrusion through innate immunity mechanisms, but mammary inflammation can also result from antigen-specific adaptive immunity. We postulated that innate and adaptive immune responses could synergize to trigger inflammation in the mammary gland. To test this hypothesis, we immunized cows with the model antigen ovalbumin and challenged the sensitized animals with either  Mastitis is the most costly infectious disease in dairy cows . VaccinaEscherichia coli, Streptococcus uberis or Staphyloccus aureus [S. aureus infection [IL-17A is produced or its encoding gene overexpressed in the bovine MG during mastitis caused by s aureus . Mammarys aureus . By usinnfection , 13. Taknfection , 15. Durnfection . In vitrnfection . This raE. coli lipopolysaccharide (LPS) or both stimuli in three different udder quarters of the immunized cows. The subsequent milk leukocytosis was monitored to reveal the potential amplification of LPS-induced cell recruitment by sensitization to ovalbumin, and milk somatic cell counts (SCC) were correlated with the production of IL-17A and IFN-γ in an ovalbumin-specific whole blood stimulation assay.The main objective of this study was to investigate whether a protein antigen and a MAMP could synergize in the induction of mammary inflammation. To test this hypothesis, we immunized cows with the model antigen ovalbumin before intramammary challenge with either ovalbumin, All animal experiments were performed with approval from the Comité d’éthique pour l’expérimentation animale du Val de Loire (CEEA VdL), protocol number 2012-10-12 V2. Animal studies were compliant with all applicable provisions established by the European directive 2010/63/UE.Lactating cows were in their first or second lactations. They had at least three healthy quarters, defined as quarters that did not shed bacteria in milk  and with somatic cell counts (SCC) less than 150 000 cells/mL milk. In most cows the four quarters were devoid of bacteria, but the shedding of coagulase-negative staphylococci from one quarter was tolerated when not accompanied by clinical signs . Cows with clinical mastitis were excluded from the selection.A total of 17 Holstein female bovine animals was used in the experiments, of which 13 were challenged through the teat canal. They were maintained in their farm  during the experiments, and remained in the herd after completion of the study. They were not isolated from their herdmates during the experiment. All cows were milked two times daily and were housed in a loose-housing cowshed. They were fed a diet of hay, silage and concentrate. Intramammary injection of antigen, LPS or both induced either a subclinical inflammation or a clinical mastitis of moderate intensity. Recovery  occurred between 24 to 48 h post-injection. Analgesics were not used because the cows did not display overt pain manifestations at milking and milk sampling. Anti-inflammatory drugs were not administered since inflammation was the reaction under scrutiny. Cows were milk sampled seven times during the intramammary challenge (see below) and blood samples were taken on five occasions.E. coli, devoid of TLR2 agonists, InvivoGen), or the combination of 0.2 μg LPS with 0.5 μg ovalbumin were infused into the lumen of different quarters of each of the three cows through the teat canal with asepsis precautions after the evening milking  by the intramuscular route. Two antigens were used to see whether low responder cows to OVA were also low responders to another unrelated antigen. A booster injection was administered 30 days later by using the same regimen except that the antigen dose was 10 μg. The cows were challenged with an intramammary infusion of either 0.5 μg ovalbumin, 0.2 μg LPS, or the combination of 0.5 μg ovalbumin with 0.2 μg LPS into three different healthy quarters. The quarters were sampled before and 8, 16, 40, 64, 88 and 160 hours post-infusion (hpi). In addition to these 10 cows, we included in the immunization schedule four non-responders to ovalbumin identified in a previous experiment [For the main experiment, 10 cows were immunized with 50 μg OVA and 50 μg hen egg lysozyme (Sigma) emulsified in Montanideperiment .ELISAs for TNF-α, CXCL8 and IL-17A were performed as described previously , 20. IFN2 for 3 days. Supernatant were then harvested and stored at -20°C in 96-well plastic storage plates (Greiner™ bio-one) until assayed for cytokine content.The antigen-specific T cell response was assessed as described . Blood wStatistical analyses were performed with the Prism software . Comparisons of paired samples were done with the Friedman’s test followed by Bonferroni post-test comparison when appropriate. Correlation analyses were done with the Spearman’s rank test. A probability level of < 0.05 was considered significant.In the preliminary experiment, the doses of either LPS or ovalbumin for the intramammary challenge were based on previous authors’ experience. The aim was to check that the pre-selected doses induced only a mild inflammatory response, and that they were suitable to disclose a potential synergistic inflammatory effect of the LPS-ovalbumin combination. To this end we used three cows that had proven in a previous experiment to be reactive to ovalbumin after sensitization . The monIn the main experiment, 10 cows were immunized with OVA and HEL . LPS indConcentrations of the chemokine CXCL8 and of the cytokines IL-17A, IFN-γ, TNF-α and IL-10 were assessed in milk samples taken at 0, 8, 16, 48 and 64 hpi from the mammary quarters infused with either 0.2 μg LPS, 0.5 μg OVA, or the combination of these two stimuli. Increases in CXCL8 concentrations coincided with the initial increases in milk cell concentrations in quarters infused with LPS, alone or with OVA Figs  and 4. IIn a previous study we had found that the ovalbumin-specific production of IL-17A and IFN-γ correlated with the milk leukocytosis induced by the infusion of ovalbumin in the mammary gland of sensitized cows, and thus that the antigen-specific WBA could be used as a predictor of mASR . In the The correlation between IL-17A and IFN-γ production in the WBA at 45 days post-first immunization was barely significant , in accordance with our previous study . ConcentAnalysis of correlations between milk SCC data shows that in quarters infused with LPS the SCC at 8 and 16 hpi correlated with the SCC in quarters infused with ovalbumin and LPS at 8 hpi but not at 16 hpi . This liIn our previous studies we had been surprised by the proportion of low or non-responders to ovalbumin, which was about one out of 3 cows , 18. ThiWith the relatively low dose of LPS used in this study, there was a clear-cut segregation between high and low responders to intramammary infusion of LPS . Milk leAttempts to develop vaccines against mastitis have primarily been focused on the induction of antibodies to bacteria and bacterial components , 21–23. The main objective of this study was to investigate whether the innate immune response triggered by MAMPs can be modulated by the adaptive immune response to a sensitizing antigen in the MG. The MG is sensitive to MAMPs such as LPS, lipoteichoic acid, peptidoglycan subunits and lipoproteins –30. MammIt is of interest that in the milk of responder cows, the synergy between responses to LPS and ovalbumin was manifest at 8 hpi, when the response to ovalbumin alone was not detected by the SCC or CXCL8 or cytokine concentration increases. This suggests that the events behind the synergy, including the antigen-specific response, had been in effect before 8 hpi. These events took place in the mammary tissue and thus escaped the monitoring of milk parameters. What was measured in milk could rather be the consequence, not the cause, of the inflammatory response. An example is CXCL8, which can be produced by MEC but also by recruited leukocytes , concentIn this study, a dose of 0.5 μg OVA induced sizeable increases in IL-17A and IFN-γ concentrations in milk. Previously we reported comparable concentrations of these two cytokines following intramammary infusion of higher amounts (10 to 25 μg) of ovalbumin in the milk of sensitized responder cows . A dose-A secondary objective of this study was to test whether the high proportion of non-responder cows is specific to this antigen or whether it is a more general phenomenon. Co-immunization with ovalbumin and HEL led also to a sizeable proportion of low antigen-specific WBA responders. There was a high correlation between the responses to ovalbumin and HEL. This tends to rule out the MHC class II gap hypothesis, although the limited number of antigens and animals calls for caution. The non-responder issue could be of importance if it occurs also with more complex vaccine preparations. Whatever it may be, it appears that the antigen-specific WBA is instrumental in detecting non-responders, and a better understanding of its underlying mechanism could constitute a lead to decipher the reasons behind the absence of response. Delayed-type hypersensitivity is under genetic control and has been shown to be heritable in cattle , which oE. coli mastitis, the low responder cows faring better than the high responders [E. coli mastitis.Besides non-responders to ovalbumin and HEL, there were also low responders to LPS among the 14 cows under experiment. The use of a low dose of LPS was probably instrumental in revealing the inter-animal difference in susceptibility. There was no correlation between the response to LPS and the response to ovalbumin in terms of milk leukocytosis, which is not surprising since the two agonists are supposed to act through innate and adaptive arms of the immune system, respectively. There was even a trend for a negative correlation between SCC induced by LPS at 8 hpi and 16 hpi and IFN-γ concentrations in the WBA . There wsponders . This insponders . It woulIn conclusion, this study shows that the innate immune response to MAMPs can synergize with the adaptive immune response to a protein antigen, resulting in an amplified milk leukocytosis at the onset of the inflammatory reaction. Enhancement of the innate immune response to bacteria by adaptive immunity is effective at the start of the inflammatory response. The magnitude of the cell recruitment is likely to be associated with qualitative modifications of the immune response, with an impact on the activities of the recruited cells, and also of the resident cells such as mammary epithelial cells. Our knowledge of these modifications is limited but we know that neutrophil bactericidal activity is augmented  and that


Asthma is a common disease causing cough, wheezing and shortness of breath. It has been shown that the lung microbiota in asthma patients is different from the lung microbiota in healthy controls suggesting that a connection between asthma and the lung microbiome exists. Individuals with asthma who are also tobacco smokers experience more severe asthma symptoms and smoking cessation is associated with improved asthma control. In the present study we investigated if smoking cessation in asthma patients is associated with a change in the bacterial community in the lungs, examined using induced sputum. We found that while tobacco smokers with asthma have a greater bacterial diversity in the induced sputum compared to non-smoking healthy controls, smoking cessation does not lead to a change in the microbial diversity. Traditionally the lungs have been considered a sterile environment. However, with the advent of culture independent techniques to study bacterial populations it has been shown that the lungs harbor a specific bacterial community . SeveralFor individuals with asthma it has been shown that the bacterial community in the lungs is different from the community in healthy controls ,6–8. SpeStudies of the lung microbiota in tobacco smoking individuals who are otherwise healthy indicate that smoking is not associated with changes in the bacterial composition compared to individuals who are non-smokers . In contWe have recently shown that cigarette smoking asthma patients who quit smoking experience an overall increase in asthma control as well as reduced airway hyper responsiveness ,13. Whetwww.regionh.dk/vek, ref. H-2-2010-140) and all asthma patients and control subjects provided written, informed consent. The randomized trial was registered at clinicaltrial.gov (NTC: NCT02233231) under the identifier: "Young smoking asthmatics".The 44 asthma patients enrolled in this study were part of a randomized clinical trial on the effect of varenicline versus placebo on cigarette smoking cessation in individuals with asthma . The stuPrior to collecting the induced sputum sample, participants washed their mouth with water to reduce oral contamination. For each patient, 1–2 ml induced sputum sample was collected and transferred directly to a sterile 15 ml tube containing 4 ml lysogeny broth (LB) medium with 27% glycerol, as we generally experience that LB preserves cultivability. The tubes were kept on ice and immediately frozen at -20°C and transferred to -80°C. Further details on the collection of induced sputum in this study have been published previously ,14.The samples were thawed at room temperature and 5 ml 0.2% dithiothreitol (DTT) solution was mixed with the sample and incubated for 10–15 minutes at room temperature to dissolve viscous sample parts. Next, the tubes were centrifuged at 7500 g for 5 minutes and the supernatant was removed. The pellet was heat inactivated for 5 minutes at 95°C in order to kill potential pathogens. The pellet was dissolved in 500 μl bead solution from the PowerLyzer™ PowerSoil® DNA Isolation Kit (MOBIO) and DNA was extracted according to the manufacture’s protocol using the Fast Prep (MPBIO) homogenizer at speed 6 for 40 seconds. The DNA was eluted in 50 μl elution buffer and stored at -20°C.For each of the 88 patient samples and the 20 control samples DNA was extracted and analyzed by 16S rRNA gene amplicon sequencing using the 454 FLX platform (Roche). All 16S libraries were constructed by amplifying the V3-V4 region of the 16S rrna gene using the 341F primer CCTAYGGGRBGCASCAG and the 806R primer GGACTACNNGGGTATCTAAT . PCR wasQuality filtering, read trimming and OTU clustering was done according to the UPARSE pipeline . BrieflyFor comparisons between asthma patients and controls and for comparisons at baseline between cigarette quitting and non-quitting asthma patients, unpaired t-test and Mann-Whitney U-test was applied for continuous variables with parametric and non-parametric distributions respectively. For categorical variables, Pearson's chi-squared test was applied. When comparing differences in change from week 0 to week 12 between quitters and non-quitters or between the varenicline and the placebo group, unpaired t-test and Mann-Whitney U-test using the paired differences (change from week 0 to week 12) was applied for paired differences with parametric and non-parametric distributions respectively. In the comparison of the quitters at week 0 to week 12, a paired t-test and a Wilcoxon Signed-Rank Test was used for continuous variables with parametric and non-parametric distributions respectively. The trimmed greengenes database includes 277 different bacterial families. Since our analysis of differences in abundance of specific families was exploratory and not driven by pre-specified hypotheses, all analysis regarding specific families were adjusted for 277 multiple comparisons using the Bonferroni method.Of the 44 included asthma patients, 25 had quit cigarette smoking at week 12 while 19 had not. Of the 25 who quit cigarette smoking 24 had already stopped at week 6, highlighting that smoking cessation for the most part occurred early in the intervention. Detailed baseline characteristics for the 44 patients according to smoking status at week 12 has been published previously [In total, 88 16S rRNA gene amplicons from the asthma patients and 20 amplicons from the healthy controls were sequenced. On average each amplicon contained 7844 sequences . ClusterAt week 0 the samples from the asthma patients contained a total of 378 different OTUs with each sample on average containing 123 OTUs (sd = 25) . Taxonom−7 and P = 6 * 10−6, respectively, unpaired t-test)  . While t−6, Mann-Whitney U-test) and Spirochaetaceae (P = 1 * 10−5) were significantly more abundant in the asthma patients compared to the controls after correction for 277 multiple comparisons using the Bonferroni-method . However, as these families contain several species commonly found in the oral and lung flora, including opportunistic pathogens, it is difficult to comment on the clinical implications of this finding.To further explore the difference between the controls and the patient group we identified the ten bacterial families with the largest absolute difference in mean abundance between the two groups . Two of The longitudinal design of our study allowed us to compare changes in the bacterial communities in the lungs within the same patients before and after cigarette smoking cessation. This helped eliminate inter-patient variation, which could otherwise mask the changes. At week 12, 25 patients had quit cigarette smoking while 19 were still smoking . When coAs all patients attempted smoking cessation we cannot rule out that the non-quitter group displayed reduced smoking frequency potentially masking the effect of smoking cessation on the diversity in the microbiome. To reduce this impact we investigated if there was any change in the bacterial diversity within the quitter group alone. We found no significant change in the number of OTUs or the Shannon index from week 0 to week 12 . While tTo explore if specific bacterial families were affected by smoking cessation we identified the five families with the largest mean change in the quitter group. For these families we compared the change (i.e. from week 0 to week 12) in the quitter group to the change in the non-quitter group (Mann-Whitney U-test of the within-patient change in the quitter group compared to the within-patient change in the non-quitter group) . For eacWe have recently shown that cigarette smoking cessation is associated with improvements in asthma control in cigarette smoking asthma patients . In the We found no significant difference in the changes in the bacterial communities between asthma patients who quit cigarette smoking and those who did not. However, as both groups attempted smoking cessation we cannot rule out that the patients in the non-quitter group changed their smoking habits in terms of the amount of daily use or intermittent quitting. Consequently, we investigated the change within the group of patients that successfully quit cigarette smoking and found no association between smoking cessation and the bacterial community. This suggests that there is no strong association between smoking cessation and changes in the sputum microbiome. Yet, it is still possible that a larger sample size or a longer follow-up period would have detected a change.We did find that bacterial diversity, measured both by the number of unique OTUs and the Shannon index, was significantly higher in the cigarette smoking asthma patients compared to the healthy non-smoking controls. This corroborates previous findings showing that the bacterial diversity in the lungs of asthma patients is more diverse than that of healthy controls ,8. Howevet al. have found that the bacterial diversity in oropharyngeal samples from asthma patients is lower than that of healthy controls [In contrast to our and others findings ,8, Park controls . HoweverAt the bacterial family level we found two families to be significantly enriched in the sputum of cigarette smoking asthma patients compared to healthy controls; Streptococcaceae and Spirochaetaceae. Yet, it is difficult to evaluate the clinical implications of this finding as both families contain species commonly found in the oral cavity and upper respiratory tract. Furthermore, as we did not specify any hypothesis with respect to changes in specific bacterial families the analysis of the changes in bacterial families required correction for multiple hypotheses testing, which greatly reduces statistical power. As a result, it is difficult to interpret the non-significant findings with a low p-value in the families in In the present study, samples were collected using induced sputum, which represents a non-invasive alternative to bronchoalveolar lavage (BAL). Samples were transferred to a LB + glycerol medium and immediately stored on ice. However, the nutrient rich medium could in theory have affected the bacterial population. In order to reduce oral contamination participants washed their mouth with water before giving the sputum sample. Yet, induced sputum samples will suffer from oral contamination.Studies comparing the microbiome at different sites along the aerodigestive tract find that this body site constitutes a microbiological continuum with substantial species overlap but varying abundances, ,21,23. CRecently a study comparing the lung microbiome of healthy tobacco smokers and non-smokers found no difference between the two groups . Since tS1 FigThe distribution of sequence reads per amplicon for the controls and the asthma patients at each sampling time.(PDF)Click here for additional data file.S2 FigThe distribution of OTUs per amplicon for the controls and the asthma patients at each sampling time.(PDF)Click here for additional data file.S3 FigRarefaction curves per amplicon for the controls and the asthma patients at each sampling time.(PDF)Click here for additional data file.S4 FigThe observed OTUs (A) and Shannon index (B) at week 0 and week 12 stratified by randomisation. When comparing the change in the observed OTU from week 0 to week 12 in the varenicline group to the change in the placebo group there is no significant difference (A) . Similarly, when comparing the change in Shannon index from week 0 to week 12 in the varenicline group to the change in the placebo group there is no significant difference (B) . The solid crossbars mark the mean of the distributions.(PDF)Click here for additional data file.S5 FigThe observed OTUs (A) and Shannon index (B) of the quitters at week 0 and week 12. When comparing the observed OTU at week 0 to week 12 there is no significant difference (A) . Similarly, when comparing the Shannon index at week 0 to week 12 there is no significant difference (B) . The solid crossbars mark the mean of the distributions.(PDF)Click here for additional data file.


SETD7 gene, was significantly associated with serum lycopene concentrations (p = 3.41 × 10−9). Our findings also provided nominal support for the association previously noted between SCARB1 and serum lycopene concentrations, although with a different SNP (rs11057841) in the region. This study identified a novel locus associated with serum lycopene concentrations and our results raise a number of intriguing possibilities regarding the nature of the relationship between SETD7 and lycopene, both of which have been independently associated with prostate cancer. Further investigation into this relationship might help provide greater mechanistic understanding of these associations.Dietary intake and higher serum concentrations of lycopene have been associated with lower incidence of prostate cancer and other chronic diseases. Identifying determinants of serum lycopene concentrations may thus have important public health implications. Prior studies have suggested that serum lycopene concentrations are under partial genetic control. The goal of this research was to identify genetic predictors of serum lycopene concentrations using the genome-wide association study (GWAS) approach among a sample of 441 Old Order Amish adults that consumed a controlled diet. Linear regression models were utilized to evaluate associations between genetic variants and serum concentrations of lycopene. Variant rs7680948 on chromosome 4, located in the intron region of the  The carotenoids are a group of over 600 pigments that are synthesized by plants and microorganisms. Lycopene is a red-pigmented carotenoid present in tomatoes, watermelon, papaya, and other fruits and vegetables. There is no endogenous production of lycopene or other carotenoids in animals and they are only obtained from the diet. Higher intake and circulating concentrations of carotenoids have been associated with lower risk of cancer , cardiovProstate cancer is the disease that has been studied most extensively in relation to lycopene. While the evidence is not conclusive, a number of large epidemiologic studies indicate that higher dietary intake and serum concentrations of lycopene are associated with lower risk of prostate cancer ,11,12, pFurther confounding the relationship between lycopene and human disease is the relatively poor correlation between dietary intake of lycopene in isolation and its concentrations in serum . The absSCARB1, which encodes for a cholesterol membrane transporter, were genome-wide significant in the multi-ethnic meta-analysis, though the signal was driven by participants of Hispanic and African heritage primarily. The other two associated loci in SLT3 and DHRS2, were identified in African-Americans only. No genome-wide significant associations have been identified in populations of European descent.There are two previously reported genome-wide association studies (GWAS) of lycopene. The first, a population based study in the Tuscany Chianti region of Italy, did not identify any genome-wide significant associations with lycopene ). In theThe main aim of this study was to identify novel genetic predictors of serum lycopene concentrations. We studied a population of Old Order Amish adults living in Lancaster County, Pennsylvania in whom serum lycopene concentrations were measured at the conclusion of a controlled diet. The Amish are a commonly studied population for these types of studies because, among other reasons, their relatively homogeneous genetics and lifestyles across the population increase the ability to detect genetic signals ,30,31,32etc., has been described previously [Clinicaltrials.gov (NCT00664040). Of the 868 Old Order Amish adults recruited into the HAPI Heart study, 469 were administered the controlled dietary intervention and subsequently provided a fasting blood sample at the conclusion of the 6-day diet. There were 27 samples of insufficient quality to measure serum lycopene concentrations and one sample excluded from analysis due to extreme serum lycopene concentration  that was considered an outlier. There were 308 nuclear families in our study sample, ranging from one participant to nine participants per family. The participant relatedness was as follows: 113 parent-children, 198 sibling, 105 avuncular, 33 first cousins, and six grandparents-grandchildren. The study was approved by the Institutional Review Board of the University of Maryland School of Medicine and all participants provided written informed consent.The study sample was composed of 441 Caucasian participants from the Heredity and Phenotype Intervention (HAPI) Heart Study who completed a 6-day controlled diet and from whom frozen blood samples were available for serum lycopene measurement. The HAPI Heart Study is a family study and by design included individuals from the same nuclear family. Moreover, many of the enrolled families were related to each other given the social structure of the Old Order Amish community. The design of the HAPI Heart Study, including study inclusion/exclusion criteria, demographics of the full study sample, eviously  and was A controlled diet was prepared for participants by study staff. A registered dietitian visited several Old Order Amish households to obtain diet histories and observe meals and foods that were in their homes. All meals in the controlled diet were designed to be representative of the typical diets of Old Order Amish adults and were provided to the study participants by home delivery over a period of six days. Study participants also abstained from both prescribed and over the counter medications and dietary supplements during this 6-day period. The full menus for the 6-day controlled diet that the participants in this study consumed are provided in the Compliance with the controlled diet was assessed by comparing sodium, potassium, and creatinine levels from first morning urine samples obtained: (1) prior to consuming the 6-day controlled diet that participants consumed in this study; (2) on the final day of the 6-day controlled diet that the participants consumed in this study; and (3) on the final day of a second isocaloric, 6-day controlled diet that was low in salt and consumed after the blood draw that was used to conduct the GWAS in this study. The compliance data have been reported in detail previously , but in Frozen blood samples that had been obtained after a 12-h fast were assayed for serum lycopene concentrations at Johns Hopkins University. 200 μL from each frozen blood sample were used for the reverse-phase high-pressure liquid chromatography (HPLC) assessment of serum lycopene concentrations . There wp > 10−6) were used for imputation with MACH using the HapMap CEU reference sample [A genome-wide association study assesses associations of phenotype with single nucleotide polymorphisms (SNPs) throughout the genome. Study participants were genotyped using either the Affymetrix 500k or Affymetrix 1M SNP chip v6.0 by the Genomics Core Laboratory at the University of Maryland. Genotyping calls were made using Birdseed, which is part of the Birdsuite tools . A totale sample . Resultsp < 5 × 10−8 to be statistically significant. At this genome-wide significance threshold, we estimated that our sample provided 80% power to detect SNPs accounting for 9%–10% of trait variation.Descriptive statistics were performed to characterize the study sample and determine the mean concentrations of serum lycopene. For GWAS analysis, we estimated the effect of genotype on lycopene levels, adjusting for the effects of age, sex, and body-mass index (BMI) using a general linear model. Genotype was coded as the number of copies of the reference allele , thus corresponding to an additive genetic model. The GWAS analyses were performed using the MMAP software , which a2 and over half of both the men and the women could be classified as overweight (BMI ≥ 25 kg/m2). Mean lycopene values were 39.2 μg/dL ). The heritability was estimated to be 0.38 ± 0.12.Baseline characteristics of the study sample are provided in p = 3.41 × 10−9). Each copy of the A allele was associated with a 8.6 μg/dL decrease in serum lycopene, and this locus accounted for 9.3% of the variation in lycopene levels. The minor allele at the locus (A) is common in the Old Order Amish (MAF = 0.202) as well as other populations . SETD7. Gene SETD7 encodes the enzyme histone H3-K4 methyltransferase SETD7, one of the histone methyltransferases (HMTs) enzymes. The quantile-quantile plot provided in p-values for the genome-wide association tests is consistent with that expected under the null.A Manhattan plot summarizing results of the GWAS is provided in p < 1 × 10−6 that are provided in SCARB1 (lead SNP: rs1672879) in the meta-analysis across three ethnic groups, and the other two achieving genome-wide significance in the African-American sample only (to SNPs in SLIT3 (lead SNP: rs11057841) and DHRS2 (lead SNP: rs74036811)). Notably, the associated SNPs in SCARB1 have a minor allele frequency (MAF) of only 0.03 in European-Americans and the associated SNPs in SLIT3 and DHRS2 were monomorphic in European-Americans. In the Amish, the MAF of the three SNPs were even lower (MAF = 0.015), and there was no evidence for association with lycopene levels (p = 0.26). Although not a replication, we did, however, detect nominal evidence for association of lycopene levels with a different SNP in SCARB1 .We additionally identified three other loci for which associations were observed at SETD7 locus, rs7680948, and serum lycopene concentrations in this genome-wide association study. This represents the first genome-wide significant genetic association of lycopene in a mixed-gender, Caucasian population and the first study evaluating genetic determinants of lycopene concentrations among a sample that had consumed a controlled diet.The key result from this study is the novel association observed between a common variant at the SCARB1 with low MAF in Caucasians and serum lycopene concentrations, although we did observe a nominal association of lycopene levels with a different SNP within this gene (rs11057841). Interestingly, rs11057841 has previously been associated with lipoprotein-associated phospholipase A2 (Lp-PLA2) [2 and lycopene are primarily carried throughout circulation on low-density lipoprotein (LDL) [SLIT3 or DHRS2 [We were unable to replicate an association previously noted between a SNP in Lp-PLA2) . Both Lp rs110578. Interesin (LDL) . Our dator DHRS2 , althougSETD7, lycopene, and prostate cancer. SETD7 is proliferative and anti-apoptotic in prostate cancer cells and nuclear expression is upregulated in prostate cancer tissue [SETD7 as a histone methyltransferase (HMT) may also play a role in prostate cancer. HMTs have been shown to be upregulated in prostate cancer [SETD7 may be related to prostate cancer risk through its relationship to serum lycopene concentrations, as was identified in this study. The potential protective mechanisms of lycopene against prostate cancer include regulation of the antioxidant response element, exertion of effects on VEGF signaling pathways, induction of cell cycle arrest, and mediation of apoptosis [Our results raise a number of intriguing possibilities regarding the nature of the relationships previously noted between r tissue . The acte cancer ,45 and de cancer . It is apoptosis ,47. FutuThere are several key strengths of the study. This was the first GWAS aimed at identifying genetic predictors of serum lycopene concentrations that was conducted among a sample that had consumed a controlled diet. The controlled diet and consistent lycopene, fat, and cholesterol intake among the study participants enabled us to more closely isolate the genetic contributions to the variance in serum lycopene than would have been possible on a variable diet. A related strength was that adherence to the diet was also high, as verified by urinary excretion. The diet was informed by home visits of the study population performed by a registered dietitian, was designed to be culturally-appropriate based upon the foods and beverages present in the homes during these visits, and was delivered to the homes of the study participants to encourage adherence. Conduct of this study in the Old Order Amish population was also advantageous for several reasons. To our knowledge, this is the first study to estimate heritability of serum lycopene concentrations in humans, an analysis made possible by the relationship structure of the Old Order Amish. The Old Order Amish study sample also provided a population that was relatively homogenous with respect to genetics, environmental exposures, and lifestyle habits. This homogeneity, particularly with respect to genetics, provided increased power to detect genetic variants associated with lycopene concentrations.n = 441) may have limited our ability to detect genome-wide significant associations between genetic variants and serum lycopene concentrations. Furthermore, the relatively high inter-assay CV of 17.4% for our serum lycopene measurements could have resulted in lower precision of our estimates. However, despite the relatively small sample size and relatively high inter-assay CV, our study was able to identify a novel locus associated with serum lycopene concentrations. We attribute this success in part to the aforementioned advantages of studying an Old Order Amish population as well as the controlled diet that the participants consumed prior to the fasted-state blood draw which enabled us to more closely isolate the genetic contributions to serum lycopene concentrations. A limitation of the controlled diet was its relatively short duration of six days. While the time to maximum concentration of lycopene after consumption is just six hours, lycopene has an elimination half-life of between five and nine days [SETD7 and serum lycopene concentrations, both of which have been associated with prostate cancer, may provide the rationale for further study into the specific mechanisms of this relationship, this study did not collect data on family history of prostate cancer, prostate specific antigen, or other markers of the disease and no direct inference can be made.There were also several notable limitations to this study. The relatively small sample size (ine days ,49. It iSETD7, and serum lycopene concentrations. These findings provide further support that genetics may affect serum concentrations of lycopene. Further studies are needed to clarify any potential relationships between SETD7, lycopene, and clinical endpoints such as prostate cancer.In conclusion, this study provides the identification of a novel genetic association between rs7680948, an intronic variant in