The present study employs a stereoscopic manipulation to present sentences in three dimensions to subjects as they read for comprehension. Subjects read sentences with (a) no depth cues, (b) a monocular depth cue that implied the sentence loomed out of the screen , (c) congruent monocular and binocular  depth cues  and (d) incongruent monocular and binocular depth cues . Reading efficiency was mostly unaffected, suggesting that reading in three dimensions is similar to reading in two dimensions. Importantly, fixation disparity was driven by retinal disparity; fixations were significantly more crossed as readers progressed through the sentence in the congruent condition and significantly more uncrossed in the incongruent condition. We conclude that disparity depth cues are used on-line to drive binocular coordination during reading. The question, then, is how effective is the eye movement system in coordinating the eyes to make both saccades and vergence movements concurrently to ensure that that reading can proceed normally? Furthermore, what information does the system utilize in order to do so?While reading, our eyes make coordinated sideways movements (fixation disparity (offset between the locations of gaze of the two eyes) when reading, but what about disparity in more extreme circumstances?Considerable progress has been made over the past 40 years in our understanding of the way eye movements relate to the visual, cognitive, and linguistic processes involved in reading All studies of eye movement control during reading to date, including those mentioned above, have investigated reading when the plane of the text is perpendicular to the line of sight . From research that has investigated binocular coordination during reading, it is clear that readers do not experience diplopia (double vision) and are able to read quite easily, despite the fact that we only fixate the same letter half the time. Liversedge, Rayner et al. Vergence responses ordinarily occur quite rapidly, especially following saccades In contrast to Blythe et al.’s results, there is evidence that, with practice, subjects can improve vergence responses to parafoveal stereoscopic depth cues. Eggert and Kapoula While the Blythe et al. Preferred Viewing Location, PVLcowboy were embedded in the sentences. While the rest of the sentence frame was presented binocularly, the target words were presented dichoptically such that one portion of the word was presented to the left eye only  and the other portion of the word was presented to the right eye only . In a control condition, the entire sentence and the entire target word were presented binocularly. Three outcomes were possible: (a) If each eye were making a saccade based on its own retinal input, the eyes would land in different locations , (b) If saccades were programmed based on the input of one eye then both eyes would land on the same location , (c) If saccades were programmed based on a single, unified percept landing positions would reflect the PVL of the entire word. Saccade targeting based on a unified percept is exactly what Liversedge et al. The locations of the fixations that we make when we move our eyes through text during reading are not random. Instead, readers target their saccades fairly precisely based on information that is obtained from the parafovea In the Blythe et al. retinal size; a given object, when closer to the viewer, will cover more retinal area than the same object at a further distance. This is such a strong depth cue that, when combined with a conflicting cue , it can produce a strong visual illusion . Second, binocular depth cues occur due to differences in the relative position of two similar images on the retinas of the two eyes . The magnitude of offset between the location of a given object in one retinal image relative to the other provides an estimate of depth.There are two classes of cue to depth that the visual system may use as a basis for vergence. First, monocular depth cues are those that are functional with just one eye, such as Several studies have investigated the perceptual consequences of non-linguistic stimuli with conflicting binocular and monocular depth cues . Such stimuli can give rise to perceptual ambiguity, where the apparent slant of the stimulus alternates between that dictated by perspective and that dictated by disparity In the present study, subjects were required to read for comprehension. Successful comprehension is only possible if the reader successfully fuses the two images  to produce a clear, single percept. Liversedge et al. normal, in which there were no depth cues, (b) monocular cue, in which retinal size was consistent with the text looming forwards from the screen, (c) congruent, in which both the monocular cue and retinal disparity was consistent with the text looming forwards from the screen and (d) incongruent, in which retinal size was consistent with the text looming forward from the screen, but disparity was consistent with the text receding behind the screen. Both the monocular and binocular depth cues increased in magnitude from the beginning to the end of the sentence so that the sentence appeared to extend increasingly closer or further from the plane of the screen as the subject read through it. As noted above, we recorded subjects’ binocular eye movements as they read sentences in these four conditions, to examine the behavioral response to congruent and incongruent disparity and monocular depth cues. The monocular cue condition was an important control condition as previous work has shown that fixation disparity exists for sentences presented in two dimensions, and is affected by the amplitude of the incoming saccade; the greater the amplitude of the incoming saccade, the greater the disparity observed at fixation onset We recorded the movements of each eye simultaneously to determine how the two eyes were coordinated in order to facilitate that fusion. Subjects read sentences that were presented stereoscopically see  in four We made three predictions for this experiment. First, there would be a slight tendency for the eyes to be uncrossed in the normal and monocular cue condition and this effect would be larger in the monocular cue condition due to increasing saccade length, which is known to be correlated with larger uncrossed disparities The investigation adhered to the principles of the Declaration of Helsinki, and was approved by the Ethics Committee at the School of Psychology, University of Southampton for human experimentation. Informed written consent was obtained from each subject after explanation of the procedure of the experiment.linear mixed effects (lme) models we used in the analyses (see Results section) are able to handle such missing data. The missing data in the congruent condition does not affect comparisons that do not involve the congruent condition and, in analyses that do involve the congruent condition, the concern is only about low power. Thus, one should only be concerned about real effects not yielding statistically significant results, as opposed to null effects yielding spurious significant results To provide further support for this argument, we performed the same analyses using only those subjects that experienced all conditions and the results corroborate those with our reported analysis of the full dataset. Subjects were between the ages of 22 and 35 and had normal or corrected-to-normal vision, and exhibited no reading problems. Subjects without functional stereopsis were excluded from participating in this experiment. Stereoacuity was assessed using the graded circle test. Subjects were judged to have functional stereopsis if they could correctly describe a stimulus with depth of 40 seconds of arc. Subjects were compensated £6 for every hour they took part in the experiment.Twelve subjects from the University of Southampton community participated in the experiment. Due to problems with the experimental equipment only a subset (four subjects) read sentences in the congruent condition. While the fact that there were fewer subjects in one condition is not ideal, the normal, (b) monocular cue, (c) congruent, and (d) incongruent. These experimental conditions were presented in blocks, the order of which was counterbalanced between subjects. Sentences were counterbalanced across conditions so that each sentence was seen in every condition. In the normal condition, 3.42 characters equaled one degree of visual angle, in the other three conditions, in which there was a monocular depth cue of increasing retinal size, characters per degree ranged from 5.68 to 1.99.Each subject read 12 sentences in each of the different display conditions  and the position of each eye was recorded every millisecond. In all conditions, calibration routines and data collection were conducted using in-house software. The same software was used to present the images in all conditions.http://www.orthostereo.com/geometryopengl.html; http://paulbourke.net/miscellaneous/stereographics/stereorender/The cameras were shifted apart by an intraocular distance of 65 cm and oriented in the same, parallel direction and the viewing frustums were adjusted in an asymmetric way to ensure zero disparity at the screen depth. This method was used to create stereo image pairs for each sentence.The stereo sentence bitmaps were constructed using the OpenGL framework to render two separate images for the left and right eyes, where each image was drawn from a different camera perspective. First, the text in a particular sentence was rendered, with antialiasing, to a bitmap with no distortion. This bitmap was then used as a texture map applied to a rectangular quad element that was placed in OpenGL 3D space in such a way that one edge of the quad is nearer to the camera than the other (this gives the text the appearance of ‘looming’ towards the viewer). The asymmetric frustum parallel projection approach was used to setup the stereo camera projections. Information about the methods of this approach can be found at the following websites: Sentences were presented on a 21-inch Phillips CRT monitor positioned 100 cm from the subject. FE1 shutter goggles (Cambridge Research Systems), which alternate between left-open/right-shut and left-shut/right-open at the same refresh rate as the monitor (8 ms), were used to display the images stereoscopically Subjects were first tested for stereopsis, then the two eye trackers were calibrated, and then the experimental trials began. For the initial calibration, and all checks of calibration accuracy, viewing was monocular – during calibration of the right eye tracker the left eye was occluded, and vice versa. The calibration was conducted using three points: one on the left of the screen, one on the right, and one in the center; the range of these three points was greater than the horizontal extent of the experimental stimuli. Once calibration had been completed, subjects were instructed to read the sentences normally for comprehension. During all conditions, subjects were instructed to maintain their fixation on the first word for several seconds until the experimenter told them to start reading; this allowed their percept to stabilize before beginning to read the sentence. This maintenance of fixation lasted approximately the same amount of time on each trial and was not determined by the subjects, themselves. Thus, it is not the case that this fixation indicated a requisite amount of time needed by the subjects to fuse the image, but was determined by the experimenter, irrespective of the subject’s behavioral response to disparity cues. Subjects then read the sentence and pressed a button when they had finished reading it. The button press terminated the trial. Subjects were instructed to read the sentences to the best of their ability. The accuracy of calibration was checked after every sentence. On a third of the trials, the sentence was immediately followed by a comprehension question (requiring a yes/no response). Each block took between 10 and 20 minutes to complete.p = .06). No subject reported experiencing diplopia and all subjects reported experiencing the sentences in depth in the binocular depth cue conditions .Subjects responded correctly to 77% of the comprehension questions , indicating that they read and understood the sentences. A logistic regression revealed that there were no significant differences between the monocular condition and the other conditions, except the normal condition produced marginally higher accuracy  and fixation duration . For saccade disparity: distance from the beginning of the sentence and incoming saccade amplitude (given that it is known to impact binocular coordination). All variables, aside from experimental condition, were centered so that the main effects were interpretable as the effect of the variable of interest at the average value of all other variables; interactions are not affected by centering.All measures were analyzed with linear mixed effects (lme) models. For both types of analyses experimental condition  was entered as a fixed effect and subjects and items were entered as crossed random effects. Additionally, for analyses of binocular measures, the following factors were entered as fixed effects. For fixation disparity: mean fixation duration (the average duration of each fixation on a sentence), total sentence reading time , the number of fixations, and the number of regressions (backward saccades) per sentence . There were no significant differences between the monocular cue and the congruent condition  except that there were fewer regressions in the congruent condition  than in the monocular cue condition . The incongruent condition led to longer average fixation durations  than in the monocular cue condition . However, there were no significant differences between the monocular cue and incongruent conditions in total sentence reading time, number of fixations, or number of regressions .We analyzed global reading measures to determine whether the different reading conditions affected the ease with which the text was processed see . In geneThese data, along with the comprehension accuracy data, reported above, suggest that, in general, the presence of depth cues did not impact the subjects’ cognitive processing difficulty . However, if the depth cues conflicted , subjects spent longer on each fixation, but otherwise, reading proceeded fairly normally.We examined disparity at both the start and end of fixations and vergence during saccades and fixations  of each other), 6% to be crossed and 32% to be uncrossed. At the end of fixations, we found 72%, 11% and 17%, respectively.t = 4.81, p<.001). There was less uncrossed disparity in the normal condition, which was significantly different from the monocular cue condition . This indicates that the presence of monocular cues to depth did result in a small increase in fixation disparity. This is likely due to the fact that words were increasing in size in the monocular cue condition and larger word objects allow for greater magnitude of disparity (in degrees) with the two points of fixation staying relatively near each other (in terms of characters). Therefore, on average, the system would be able to tolerate more physical disparity because the eyes would still fixate the same character, given its larger size . In the congruent condition, fixations were significantly more crossed compared to the monocular cue condition , whereas in the incongruent condition disparity was significantly more uncrossed than the monocular cue condition .In the monocular cue condition the eyes tended to be uncrossed . This may seem to contradict our claim that the increased size of the words in the monocular cue condition led to greater disparity, as word size increases in the monocular cue condition as one moves through the sentence. It is important to note, however, that words in the sentences were of variable lengths . Therefore, the degree to which words increase in size  is, to some extent, offset by variability in word length. Thus, a long word at the beginning of the sentence would subtend a larger degree of visual angle than a short word at the end of the sentence. This variability is likely the reason that, in the monocular condition compared to the normal condition, there is a significantly greater amount of uncrossed disparity, which is almost significantly (t = −1.88) modulated by distance from the beginning of the sentence.The effect of distance from the beginning of the sentence was not significant in the monocular cue or normal conditions  and the incongruent condition ; retinal disparity induced by the stereoscopic manipulation increased as distance from the beginning of the sentence increased. The direction of this disparity was opposite in the two conditions (see ps>.5).Most importantly, there was a significant effect of distance from the beginning of the sentence on fixation disparity in both the congruent . There was no significant difference between the monocular and normal condition (t<1). In the congruent condition, the effect was significantly different from the monocular condition in the opposite direction–longer saccades produced more crossed fixation disparities, and in the incongruent condition the effect was larger than in the monocular condition and in the same direction–longer saccades produced more uncrossed fixations. Thus, we replicated the finding that larger saccade amplitudes produce greater disparity, but found that our manipulations of increasing retinal size and increasing retinal disparity were more deterministic of fixation disparity than saccade amplitude when both were entered in the analysis.To test this, we conducted the same analysis with distance from the beginning of the sentence removed. This analysis revealed that saccade amplitude was related to start of fixation disparity in the monocular condition; longer saccades tended to produce more uncrossed fixation disparities . In the normal condition, fixations tend to be significantly less uncrossed than in the monocular cue condition . As mentioned before, this slight increase in uncrossed fixations in the monocular condition compared to the normal condition is likely due to increased word size . The magnitude of fixation disparity was much greater in both stereoscopic conditions and the direction of this disparity was determined by the direction of the stereoscopic manipulation; fixations were more crossed in the congruent condition  and more uncrossed in the incongruent condition  than in the monocular cue condition. Again, there were significant interactions with distance from the beginning of the sentence in both stereoscopic conditions, such that the magnitude of fixation disparity increased as the reader progressed through the sentence. In the congruent condition, fixations became more crossed as the reader progressed through the sentence , while in the incongruent condition fixations became more uncrossed as the reader progressed through the sentence . Additionally, fixation duration did not have a significant effect on end of fixation disparity .The end of fixation data were broadly similar to the start of fixation data see . In the In summary, the end of fixation data were quite similar to the start of fixation data: in the monocular cue and normal conditions, the eyes tended to be slightly uncrossed. In the congruent condition the eyes tended to be crossed and in the incongruent condition the eyes tended to be more uncrossed than in the monocular cue condition. In the monocular cue and normal conditions the magnitude of the disparity was unaffected by position in the sentence while in the congruent and incongruent conditions the magnitude of this disparity increased as the subject progressed through the sentence. Therefore, it seems that fixation disparity was, for the most part, driven by the direction of stereoscopic disparity cues and very little by the monocular cue. Finally, end of fixation disparity was unaffected by fixation duration. As with the lack of an effect of saccade amplitude in the start of fixation disparity analyses, it is likely that the non-significant effect of fixation duration is due to the fact that stereoscopic disparity has such an influence on fixation disparity that the effects of other variables do not have the opportunity to exert an influence.To illustrate the vergence responses that occurred during saccades and fixations across a trial, we plotted eye positions and vergence for example trials in each of the four conditions see . Formal The panels in this figure clearly show that the eyes are quite well aligned to the same location on the screen in both the monocular cue and normal conditions. In contrast, in the congruent and incongruent conditions, where there is disparity in the stimulus, the eyes fixate somewhat different locations on the screen . Furthermore, these figures show that vergence occurs both during saccades and fixations. Fixations are represented by relatively stable values on the y-axis (location within the sentence) over changes in the x-axis (time). Saccades are represented by large changes in the y-axis during small increases in the x-axis. In the plots of vergence there is clearly a large transient change in disparity during the saccade , proportion of saccade convergence , summed fixation vergence, and proportion of fixation convergence  occurred, values were arcsine transformed  before being entered into the model. For these analyses, each saccade and fixation was classified as being either convergent (in which case the point of fixation moved closer to the subject) or divergent (in which case the point of fixation moved further away from the subject). Those that did not contain any detectable vergence (2.3% of saccades and 3.5% of fixations) were excluded from the analyses. We analyzed the cases in which a convergent movement was observed, and the intercept was compared to 50% . Given that the analysis is based on the relative proportions of convergence and divergence, and that each saccade/fixation was classified as falling into one of these two categories, then proportions of convergence and divergence are dependent – if one increases, then the other must decrease. For this reason, we analyzed only one of the two categories (the proportion of convergence). Obviously, the effects in the convergence analyses necessarily hold true for the divergence analyses .t = −6.24, p<.001), indicating that, on trials with longer saccades there was more divergence exhibited during saccades. This may appear to contrast with the start of fixation disparity analyses where saccade amplitude . Summed saccade vergence in the normal condition was not significantly different from the monocular cue condition, showing that the eyes also diverged during saccades . In contrast, in the congruent condition (where the sentence appeared to be looming out from the screen) summed saccade vergence was significantly greater than in the monocular condition–there was significantly less divergence during each trial . The reduced divergence appears to have occurred as a result of the disparity cue suggesting that the sentence was looming towards the reader, in which case the subject should make more convergent movements. In line with this suggestion, in the incongruent condition (where disparity cues suggested that the sentence was receding behind the screen) summed vergence was significantly smaller than in the monocular condition–there was significantly more divergence during each trial . Again, it appears that disparity impacted the amount and nature of vergence observed.As expected, in the monocular cue condition, the eyes tended to diverge during saccades . The normal condition did not significantly differ from the monocular cue condition in the proportion of saccade convergence . Consistent with our analysis of the summed saccade vergence, the congruent condition had a significantly larger proportion of saccade convergence than the monocular cue condition  and the incongruent condition had a significantly smaller proportion of saccade convergence .With respect to our analysis of the proportion of saccade convergence (as opposed to divergence), in the monocular cue condition we found that convergence occurred in significantly fewer than half of the saccades made  or less convergence  respectively. This was observed in terms of both the likelihood of making a direction-appropriate movement, as well as the magnitude of that movement.t = 9.15, p<.001). As with the effect of saccade amplitude on vergence during saccades, it is likely that the significant effect of fixation duration in these analyses  is due to the fact that location through the sentence is not a predictor entered in the model. In the monocular cue condition, the eyes produced a significant amount of convergence during fixations . Summed fixation vergence in the normal condition was not significantly different from the monocular cue condition . In the congruent condition summed fixation vergence was significantly greater than in the monocular cue condition  and in the incongruent condition it was significantly smaller than in the monocular cue condition .For the summed fixation vergence measure we entered summed fixation duration as a predictor in the analysis, which produced a strong positive effect on fixation vergence, with longer fixations exhibiting more convergence . In the normal condition there was a significantly smaller proportion of fixation convergence than in the monocular cue condition . This difference is consistent with our analyses of fixation disparity, showing a tendency for larger uncrossed fixation disparities in the monocular condition compared to the normal condition. The congruent condition did not differ from the monocular cue condition  but the incongruent condition had a significantly smaller proportion of fixation convergence . Thus it seems as if the eyes tend to converge during fixations except in the incongruent condition .As in our analysis of vergence during saccades, all fixations were classed as being convergent or divergent, and we then analysed the proportion of fixation convergence compared to 50%. There was a significant proportion of fixations that exhibited convergence in the monocular cue condition , it seems as if the disparity cue in the stimuli that we used was primarily responsible for changing the vergence response. While the eyes tend to diverge during saccades under normal non-disparate viewing conditions they do so more when disparity cues indicate divergence is necessary and do so less when disparity cues indicate that convergence is necessary. Consequently, under normal reading conditions, the eyes tend to compensate for the divergence during saccades by converging during fixations. Furthermore, when disparity cues indicate that convergence is necessary the eyes converge more than under normal circumstances  and when disparity cues indicate that divergence is necessary the eyes converge less than under normal circumstances.As can be seen in The present data suggest that readers easily coordinate their eyes to fuse the percept of a sentence presented in depth in order to read it for comprehension. The results of the comprehension accuracy and global monocular reading measures reveal that, for the most part, readers did not have more difficulty reading sentences when they contained a disparity depth manipulation than when they did not. However, when the disparity and monocular depth cues conflicted, a situation that readers almost never encounter, readers incurred difficulty, reflected in longer average fixation durations. These data suggest that, on each fixation, readers spent more time but otherwise reading behavior was not affected. Thus, when readers encounter sentences with either no or normal depth cues, reading progresses unaffected. Under all experimental conditions, readers were able to process the sentences such that they could respond accurately to comprehension questions.In terms of a binocular response to apparent depth, eye movements seem to primarily be driven by disparity depth cues, obtained in both foveal and parafoveal vision. First, the vergence response exhibited in response to the stereoscopic manipulation was compatible with the disparity depth cue (stereoscopic offset), even when that cue conflicted with a monocular depth cue . Second, these vergence responses occurred not only during fixations, but also during saccades and therefore must be responding to disparity detected in the parafovea. These data differ from those found by Blythe et al. As shown in The present study is important in that it suggests that the vast amount of knowledge we have gained over the past few decades about eye movements during reading  may be, for the most part, generalized to three-dimensional reading situations. As mentioned before, there are many situations in which the text changes its distance from the reader as the sentence progresses. We can now make inferences about these reading situations, based on data from standard reading studies, because the present experiment suggests that readers (a) do not incur more difficulty reading three dimensional text than two dimensional text, (b) make vergence responses to stereoscopic depth easily and immediately when cues are consistent, and (c) incur negative consequences in the presence of conflicting depth cues . In short, the visual and eye movement systems are quite capable of accommodating variable and even conflicting visual input for the cognitive and linguistic processes necessary for reading to proceed almost unaffected.


Colonoscopy is widely regarded to be the gold standard for colorectal cancer (CRC) detection. Recent studies, however, suggest that the effectiveness of colonoscopy is mostly confined to tumors on the left side of the colon , and that the technology has poor tumor detection for right-sided  lesions. A minimally invasive test that can detect both left-sided and right-sided lesions could increase the effectiveness of screening colonoscopy by revealing the potential presence of neoplasms in the right-sided “blind spot”.We previously reported on a seven-gene, blood-based biomarker panel that effectively stratifies a patient’s risk of having CRC. For the current study, we assessed the effectiveness of the seven-gene panel for the detection of left- and right-sided CRC lesions. Results were evaluated for 314 patients with CRC  and 328 control samples. Blood samples were obtained prior to clinical staging and therapy. Most CRC subjects had localized disease ; regional (stage III) and systemic (stage IV) disease represented 32% and 9%, respectively, of the study population.The panel detected left-sided  and right-sided  lesions with an overall sensitivity of 78% (215/316) at a specificity of 66% (215/328). Treatable cancer (stages I to III) was detected with left-sided lesion sensitivity of 76% (138/182) and right-sided sensitivity of 84% (80/95).This seven-gene biomarker panel detected right-sided CRC lesions across all cancer stages with a sensitivity that is at least equal to that for left-sided lesions. This study supports the use of this panel as the basis for a patient-friendly, blood-based test that can be easily incorporated into a routine physical examination in advance of colonoscopy to provide a convenient companion diagnostic and a pre-screening alert, ultimately leading to enhanced CRC screening effectiveness. Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer deaths in the United States and Canada. The disease is expected to be diagnosed in approximately 142,820 Americans in 2013, and an estimated 50,830 people are expected to die of CRC in that year . The independent blind test set confirmed performance .Details of the characterization and validation of the seven-gene panel to identify CRC have been described previously . In thatFor the present study we re-analyze the previously reported data in order to determine the ability of the seven gene panel not only to identify the presence of CRC but also to identify cancer stages and left- and right-sided colon cancer.The training set data was used to determine the best coefficients for a logistic regression model using 6 ratios of the 7 genes most discriminative for CRC. This model was then used to predict the CRC risk for the test set samples.Breaking the data down by cancer stages, we were able to find the same predictive values for left- and right-sided cancers as for CRC detection as in the original paper Table .In this study, CRC detection sensitivity was generally higher for right-sided cancer except in the case of TNM stage I in the test set. However, this finding may be simply a sampling issue. To resolve this question, we combined all training and test set samples and performed 2-fold cross validation, iterated 1000 times. This process partitions the samples into 2 halves such that that the coefficients of the model are fitted to the training half and applied to the test half.The results for all the test halves after 1000 permutations represent a less biased estimate of the performance of the gene panel. As expected, the lower sensitivity for right-sided TNM I as compared with left-sided TNM I cancers is no longer observed in the cross-validated results. Overall, right-sided lesions are detected at a higher sensitivity than left-sided lesions; however, there are fewer right-sided samples, so the observed higher sensitivity may not be statistically significant. As can be seen from the box and whisker plots of the distribution of the prediction scores, the 98% confidence intervals show considerable overlap both across all TNM stages and for left and right sided cancers  and right-sided  lesions with an overall sensitivity of 78% (248/316) at a specificity of 64% (210/328). Treatable cancer (stages I to III) was detected with a left-sided lesion sensitivity of 76% (138/182) and a right-sided sensitivity of 83% (79/95).In several studies we have shown that gene signatures obtained using blood mRNA can identify a variety of conditions occurring in various sites throughout the body, including heart failure , inflammWe have also recently reported a blood test based on the performance characteristics of a seven-gene panel that enables us to assess a patient’s current risk of having CRC . As a blIn this study we use the same seven-gene panel to address another issue limiting the effectiveness of colonoscopy: the right-sided bias observed in such technology. CRC can arise in either the right, proximal colon or the left, distal colon. The former includes the cecum, ascending colon, hepatic flexure and transverse colon, and the latter the descending and sigmoid colon and rectum. Colonoscopy tends to bias towards detection on the left side, for reasons both technical and biological. The blood-based test for CRC reported in this study would have the effect of reducing such bias, thus potentially increasing detection rates for right sided lesions.. For these patients, test sensitivity is 76% for left-sided cancers and 84% for right-sided cancers. TNM IV stage patients are likely to be diagnosed by conventional means and are less likely to benefit much from intervention.This pre-screening test is mainly intended for detection of TNM I to TNM III patientsThis study finds that detection of CRCs using mRNA biomarkers from whole blood is equally sensitive to treatable TNM I – III lesions located throughout the colon Figure . These fCRC: Colorectal cancer; BMI: Body mass index; AUC ROC: Area under the receiver-operating-characteristic curve.Samuel Chao, Gailina Liew and Choong Chin Liew are all employed by GeneNews Ltd, Ontario, Canada, who funded this study. Gailina Liew is President and COO and Choong Chin Liew is Chief Scientist of GeneNews; Wayne Marshall was CEO of the company when the research was carried out. Robert Burakoff has no competing interests to declare.CCL, WM and RB conceived and designed the study; SC and JY provided data analysis; GL and SC drafted the manuscript. All authors read and approved the final manuscript.


The translational axis is one of the most important subsystems in modern machine tools, as its degradation may result in the loss of the product qualification and lower the control precision. Condition-based maintenance (CBM) has been considered as one of the advanced maintenance schemes to achieve effective, reliable and cost-effective operation of machine systems, however, current vibration-based maintenance schemes cannot be employed directly in the translational axis system, due to its complex structure and the inefficiency of commonly used condition monitoring features. In this paper, a wavelet bicoherence-based quadratic nonlinearity feature is proposed for translational axis condition monitoring by using the torque signature of the drive servomotor. Firstly, the quadratic nonlinearity of the servomotor torque signature is discussed, and then, a biphase randomization wavelet bicoherence is introduced for its quadratic nonlinear detection. On this basis, a quadratic nonlinearity feature is proposed for condition monitoring of the translational axis. The properties of the proposed quadratic nonlinearity feature are investigated by simulations. Subsequently, this feature is applied to the real-world servomotor torque data collected from the X-axis on a high precision vertical machining centre. All the results show that the performance of the proposed feature is much better than that of original condition monitoring features. The value of eigenvalue is proportional to the amount of correlation in the direction of their associated eigenvectors, and the maximum eigenvalue linearly increases with the increase of the correlation strength in a cluster. Therefore, the single-value MEBRWB feature can be defined as a global phase coupling feature of the BRWB.At the same time, a BRWB-based quadratic nonlinearity feature, namely the maximum eigenvalue of the BRWB (MEBRWB feature) ,34, is p3.2.n epochs.Step 1: Divide the servomotor torque signal into PFLF  length often covers all characteristic frequencies of translational axis system, the torque signal is divided into n epochs with each period equal to1/PFLF, with the overlap of 75% [Generally, in computing of BRWB, the signal with long data record length is firstly divided into a series of epochs, and then, the reliable bicoherence estimation of the signal is obtained by averaging the BRWB of all epochs. In this study, the BRWB of torque signal is calculated based on smallest meaningful signal segment, due to its non-stationary property. Because that the signal segment of 1/p of 75% .ψW of each epoch by Step 2: Compute the wavelet transform ω0 to 6 to satisfy the admissibility condition, and takes σ within 3 to 8 to obtain the appropriate half power time-width of the mother wavelet.For the torque signals, impulse components often correspond to the degradation of the translational axis system, thus the basic wavelet used for feature extraction should be similar to an impulse . For thiφ of each epoch by using Step 3: Calculate the biphase R within and R to generate a new biphase R φ.Step 4: Generate a random variable iRφe is generated to damage the phase component dependent condition of each epoch by using the new biphase R φ. At the same time, the biphase randomization term is also used to eliminate the spurious bicoherence coming from non-QPC waves.In practice, signals often have very long coherence time, which makes the biphase component dependent over each epoch. The biphase component dependent over each epoch may cause spurious bicoherence. In this step, a biphase randomization term Step 5: Calculate the wavelet bispectrum by iRφe generated by Step 4 is introduced for the wavelet bispectrum calculation, and the result is retained as the sample for the reliable bispectrum estimation. Actually, k data samples can be obtained after k times of cycles of the Step 4 and Step 5. k is taken 100 to keep the balance between a satisfied calculation result and an acceptable calculation time. Thus, 100 data samples can be obtained for reliable bispectrum estimation of this signal epoch, and each data sample is calculated by different biphase randomization term iRφe.In this step, the biphase randomization term Step 6: Estimate the biphase randomization wavelet bispectrum of each signal epoch.n BRWB data samples can be obtained for reliable BRWB estimation.In this step, the biphase randomization wavelet bispectrum of this signal epoch is estimated by ensemble average operation of the 100 data samples obtained in Step 5. The spurious bispectrum coming from non-QPC waves can be eliminated by the ensemble averaging operation. After normalization, the BRWB of this signal epoch can be obtained, and the result is retained as the samples for reliable BRWB estimation. From this step, Step 7: Estimate the BRWB by n signal epochs are used for BRWB estimation, here, the reliable BRWB is estimated by averaging n BRWB data samples obtained in Step 6. The biphase component of each BRWB sample has been made independent over each other in previous steps. Thus, by the average operation, the spurious bicoherence coming from long coherence time waves can be eliminated automatically. Commonly, 10–20 signal epochs are sufficient to obtain a reliable BRWB estimation [Since timation .Step 8: parameter estimation by The SBRWB feature is calculated by The estimation of the BRWB, the SBRWB and MEBRWB features is detailed in the following steps as illustrated in 4.1f and 2f are the coupled frequency, φ1 and φ2 are the initial phase distributed within  is white Gaussian noise with zero-mean and unit variance, T is the period of the signal and A is the coupling coefficient. The simulation signal in 1f = 80 Hz, 2f = 270 Hz, A = 1 and T = 0.25 s with the sampling rate 1,000 Hz, and its wavelet scalogram is shown in According to the QPC model introduced in A from 0 to 1. For comparison, the maximum eigenvalue of the traditional WB (MEWB feature) and the proposed MEBRWB feature are estimated using the same data. By changing the coupling coefficient A from 0 to 1, the estimation of the MEWB feature and the proposed MEBRWB feature at different SNR levels are obtained and plotted in In this signal, the global QPC energy of the simulation signal is adjusted by increasing the coupling coefficient A. Thus, the MEWB feature is not able to detect the coupling components at each SNR levels, when phase-coupled and non-phase-coupled are mixed together. In contrast, as shown in It can be seen from 5.5.1.The proposed method is applied to monitor the translational axis of a high precision vertical machining centre. Experiments are conducted on the X- axis of this machining centre in an in-service environment, and the experimental time is the whole maintenance period of the X-axis. The experiment system is shown in As shown in 5.2.To describe the degradation progress, the proposed BRWB is used to detect the quadratic nonlinear phase coupling frequencies band of the torque signature. Here, the bandwidth from 0–500 Hz is selected for the bicoherence calculation. To further investigate the interactions, the phase coupling distribution of each data sample is estimated by using the SBRWB feature, as shown in For convenience, the single-value MEBRWB feature of the eight torque data samples is calculated to monitor the condition of X-axis. The MEBRWB feature is compared with other commonly used feature during the whole maintenance period of the X-axis, as shown in Results show that the commonly used features don't perform well in describing the progress of X-axis degradation. By contrast, as shown in 6.(1)A BRWB is established to overcome the problem of current WB, which can eliminate the spurious peaks coming from long coherence time waves and non-QPC waves efficiently. Based on the proposed BRWB, a quadratic nonlinearity MEBRWB feature is proposed for translational axis condition monitoring. Numerical example results show that the global QPC of the simulation signal can be tracked approximately linearly by the proposed feature, especially when the SNR is greater than 3 dB.(2)The proposed quadratic nonlinearity MEBRWB feature is also used to study the torque data collected from a high precision vertical machining centre. Experimental results illustrate the robust experimental performance of the proposed feature, compared with commonly used features. The advantage of MEBRWB feature is that it can exploit the true global QPC of the signal at different frequencies, which contains useful additional information for detection of quadratic nonlinear phenomena induced by mechanical faults. Potentially, this single-valued feature can be used in prognostic models for remanding useful life estimation of mechanical components.A BRWB based quadratic nonlinearity feature is established for translational axis condition monitoring of the machine tools, which shows that it is possible to perform condition monitoring by using servomotor torque signature. Some conclusions are drawn as follows:


NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis.in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling.Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit), an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFκB signaling will not be limited to FGFR4 alone. NFκB is a transcription factor of pivotal importance as a regulator of genes that control cell differentiation, survival, and inflammatory responses in mammalian cells. Thus, NFκB has been the subject of intense research to identify clinically useful inhibitors, and to understand the intersection of NFκB signaling with signaling pathways that are important in cancer cell biology. Upon activation with TNFα, IKKβ phosphorylates IκB, the inhibitor of NFκB, which targets it for proteasomal degradation. Subsequently, NFκB is released from sequestration in the cytoplasm, permitting translocation of NFκB dimers into the nucleus where they activate the transcription of target genes Members of the FGFR family of receptor tyrosine kinases are of tremendous significance in many aspects of normal development and, additionally, have been implicated in a variety of human cancers, such as FGFR4 with regards to prostate cancer Using the intracellular domain of FGFR4 as bait, we conducted a yeast two-hybrid assay To confirm the interaction of FGFR4 and IKKβ by coimmunoprecipitation using full-length proteins, human IKKβ was co-expressed with FGFR4 in HEK293 cells. IKKβ interacted with wild-type FGFR4 (FGFR4-WT), as well as with a constitutively-activated mutant of the receptor (FGFR4-K645E) . InteresWe also utilized mass spectrometry to characterize proteins recovered in IKKβ immunoprecipitates. Following expression of both the activated FGFR4-K645E and IKKβ in HEK293 cells, IKKβ immunoprecipitates were analyzed by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS) These results indicate a physical interaction between the intracellular domain of FGFR4, a receptor tyrosine kinase, and IKKβ, an important regulatory protein in NFκB signaling. The interaction described here of FGFR4 with IKKβ, or indeed with any protein involved in NFκB signaling, has not been previously reported.The primary mode of IKKβ regulation is through phosphorylation of serine residues, which can be either activating as when Ser177 and Ser181 are phosphorylated, or inhibitory if phosphorylated on C-terminal residues By SDS PAGE, IKKβ migrates at ∼87 kDa while the lower, unmodified band of FGFR4 almost comigrates at ∼85 kDa. To ensure that the tyrosine phosphorylation observed was on IKKβ and not autophosphorylation of FGFR4 , cells wThese experiments thus provide an explanation as to why tyrosine phosphorylation of IKKβ may not have been previously reported, due to the presence of a Tyr-containing epitope tag on the most commonly used IKKβ vectors Utilizing indirect immunofluoresence, we monitored changes in NFκB translocation to the nucleus in TNFα stimulated cells expressing FGFR4 proteins. In starved unstimulated cells, NFκB was observed to be predominantly cytoplasmic , presumain vitro kinase assays utilizing GST-IκB(1–54) as the substrate (1–54) phosphorylation was visualized and quantified , a significant reduction (approximately 25%) was observed in IKK complex activity. This result demonstrates that activation of the endogenous FGFR4 pathway, in the absence of overexpressed or transfected FGFR4, is sufficient to negatively regulate NFκB signaling. This negative regulation was further enhanced when cells, stimulated with TNFα+FGF19, were expressing excess FGFR4-WT (Lane 7). The inhibitory effects of FGF19 were reversed, however, when cells stimulated with TNFα+FGF19 were expressing FGFR4-KD (Lane 8). Thus, in this assay, the kinase-dead receptor exhibited a dominant-negative effect.Since previous research has implicated FGFR4 in prostate cancer progression, we sought to examine the effect of FGFR4 activation on NFκB signaling in DU145 prostate cancer cells We also examined the interaction of endogenous IKKβ and FGFR4 in DU145 cells. As shown in Using multiple assays, these experiments thus demonstrate that stimulation of the endogenous FGFR4 receptor in DU145 cells exerts an unequivocal negative regulatory effect on TNFα-stimulated outcomes.Next we examined the effect of FGF19 treatment on TNFα-induced apoptosis in the DU145 prostate cancer cell line. Since this cell line has previously been found to be resistant to apoptosis induced by TNF-family ligands, we utilized trichostatin A (TSA), a histone deacetylase inhibitor, to sensitize the cells to TNFα Changes in global gene expression were quantified by microarray analysis using DU145 cells treated with TNFα, FGF19, or both, and harvested at 1.5 h. Using Mock (−FGF19/−TNFα) as the control condition, 1148 out of 24,220 probesets satisfied a corrected p-value cut-off of 0.015 using ANOVA analysis; furthermore, of these, 307 satisfied a fold-change cut-off of 2.0. These results are presented graphically in the heat map shown in The microarray expression data were reanalyzed using the same statistical cutoff as before, but with the [+TNFα] as the control condition. Approximately 260 probesets exhibited a fold change cut-off of 2.0 or more  as the readout. We show that expression of FGFR4-WT or an activated FGFR4 K645E mutant, but not kinase-dead FGFR4, leads to a decrease in the in vitro kinase activity of endogenous IKKβ complexes  (http://www.atcc.org/).HeLa and HEK293 cells were grown in DMEM with 10% FBS and 1% Pen/strep; DU 145 cells were grown in RPMI1640 with 10% FBS and 1% Pen/strep. HeLa and DU145 cells were maintained in 5% CO(1–54) plasmid was provided by Prof. Alexander Hoffmann (UCSD).The full-length FGFR4-WT and constitutively active FGFR4-K645E were described previously Antibodies were obtained from the following sources: FGFR4 (C-16), IKKβ (H-4), IKKβ (10AG2), NFκB p65 (F-6), β-tubulin (H-235), IKKγ (FL-419), normal mouse IgG (sc-2025) from Santa Cruz Biotechnology; phospho-p44/42 MAPK  and cleaved PARP (Asp214) from Cell Signaling; MAPK (ERK1+ERK2) from Zymed; 4G10 (antiphosphotyrosine) from Upstate Biotechnology; horseradish peroxidase (HRP) anti-mouse, HRP anti-rabbit from GE Healthcare; fluorescein-conjugated anti-mouse from Sigma and rhodamine-conjugated anti-rabbit from Boehringer-Mannheim. FGF19 and TNFα were obtained from R&D. mSin3A antibody  was a gift from Dr. Alexander Hoffmann. Poly was obtained from Sigma. Trichostatin A (TSA) was a gift from Dr. Leor Weinberger (UCSD). Techniques for immunoprecipitation and immunoblotting were as described previously Saccharomyces cerevisiae strain L40 generated by Dr. Stan Hollenberg was transformed with derivatives of pBTM116 (constructed by Dr. Paul Bartel and Dr. Stan Fields). A LexA bait plasmid was constructed containing the juxtamembrane and intracellular region of FGFR4 (amino acids 373–803), fused in frame with LexA in pBTM116. This was screened against a 9.5 d.p.c. mouse embryonic cDNA library encoding fusion proteins with the transactivation domain of pVP16, kindly provided by Dr. Stan Hollenberg. Controls for two-hybrid assays, LexA-lamin as a negative control, and VP16-PLCγ as a positive control, were previously described The yeast two-hybrid assay was conducted as described Techniques for indirect immunofluorescence have been previously described in vitro kinase assay utilizing GST-IκB(1–54) as the substrate In vitro kinase assays containing 1 µCi [γ-32P]-ATP in a total of 20 µM ATP were incubated at 30°C for 30 min, separated by 10% SDS-PAGE, exposed to film or phosphorimager screen, and quantitated.HEK293 or DU145 cells were transfected as indicated prior to overnight starvation in DMEM, then treated with 25 ng/ml FGF19 for 10 min and/or followed by 10 ng/ml TNFα for 10 min. Cells lysates were prepared, immunoprecipitated with IKKγ antibody, collected on Protein A-Sepharose beads, and subjected to 32P-labeled 30 bp double-stranded oligonucleotide (GGGACTTTCCGCTGTCTACTTTAGCTTGCTACAA) containing a consensus κB-site in 6 µl binding buffer ). Complexes were resolved on a non-denaturing 5% acrylamide gel containing 5% glycerol, and visualized and quantified using a Phosphorimager (Bio-Rad). Experimental details and probe specificity have been described EMSA assays were as described elsewhere DU145 cells were plated on 10 cm dishes. Upon reaching 80% confluency, cells were starved overnight and treated the next day with 50 ng/ml FGF19 and 1 µg/ml heparin for 10 min prior to the addition of 10 ng/ml TNFα for 30 min. Cell lysates were fractionated as for EMSA.6 per 15 cm dish, 10 dishes total), 1 day prior to transfection with expression plasmids for both the activated FGFR4-K645E and IKKβ. After an additional 24 h, cell lysates were prepared as described HEK293 cells were plated (3×105 per 10 cm dish), and the following day cells were starved for 24 h. Cells were treated with 50 ng/ml FGF19 and 50 µg/ml heparin for 10 min prior to the addition of 10 ng/ml TNFα for 1.5 h. RNA was isolated using RNA-BEE (Tel-Test) per manufacturer's protocol. RNA was analyzed by the UCSD Moores Cancer Center Microarray Shared Resource using Affymetrix GeneChip Human Gene 1.0 ST Arrays (# 901085). Duplicate samples were analyzed in duplicate microarrays, and data were further analyzed by VAMPIRE and GeneSpring. All data is MIAME compliant and the raw data have been deposited in the Gene Expression Omnibus (GEO) (accession number GSE22807).DU145 cells were plated (8×10Table S1Microarray Expression Data of DU145 Cells.(0.03 MB PDF)Click here for additional data file.Table S2Microarray Expression Data of DU145 Cells Using TNFalpha as Control.(0.12 MB PDF)Click here for additional data file.


The Wnt/β-catenin signaling pathway plays a critical role in directing cell fate during the embryonic development of animals and humans. To investigate the effects of the Wnt/β-catenin signaling pathway on gastrointestinal development and differentiation, we studied the expression pattern of β-catenin, a key component of the pathway, in the gastrointestinal tissues of embryonic and perinatal rats. Immunohistochemistry was used to examine the expression levels of β-catenin in Sprague Dawley (SD) rat embryos at days 13, 18 and 21 and in SD rats at 1, 3, 7 and 28 days of age. We observed that the expression of β-catenin was greater and more diffuse in the gastrointestinal tissues of rat embryos at days 18 and 21 of gestation and in SD rats at days 1 and 3. In conclusion, our data suggest that β-catenin also plays an important role in the development of gastrointestinal tissues during the middle and late embryonic periods and the early postnatal period. The Wnt gene family encodes evolutionarily conserved proteins that regulate processes in adults and in embryonic development –4. The WThe Wnt/β-catenin signaling pathway is topic of great interest in biomedicine. Numerous studies have confirmed that the key activator of the Wnt/β-catenin signaling pathway is the nuclear translocation of β-catenin. As a key signaling protein, its accumulation in the cytoplasm and translocation into the nucleus mark the activation of this signaling pathway ,11. HoweSD rats  were purchased from the animal center of the Cancer Institute of the Chinese Academy of Medical Sciences , and bred at the facility of laboratory animals in Tianjin University . All experimental procedures were carried out according to the regulations and internal biosafety and bioethics guidelines of Tianjin Medical University and the Tianjin Municipal Science and Technology Commission . The rats were housed together each night and vaginal secretions were obtained the next morning. Day 0 of gestation was marked as the day when sperm was discovered. Since the average fertility cycle of rats is 28 days, the integrated embryos were removed by cesarean section from anesthetized female rats at days 13, 18 and 21 of gestation and then fixed in 4% paraformaldehyde. Additionally, the gastrointestinal tissues of rats at days 1, 3, 7 and 28 of age were removed and fixed in 4% paraformaldehyde.2O2 for 10 min and incubated with the appropriate antibody  overnight at 4°C. Biotinylated secondary antibody  was added at room temperature for 1 h, which was followed by incubation with ABC-peroxidase for an additional hour. After washing with Tris buffer, the sections were incubated with 3,3′-diaminobenzidine  for 5 min, rinsed in water and counterstained with hematoxylin.After the embryos and the gastrointestinal tissues of rats were fixed, paraffin-embedded tissue sections were used for the examination of β-catenin expression. The sections were dewaxed, treated with 3% Het al. To compare the expression-positive rates of β-catenin at various time points, a χ2=408.73, P<0.001). The positive rates were higher in the latter samples. Secondly, in the nuclei of the samples taken at the 7 chosen time points, the expression of β-catenin was only observed in the rats at day 21 of gestation and days 1, 3 and 7 of age, which corresponded to positive rates of 12%, 81%, 86% and 90%, respectively. Statistical comparisons between any two samples showed that the differences between the sample at day 21 of gestation and the latter three samples were statistically significant , whereas the differences between the samples taken at days 1, 3 and 7 of age were not statistically significant (P>0.05). The positive rate of the sample taken at day 21 of gestation was low, whereas the rates of the latter three samples were high . The positive rate was higher in the latter samples. β-catenin expression was also observed in the nuclei of intestinal samples taken from rats at days 18 and 21 of gestation and days 1, 3 and 7 of age. Using statistical analysis, we identified that the differences between the sample taken at day 7 of age and each of the other four samples were statistically significant , whereas the differences among days 18 and 21 of gestation and days 1 and 3 of age were not (P>0.05). The positive rates were low for the samples taken at day 7 of age, whereas the rates were high for the samples taken at the other four time points  et al. iii) Thiability . Researciability ,20. Undeiability . When thXenopus development, β-catenin is distributed mainly in the cytoplasm of dorsal cells, whereas during the 16-cell stage to 32-cell stage; it is mainly distributed in the nuclei of dorsal cells. However, if the β-catenin in the rat embryo is knocked out, the development of the embryonic primary body axis fails. Few studies have systematically investigated whether β-catenin plays an important role during the middle and late embryonic periods and the early postnatal period.A previous study suggests that Wnt/β-catenin plays an important role in the early development of the embryo . For exaIn the longitudinal sections of gastric tissues from the primitive gut shown in In summary, we examined the development and differentiation of gastrointestinal tissues in the middle and late embryonic periods and the early postnatal period. We observed that the expression of β-catenin in the gastrointestinal tissues was present at day 13 of gestation, activated from day 18 of gestation to day 3 of age, and began to decrease at day 7 of age. These data suggest that β-catenin plays an important role by promoting the development of gastrointestinal tissues in the middle and late embryonic periods and the early postnatal period. However, after tissues and organs mature, β-catenin is maintained at a low level in the cytoplasm so it is not able to promote development. The results of these experiments suggest that β-catenin plays an important role in gastrointestinal tissue development during the middle and late embryonic periods and the early postnatal period, which provides the necessary experimental evidence for further studies concerning the mechanism of this signaling pathway, tumorigenesis and tumor treatment.


Arabidopsis thaliana (Arabidopsis) NON-EXPRESSOR OF PR1 (NPR1) is a transcription coactivator that plays a central role in regulating the transcriptional response to plant pathogens. Developing flowers of homozygous npr3 mutants are dramatically more resistant to infection by the pathogenic bacterium Pseudomonas syringae, suggesting a role of NPR3 as a repressor of NPR1-mediated defense response with a novel role in flower development.NPR3 gene from the tropical tree species Theobroma cacao (TcNPR3). Like in Arabidopsis, TcNPR3 was constitutively expressed across a wide range of tissue types and developmental stages but with some differences in relative levels compared to Arabidopsis. To test the function of TcNPR3, we performed transgenic complementation analysis by introducing a constitutively expressing putative TcNPR3 transgene into an Arabidopsis npr3 mutant. TcNPR3 expressing Arabidopsis plants were partially restored to the WT pathogen phenotype . To test TcNPR3 function directly in cacao tissues, a synthetic microRNA targeting TcNPR3 mRNA was transiently expressed in cacao leaves using an Agrobacterium-infiltration method. TcNPR3 knock down leaf tissues were dramatically more resistance to infection with Phytophthora capsici in a leaf bioassay, showing smaller lesion sizes and reduced pathogen replication.We report here the characterization of a putative TcNPR3 functions similar to the Arabidopsis NPR3 gene in the regulation of the cacao defense response. Since TcNPR3 did not show a perfect complementation of the Arabidopsis NPR3 mutation, the possibility remains that other functions of TcNPR3 remain to be found. This novel knowledge can contribute to the breeding of resistant cacao varieties against pathogens through molecular markers based approaches or biotechnological strategies.We conclude that  To effectively combat a wide range of pathogens, plants are able to induce a complex network of defense responses against invasions . Upon acPR gene expression in the SA-dependent signaling pathway [Arabidopsis thaliana (Arabidopsis) npr1 mutants exhibit higher susceptibility to pathogen infection and impaired expression of PR genes [The most well-studied regulator of systemic acquired pathogen resistance (SAR) is Non Expressor of PR1 (NPR1), a transcription coactivator that regulates  pathway ,8. ArabiPR genes . AdditioPR genes , SA InduPR genes  and TGA PR genes .Several negative regulators of the plant defense response have also been discovered in Arabidopsis -18. The The functions of the Arabidopsis NPR1 paralogs, NPR2, NPR3 and NPR4, are partially known. Various lines of evidence demonstrate that they all act as components of the SA signal transduction pathway -24. NPR3Theobroma cacao (cacao), is caused by the fungal pathogen Moniliophthora perniciosa, is a serious disease in Latin America. Indigenous to the Amazon rainforest, it now occurs in most cocoa growing regions in South America and has became a serious threat to world cocoa production [Understanding the mechanisms regulating the defense response has profound implications for agriculture. Such knowledge can be used to design breeding strategies, or to create transgenic plants with enhanced disease resistance against the plethora of plant pathogens. One such disease, witches’ broom disease of oduction ,26. Termoduction . The genoduction  and a deoduction -32. In aTcNPR1 gene [npr1-2 mutant phenotype in transgenic Arabidopsis. In addition, a phylogenetic analysis of all four of the NPR1-like gene family members in cacao was presented in the supplementary material of Agrout et al. [Theobroma cacao was described.Several homologs of Arabidopsis defensive proteins were previously identified in cacao after SA/MeJA treatment ,34 suggePR1 gene  and demot et al. , in whicTheobroma cacao gene Tc06t011480 (TcNPR3) that is homologous to the Arabidopsis NPR3 gene At1G5G45110. The TcNPR3 gene was capable of partial restoration of the Arabidopsis npr3-3 mutant disease resistant flower phenotype. Moreover, micro-RNA-mediated knock down of TcNPR3 expression in cacao leaves was strongly correlated to decreased susceptibility to infection by the cacao pathogen Phytophthora capsici. Based on our results, we conclude that TcNPR3 shares at least some of the central features of Arabidopsis NPR3, and plays a role as a negative regulator of the defense response.In this manuscript, we present the isolation and functional analysis of the http://esttik.cirad.fr/) houses EST discovery data from fifty-six different cDNA libraries constructed from different organs, genotypes and in varying environmental conditions [Theobroma cacao was sequenced and analyzed from a Belizean Criollo genotype (B97-61/B2) and can be accessed via the Gbrowse website (http://cocoagendb.cirad.fr) [Cacao transcriptomes: The Esttik database (nditions . 454 tranditions . Cacao girad.fr) .TcNPR3 gene was identified by screening BAC filter arrays constructed using genomic DNA of genotype LCT-EEN 37 obtained from the Clemson University Genomic Institute (http://www.genome.clemson.edu/) using a partial TcNPR1 sequence as a radio-labeled hybridization probe. Based on its partial sequence, primers were designed to clone the full-length cDNA of TcNPR3 from genotype Scavina6 (SCA6). Total RNA was isolated from cacao SCA6 stage C leaves as previously described in [TcNPR3 cDNA (starting at the ATG codon and ending at the stop codon of the predicted CDS) was cloned by PCR using 1 μl of ½ diluted cDNA as template and 5 μM of primers were used to include KpnI and NotI restriction sites at the 5′- and 3′-ends respectively . All synthetic oligonucleotides were purchased from Integrated DNA Technologies, Coralville, ID. PCR was performed using the following conditions: 94°C for 2 min., 32 cycles of 94°C for 30 sec., 60°C for 30 sec., followed by a 5 min. final extension at 72°C. PCR product was resolved on 1% agarose gels, purified with the GENECLEAN II Kit  and cloned into the pGEM T-Easy vector . Forward and reverse sequencing was performed at the Penn State Genomics Core Facility to verify the sequence. The resulting clone was designated as pGEM-TcNPR3. The full-length TcNPR3 cDNA sequence was used to identify the TcNPR3 genomic locus in the genome assembly (Tc06t011480) using blastn [Unless otherwise indicated, all chemicals and supplies were obtained from Sigma-Aldrich, St. Louis, MO. A partial sequence of the ribed in . Cacao cribed in . A full-g blastn .TcNPR3 gene structure was annotated by alignment of the full-length cDNA with the genomic sequence using SPIDEY (http://www.ncbi.nlm.nih.gov/spidey/). Arabidopsis NPR3 protein sequence was retrieved from TAIR (At5g45110) and compared to the conceptual translation of the TcNPR3 sequence using MUSCLE [http://smart.embl-heidelberg.de/).The g MUSCLE . PutativRT-Q-PCR was performed to measure the relative expression of TcNPR3 in different cacao tissues. Basically, total RNA was extracted from leaf stages A, C and E, open flowers, un-open flowers, roots, pod seeds and pod exocarps. Three biological replicates were collected for each tissue. Cacao cDNA was synthesized in a final volume of 25 μl from 2 μg of total cacao RNA using M-MLV reverse transcriptase  following the protocol given in .The primers to detect TcNPR3 transcripts were designed based on the coding sequence of TcNPR3 . The 10 μl quantitative RT-Q-PCR mixture contains 4 μl diluted-cDNA (1:50), 5 μl SYBR Green PCR Master Mix (Takara), 0.2 μl Rox, and 0.4 μl each 5 μM primers. Each reaction was performed in duplicates in Roche Applied Biosystem StepOne Plus Realtime PCR System under the following program: 15 min at 94°C, 40 cycle of 15 sec. at 94°C, 20 sec. at 60°C, and 40 sec. at 72°C. The specificity of the primer pairs were examined by RT-Q-PCR visualized on the 2% Agarose Gel and dissociation curve. Based on consistent expression level in the different samples, the cacao tubulin1  gene was used as a control and used to normalize expression data (TUB1-5′: GGAGGAGTCTCTATAAGCTTGCAGTTGG and TUB1-3′: ACATAAGCATAGCCAGCTAGAGCCAG).TcNPR3 coding sequence (p35S: TcNPR3) was created as follows. The TcNPR3 coding sequence was excised from pGEM-TcNPR3 vector and cloned into KpnI and NotI sites of an intermediate cloning vector (pE2113) between the very strong E12-Ω promoter [TcNPR3 gene cassette was excised from pE2113 using EcoRI and PvuII and ligated into the EcoRI and SmaI sites of pCAMBIA-1300 [TcNPR3. The binary vector p35S:TcNPR3 containing a plant selectable hygromycin resistance gene was introduced into Agrobacterium tumefaciens strain AGL1 by electroporation as previously described [npr3-3 mutants with p35S:TcNPR3 [npr3-3 mutant and five individual transgenic lines were collected. RNA was extracted from each genotype using RNeasy plant mini kit . cDNA was synthesized using M-MLV reverse transcriptase  as in [TcNPR3 expression. AtUbiquitin served as a cDNA loading and normalization control. Following primers and conditions were employed: TcNPR3-RT5′: TGCTTGTCGACCCGCCATCAATTT; TcNPR3-RT3′: AGGTTGTCTCAGCATGTGCTATGTCC . Ubiquitin-5′: ACCGGCAAGACCATCACTCT; Ubiquitin-3′: AGGCCTCAACTGGTTGCTGT . The PCR products were resolved on 1% agarose gels.A T-DNA binary vector designed for overexpression of the promoter  and a 35BIA-1300 . Ligatioescribed . The floS:TcNPR3 . Seeds w2light intensity (Octron 4100 K Ecologic bulbs). Pseudomonas syringae pv. tomato DC3000 cultures were grown on pseudomonas agar with kanamycin (25 ng/μl) and rifampicin (100 ng/μl) at 28°C for two days. The pathogen infection and bacterial bioassay was conducted as previously described in [Plants were grown in a Conviron growth chamber at 22°C, with 16 h light/8 h dark cycle 60% humidity and 200 μM/mribed in .http://wmd3.weigelworld.org/cgi-bin/webapp.cgi?page%20=%20Designer;project%20=%20stdwmd) by choosing cacao EST TcaGI-3.0 database. The miRNA targets 373 bp upstream of the stop codon. The primer sequences were: .A 21 nucleotide microRNA of TcNPR3 was designed using the web-based designer at WMD3 , also as described in ,45, withTcNPR3 microRNA) were transformed in cacao leaves by Agrobacterium (AGLI strain) vacuum infiltration. Agrobacterium was grown in 523 medium overnight to OD600 = 1.0 and induced as described in Maximova et al., [-2 sec.-1and 14 h daylight. After two days, two replicate leaf pieces per vector were quickly frozen on liquid nitrogen. Total RNA was extracted as previously described [TcNPR3 to TcActin was measured by RT-Q-PCR using Takara SYBR premix EX TaqII kit (Clontech) according to the user manual with 1:200 dilution of the cDNA template. For each measurement, two biological replicates, each with two technical replicates were performed for both genes using the primers described above.Vectors pGH00.0126 (control) and pGS12.0225  for two days at 27°C, 12 h daylight. Attached leaf pathogen bioassay was performed as previously described [Phytophthora capsici mycelium and the left half was inoculated with sterile agar plugs as negative control. Inoculated leaves were incubated at 27°C and 12 h day/light cycle for three days before the evaluation of disease symptoms. Photographic images of the leaves were taken with a Nikon D90 camera and average lesion sizes were determined using ImageJ software tools (Imagej.nih.gov). Average lesion sizes are calculated from 18 measurements  and significance was determined by single factor ANOVA. To measure pathogen replication as a proxy of virulence, the ratio of Phytophthora DNA to cacao DNA was measured by RT-Q-PCR as follows. Tissue samples including the lesions (1.4 cm2 surrounding the inoculation site) were excised from the infected leaves and used for genomic DNA extraction using a Tissue Lyzer homogenizer and the DNeasy plant mini kit (Qiagen). The relative amount of Phytophthora capsici genomic DNA in leaf disks was measured by amplification of a PcActin gene (primer set F: GACAACGGCTCCGGTATGTGCAAGG and R: GTCAGCACACCACGCTTGGACTG) and TcActin7 (Tc01t010900) (primer set F: AGCTGAGAGATTCCGTTGTCCAGA and R: CCCACATCAACCAGACTTTGAGTTC) were used as pathogen and host targets respectively. DNA RT-Q-PCR was preformed as described in Wang et al. [P. capsici DNA to cacao DNA in the infected tissues was calculated as two to the power of the difference between Ct numbers (2(Ct-P. capsici - Ct-T.cacao)).Cacao pathogen escribed  on the rg et al. , using ahttp://cocoagendb.cirad.fr/gbrowse/cgi-bin/gbrowse/theobroma/) under the following accession numbers: At5g45110 (NPR3), At3g52590 (ubiquitin), BT031870.1(Phytophthora Actin), Tc06t011480/ JX983187 (TcNPR3), Tc01t010900 (TcActin) and Tc06g000360 (TcTUB1).Sequence data from this article can be found in the Arabidopsis Genome Initiative, GenBank databases or cacao genome browser  stage C leaves. A fragment of 1764 bp was isolated, cloned into pGEM vector and sequenced to reveal an intact coding sequence of the expected length and with high homology to the Arabidopsis NPR3 gene.Initially, a partial TcNPR3 gene was identified by searching a cacao genome database (http://cocoagendb.cirad.fr/) [TcNPR3 gene (Tc06t011480) was deduced by comparing the full-length cDNA and genomic sequences using SPIDEY software tool (http://www.ncbi.nlm.nih.gov/spidey/index.html) [NPR3   using thrad.fr/) . The strex.html) , which rne Tc06t01480 was TcNPR3 predicted transcript resulted in a putative protein sequence consisting of 587 amino acid residues, only one amino acid longer than Arabidopsis NPR3. Alignment of the TcNPR3 and AtNPR3 protein sequences demonstrated that they are highly similar to each other (60% identity and 77% similarity). Arabidopsis and cacao NPR3 share key structural features . Its expression was high in roots, exocarps and mature leaves (stage E).RT-Q-PCR was performed to investigate the expression level of d Figure , similarPR3 gene . TcNPR3 npr3 mutant phenotype includes a enhanced level of floral disease resistance and significantly reduced whole plant fitness [TcNPR3, we introduced the cacao NPR3 CDS under the control of a constitutive promoter into Arabidopsis npr3-3 mutant transgenic plants, and tested its ability to complement the npr3 mutant phenotype.Our earlier work has shown that the Arabidopsis  fitness . To explPseudomonas syringae pv. tomato DC3000 (P.s.t.), floral infection resulting in shortened siliques and reduced seed production, the primary phenotype of Arabidopsis npr3-3 mutant is normal silique and seed development regardless of the inoculation of pathogen [TcNPR3 overexpression in the npr3-3 mutant can complement this phenotype, the floral infection assay was carried out with five individual TcNPR3 transgenic lines, Col-0 and npr3-3 mutant Arabidopsis plants. We inoculated young developing flowers with P.s.t and disease symptoms and bacterial growth were assayed. In water-treated inflorescences, we did not observe any phenotypic differences in any of the different lines  showed a level close to that of Col-0. The other two lines (line 1 and line 2) also supported significantly more bacteria than npr3-3 mutant though the level was not as high as in Col-0. In all, four out of five transgenic lines exhibited significantly higher bacteria levels than in npr3-3 mutant, again suggesting that TcNPR3 can at least partially complement npr3-3 mutant phenotype. The TcNPR3 protein only partially complemented the npr3 mutant, most likely because of the heterologous nature of the interactions between TcNPR3 and the Arabidopsis defense response machinery. It is also possible that TcNPR3 has functions that differ from those of AtNPR3. To gain further evidence to support these conclusions, we tested TcNPR3 function directly in T. cacao, using a transient expression system.To further evaluate the effect of the 0 Figure . AlthougTcNPR3 is expressed at moderate to high levels in leaves depending on developmental stage  encodes the functional ortholog of the Arabidopsis NPR3 gene. Two independent experiments testing TcNPR3 function were consistent with a role in suppression of the NPR1-dependent defense response pathway  biotrophic pathogens.The pathogen used in our experimental bioassays, orldwide -56. Our npr3 knockout mutant exhibits a high level of pathogen resistance in developing flowers compared to Col-0, WT plantlets [NPR3 gene expression is low in seeds, moderate in both floral tissues and young leaves, but elevated in the exocarps and stage E leaves  which can infect developing flower cushions and cause hypertrophy and abnormal fruit development.In Arabidopsis, the lantlets . This les Figure . In contThese findings could be used to guide the screening of cacao germplasm for novel sources of disease resistance that could be incorporated into breeding programs. Screening of cacao varieties for lines expressing low levels of NPR3 transcript could be a rapid method for discovery of potentially resistant varieties. By defining the specific genes and mechanisms involved in cacao disease resistance, and translating these discoveries into tools for germplasm screening and breeding, it will be possible to pyramid multiple genes for resistance into the elite cacao genotypes of the future.npr3 mutant allowed us to rapidly confirm its function in the negative regulation of defense response in floral tissues. Moreover, the knock down of TcNPR3 in cacao leaves via cacao transient transformation resulted in decreased resistance to cacao pathogen Phytophthora capsici in detached leaf bioassays. Taken together, we conclude that TcNPR3 is a functional ortholog of Arabidopsis NPR3 gene. This fundamental finding can potentially help us to breed cacao varieties with enhanced disease resistance by traditional breeding and/or transgenic manipulations, which will benefit farmers, developing countries, the environment and the chocolate industry.The isolation of NPR3 from cacao and its heterologous complementation in Arabidopsis No financial or non-financial competing interests exist related to the subject matter of this manuscript. No reimbursements, fees, funding, or salary were received from any organization that may in any way gain or lose financially from the publication of this manuscript, at any time. All costs of this research and its publication charges were funded by grants from the Penn State Endowment in the Molecular Biology of Cacao. None of the authors hold stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, at any time. The authors are not currently applying for any patents relating to the content of the manuscript, nor have they received reimbursements, fees, funding, or salary from any organization that holds or has applied for patents relating to the content of the manuscript, There exist no non-financial competing interests  to declare in relation to this manuscript.ZS, SM and MG contributed to the design of all experiments, ZS was involved in implementation of most experimental plans and data acquisition, YZ contributed to the RT-Q-PCR experiment and data analysis of TcNPR3 expression across tissues, ZS and SM contributed to analysis and interpretation of all data as well as manuscript drafting and revision. MG conceived the overall plan of study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Protein arginine methyltransferase 5 (PRMT5) plays multiple roles in a large number of cellular processes, and its subcellular localization is dynamically regulated during mouse development and cellular differentiation. However, little is known of the functional differences between PRMT5 in the cytoplasm and PRMT5 in the nucleus. Here, we demonstrated that PRMT5 predominantly localized in the cytoplasm of prostate cancer cells. Subcellular localization assays designed to span the entire open-reading frame of the PRMT5 protein revealed the presence of three nuclear exclusion signals (NESs) in the PRMT5 protein. PRMT5 and p44/MED50/WD45/WDR77 co-localize in the cytoplasm, and both are required for the growth of prostate cancer cells in an PRMT5 methyltransferase activity-dependent manner. In contrast, PRMT5 in the nucleus inhibited cell growth in a methyltransferase activity-independent manner. Consistent with these observations, PRMT5 localized in the nucleus in benign prostate epithelium, whereas it localized in the cytoplasm in prostate premalignant and cancer tissues. We further found that PRMT5 alone methylated both histone H4 and SmD3 proteins but PRMT5 complexed with p44 and pICln methylated SmD3 but not histone H4. These results imply a novel mechanism by which PRMT5 controls cell growth and contributes to prostate tumorigenesis. PRMT5 is crucial for mouse embryonic development Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues within target proteins p21 and p27 gene expression; downregulation of cyclin A, cyclin B, and CDK2 gene expression; and cell cycle arrest at the G1/G0 phase We purified and cloned a novel androgen receptor (AR)-interacting protein, designated p44 PRMT5 forms a stoichiometric complex with p44/MEP40/WD45/WDR77 in various cells Our research did not involve human participants and animals, only the use of human prostate cancer tissues. The patients cannot be identified, directly or indirectly, through identifiers linked to the subjects. Thus, the research project was exempt under Exemption 4 (45 CFR Part 46) and an ethics statement is not required.Benign and cancerous prostate tissues were derived from radical prostatectomy specimens of 19 patients with prostate cancer treated at New York University Medical Center, and the study protocol was approved by its institutional review board. Patient identities were removed from all samples and an exemption from the need for consent was granted by the institutional review board of New York University School of Medicine, so no informed consent was needed. The tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Immunohistochemical analysis was performed on the 19 human prostate cancer samples as described previously Cultured cells were grown on chamber slides and fixed with cold methanol (–20°C) for 10 min. Nonspecific proteins were blocked in 4% fish gelatin in PBS for 20 min. Overnight incubation at 4°C with primary antibodies was performed followed by a 1-h incubation with anti-mouse or anti-rabbit IgG antibody labeled with Alexa 595  at room temperature. The samples were washed in PBS and then counterstained with TOPRO 3, Far-red, or Sytox green (Molecular Probes) for 10 min at room temperature, mounted in Histogel (Linaris Histogel), and analyzed directly by fluorescence confocal microscopy. For double staining, anti-PRMT5 and anti-p44 or anti-PRMT5 and anti-coilin  were incubated with cells overnight at 4°C. The secondary antibodies  were used.LNCaP, PC3, and Cos 7 cells were cultured in RPMI 1640 medium (Cellgro) with 10% (v/v) fetal bovine serum (HyClone). For the cell growth assay, cells  were plated onto 24-well plates, and cell numbers were counted every day for 7 days.5) using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. The transfected cells were fixed with cold (–20°C) methanol for 10 min, stained with TO-PRO 3 (10 micrograms/ml) (Molecular Probes), mounted in Histogel (Linaris), and analyzed directly by fluorescence confocal microscopy.The PRMT5 cDNA fragments were amplified from the pcDNA-PRMT5 construct 5′-GGGAACTAGATGAGAATGA-3′), PRMT5 shRNA (target sequence: 5′-GGATAAAGCTGTATGCTGT-3′), and a nontargeting shRNA (NT-shRNA) (target sequence: 5′-TTCTCCGAACGTGTCACGT-3′) were designed with a hairpin and sticky ends (ClaI and MluI). The oligonucleotides were annealed into the lentiviral gene transfer vector, pLVTHM, using the ClaI and MluI restriction enzyme sites. The DNA constructs were sequenced to test for proper insertion and length of the inserts. The lentivirus was then produced by transfecting human embryonic kidney cells  with the sequence-verified PLVTHM vector, the packaging plasmid (MD2G), and the envelope plasmid (PAX2), which are required for viral production. Three days later, the viral supernatant was collected and filtered to remove cellular debris. LNCaP cells (1×105) were plated onto six-well plates and transduced with lentivirus vector particles. After 16 h, the virus-containing medium was removed and replaced with normal growth medium. Three days after infection, cells split at 1∶6 and were grown for 3 days. Whole-cell lysates (5 micrograms of protein) made from the infected cells were analyzed by Western blot.P44 shRNA (p44-shRNA) . The recombinant lentivirus was produced with 293T as described above. To rescue PRMT5 or p44 expression, LNCaP PRMT5-shRNA or p44-shRNA cells were plated onto six-well plates and transduced with the virus containing either the nontargetable PRMT5 or p44 expression vector or empty vector. After 48 h, the cells were re-plated, and the PRMT5 or p44 expression was confirmed by Western blot.To create nontargetable PRMT5 and p44 expression vectors, the nucleotide sequences targeted by shRNAs were mutated using an oligo-direct mutagenesis kit. The target sequence Cytoplasmic and nuclear fractions were prepared from cultured cells by using the Nuclear Extract Kit  as described previously PRMT5 and p44 were detected in total cell extracts (5 micrograms) by 10% SDS-PAGE and transferred to an Immobilon-P transfer membrane (Millipore). The membranes were washed in Tris-buffered saline with Tween 20  and blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. The blots were then probed overnight with primary antibodies at dilutions of 1∶2,000 (anti-p44), 1∶1,000 (anti-PRMT5), 1∶1000 , 1;500 , and 1∶1,000 . After 1.5 h of incubation with horseradish peroxide-conjugated secondary antibody, immunoreactive proteins were detected by enhanced chemiluminescence using the ECL detection system per the manufacturer's instructions . Protein concentrations were determined by using the Bradford protein assay (Bio-Rad).6) were transfected with 6 micrograms of pcDNA-f:p44, -f:p44(26–27AAA), -f:p44(29–31AAA), -f:p44(35–37AAA), or -f:p44(42–44AAA) with Lipofectamine 2000. The whole-cell lysates were prepared from the transfected cells 48 h after transfection and incubated with 15 microliters of M2 agarose (Sigma) for 2 h at 4°C in a final volume of 0.5 ml containing 20 mM HEPES (pH 7.9), 0.2 mM EDTA, 20% glycerol, 2 mM DTT, 300 mM KCl, and 0.1% NP40. The beads were washed five times (1 ml each) with the incubation buffer. The bound proteins were eluted with 30 microliters of FLAG peptides (0.2 mg/ml) for 30 min at 4°C and analyzed by Western blot with anti-PRMT5 antibody.PC3 cells  with an amino-terminal His6 tag, and the p44 or pICln coding region was cloned into the second multiple cloning site of the same vector with a N-terminal FLAG-epitope tag. For PRMT5 co-expression with p44 and pICln, PRMT5 was cloned into pACYCDuet with an amino-terminal His6 tag, the pICln coding region was cloned into the second multiple cloning site of the same vector, and the coding region of p44 was cloned into a pET vector with an N-terminal FLAG-epitope tag. Proteins were expressed in BL21(DE3) cells at 30°C for 3 h after induction with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside. Cells were lysed by sonication three times for 5 min each in lysis buffer . The proteins were purified using Ni-NTA agarose (Qiagen) according to the manufacturer's protocol with imidazole elution and subsequently purified on the M2 agarose (Sigma-Aldrich) with FLAG peptide elution for PRMT5-containing complexes. Histones were purified from HeLa cells as described previously PRMT4, p44, pICln, or SmD3 cDNA was cloned into pET15d (Novagen) to be expressed as an amino-terminal His3H]adenosymethionine (PerkinElmer) were incubated in 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, and 1 mM EDTA at 30°C for 1 h. Reactions were boiled in SDS sample buffer and separated on a 15% polyacrylamide gel. Gels were fixed for 30 min in 40% methanol-10% acetic acid, incubated in 20 ml of Amplify (Amersham Life Science) for 10 min, dried, and exposed to x-ray film at −80°C.Methylation reactions were performed as previously described with a few modifications The subcellular localization of PRMT5 is dynamically regulated during mouse development Western blot of the cytoplasmic and nuclear fractions confirmed its subcellular localization -tagged PRMT5 (GFP-PRMT5) was transiently expressed in PC3, LNCaP, and Cos 7 cells, and the resulting GFP-PRMT5 fusion protein had a predominant cytoplasmic localization , panel dTwo protein fragments, PRMT5(1–324) and PRMT5(325–637), were found within the cytoplasm in 100% of transfected cells , suggestFurther deletion analysis identified the other two NES sequences in the C-terminal part of PRMT5. The region spanning amino acid residues 500 to 560 localized to the cytoplasm in 100% of transfected cells  to alanines in the p44 protein and examined the consequence of these mutations on PRMT5-driven cytoplasmic translocation of GFP-p44 (35RYR37 to 35AAA37 and 42LLL44 to 42AAA44) abolished PRMT5-driven cytoplasmic translocation of GFP-p44  in p44 abolished its interaction with PRMT5  targeted against the PRMT5 sequence. To test whether the shRNA could suppress PRMT5 expression, we infected LNCaP cells with the lentiviral vector transducing a DNA segment specifying such shRNA sequence. As shown in LNCaP cells expressing NT, PRMT5, or p44 shRNA were plated onto 24-well plates, and cell numbers were counted every day. Silencing PRMT5 or p44 expression strongly inhibited the growth of LNCaP cells . LNCaP cWe noticed that the mutant p44 proteins that lack the interaction with PRMT5 were expressed at lower levels than the wild-type p44 protein , and silPRMT5 or p44 was silenced in LNCaP cells, and at the same time the shRNA-resistant p44 or PRMT5 was expressed via lentivirus. As demonstrated above, p44 or PRMT5 shRNA expression strongly downregulated both PRMT5 and p44 protein levels . The lenTo investigate the function of PRMT5 in the nucleus, we targeted PRMT5 into the nucleus of LNCaP cells by fusing a strong nuclear localization signal (NLS) Several groups have shown that PRMT5 interacts with p44 and pICln 3H]adenosymethionine. SmD3 contains multiple RG repeats in its carboxyl terminus that are methylated by PRMT5 in vivo and in vitroTo test the methyltransferase activity, we incubated purified PRMT5 or PRMT5-containing complexes with SmD3 or histone substrate in the presence of S-[methyl-To evaluate p44 and PRMT5 nuclear and cytoplasmic subcellular localization in areas of benign, premalignant , and cancer tissues, we performed immunohistochemical staining for p44 and PRMT5 in 19 samples derived from patients with prostate cancer. Both p44 and PRMT5 were negative in stromal cells . As obseBy using a subcellular localization assay, we found that PRMT5 contains three strong NES sequences that determine its predominant cytoplasmic localization in prostate cancer cells. The functional activity of PRMT5 is controlled by its subcellular localization. When localized in the cytoplasm of prostate cancer cells, PRMT5 is essential for cell growth; in contrast, the nuclear PRMT5 suppresses cell growth. PRMT5 and p44 are co-expressed in the cytoplasm, and both are required for the growth of prostate cancer cells. In addition, we demonstrated that PRMT5 and p44 localized in the nucleus in benign prostate epithelium but localized in the cytoplasm in prostate premalignant and cancer tissues. These results imply a novel role for PRMT5 and p44 in the control of cell growth and in prostate tumorigenesis.Mammalian PRMT5 primarily localizes to the cytoplasm in somatic cells The p44 protein localizes to both the cytoplasm and nucleus of prostate cancer cells when expressed as a GFP-fusion protein since it contains both NESs and NLSs Silencing PRMT5 or p44 by shRNA significantly decreased both PRMT5 and p44 proteins in the cytoplasm and had little effect on their expression in the nucleus of LNCaP cells. The slow turnover of proteins in the nucleus might account for this different effect of shRNAs on cytoplasmic and nuclear p44 and PRMT5 proteins. Silencing p44 or PRMT5 expression dramatically inhibited the growth of LNCaP cells. The growth effect of PRMT5 is dependent on its methyltransferase activity. Given the fact that p44 and PRMT5 form a stoichiometric complex and are co-expressed in the cytoplasm of LNCaP cells, it is more likely that p44 and PRMT5  in the cytoplasm function as a unit (complex) in the control of cell growth via methylating substrates.in vitro and in living cells in vivo. Thus, COPR5 may be an important chromatin adaptor for PRMT5 to function in the nucleus.On the other hand, the forced nuclear localization of p44 Our studies revealed distinct subcellular localization of p44 Taken together, the results from this study provide insight into the functional roles of PRMT5 in the control of cell growth and in prostate tumorigenesis.Figure S1PRMT5 promotes p44 cytoplasmic translocation independent of its methyltransferase activity. (A) Cells were transfected with pcDNA-GFP-p44 and pcDNA-PRMT5 or pcDNA-PRMT5mt and the GFP-p44 subcellular localization was observed under a confocal microscope. (B) Western blot analysis of PRMT5 and PRMT5mt expression in the transfected cells.(TIF)Click here for additional data file.Figure S2The amino acid residues in p44 are required for PRMT5-promoted p44 cytoplasmic translocation. Cells were transfected with pcDNA-PRMT5 and pcDNA-GFP-p44 or pcDNA-GFP-p44 truncations, and the GFP-p44 or GFP-p44 truncation subcellular localization was observed under a confocal microscope.(TIF)Click here for additional data file.Figure S3PRMT5 expression alone is not sufficient to support growth of LNCaP cells. (A) Western blot of whole-cell lysates derived from LNCaP cells expressing NT shRNA, p44 shRNA, PRMT5 shRNA, p44 shRNA plus PRMT5, PRMT5 shRNA plus p44 with anti-PRMT5, -p44, or -actin antibody as indicated. (B) Growth curves of LNCaP cells expressing NT shRNA, p44 shRNA, PRMT5 shRNAs, p44 shRNA plus PRMT5, or PRMT5 shRNA plus p44.(TIF)Click here for additional data file.