A key question in neuroscience is how cortical organisation relates to experience. Previously we showed that amputees experiencing highly vivid phantom sensations maintain cortical representation of their missing hand . Here, we examined the role of sensory hand experience on persistent hand representation by studying individuals with acquired and congenital hand loss. We used representational similarity analysis in primary somatosensory and motor cortex during missing and intact hand movements. We found that key aspects of acquired amputees’ missing hand representation persisted, despite varying vividness of phantom sensations. In contrast, missing hand representation of congenital one-handers, who do not experience phantom sensations, was significantly reduced. Across acquired amputees, individuals’ reported motor control over their phantom hand positively correlated with the extent to which their somatosensory hand representation was normally organised. We conclude that once cortical organisation is formed, it is remarkably persistent, despite long-term attenuation of peripheral signals. A fundamental organising principle in the primary somatosensory cortex (SI) is somatotopic mapping, where adjacent body parts are represented more proximally on the cortical sheet than those further apart . In the We  and otheOur previous findings demonstrate the stability of SI hand organisation despite decades of amputation. It remains unknown, however, whether hand representation after amputation reflects phantom sensations, and as such only persists in individuals with highly vivid phantom sensations. Here, we asked whether persistent representation of a missing hand reflects an organisational principle in the sensorimotor cortex, and thus will even be observed in amputees with little phantom sensations. To address this question, we measured cortical hand representation in 18 acquired amputees with varying vividness of their phantom sensations (hereafter amputees). To test the idea that the development of a hand representation requires sensory experience, we also tested 13 individuals missing one hand from birth . All participants underwent fMRI while performing a visually cued motor task involving individual digit movements (of both the missing hand and the intact hand). Activity patterns in the missing hand of SI and M1 were analysed using representational similarity analysis . We hypoWe first focused our analysis on the representation of the missing hand, as revealed by instructing individuals to move individual digits of their missing hand (or nondominant hand in controls). We interrogated fMRI activity in the SI hand area contralateral to the missing/nondominant hand  definition). We examined univariate task-related activity, as quantified by averaging the BOLD response across all the digit conditions within the missing hand ROIs  with a dataset of hand RDMs in 2-handed controls drawn from a different study  and controls . We note that by splitting the data we are reducing the effectiveness of our analysis. Nevertheless, the relative reduction in split-half consistency indicates that there is no strongly consistent digit information in the missing hand area of congenital one-handers during this task.Although the inter-digit representational structure of congenital one-handers is atypical with respect to canonical hand representation, it is possible that it is still consistent within participants. To explore this idea, we split individual participants’ data to odd and even scans. For each participant, we calculated an RDM in the missing/nondominant hand area using the odd and even runs, and correlated the two RDMs. The correlation between odd and even RDMs was significantly lower in congenital one-handers (rho = -.02) compared to both amputees  and the intercept . This regression was submitted to a bootstrapping analysis, allowing us to estimate the consistency of the final model . This bootstrapping analysis returned kinaesthesia as the final variable in 96.3% of the iterations . The proportion of the other included factors in the final model was: typicality of the intact hand (7.0%); time since amputation (9.5%); age at amputation (9.6%); vividness of nonpainful phantom sensations ; phantom limb pain, acute: 22.7%; chronic: 10.1%).Next, we evaluated whether the consistency of hand representation in SI during missing hand movements correlates with amputees’ subjective reports of phantom sensations. We first carried out an exploratory forward stepwise regression with typicality as the dependent variable. The following factors were tested as independent variables: kinaesthesia of phantom sensations - the number of phantom digits perceived as independently moving during the phantom movement task; vividness of nonpainful phantom sensations as experienced both during the study and chronically; intensity of phantom limb pain, as experienced both acutely during the study and chronically; time since amputation; age at amputation, and; typicality of the intact hand . The final model . This analysis suggests that although better recruitment of M1 is expected in individuals with clearer kinaesthetic sensations, the strong correlation between kinaesthesia and typicality in SI does not merely reflect information in M1.Further analysis confirmed that the correlation between kinaesthesia and typicality remained significant in SI after accounting for typicality in M1 (mean rho = 0.44) as a covariate  dependent variable . These results predict that even amputees who do not experience any phantom sensations will retain some typical missing hand representation. To test this prediction directly, we examined the three amputees in our dataset showing weak to no chronic phantom vividness (below 10/100). Despite not being able to experience clearly their phantom hand when performing the phantom movements task, these individuals showed high typicality  = 0.83). Moreover, when comparing their typicality to that found in the congenital one-handers (who were arguably better matched to this sub-group in terms of task demands), the amputees with diminished phantom sensations showed significantly stronger correlations with the canonical hand structure . Typicality was not different between these three amputees and controls . Together, these additional analyses confirm that the representational structures’ typicality in SI of amputees is still present in those with little to no phantom or kinaesthetic sensations.Regardless of the positive relationship between kinaesthesia and typicality, amputees with little to no kinaesthetic sensations still showed missing hand representation. As stated above, the regression line between kinaesthesia and typicality had an intercept of βWhile the task involving phantom hand movements was suitable to test the persistence of missing hand representation in individuals with phantom sensations, it was not designed to rule out the existence of missing hand representation in congenital one-handers. Indeed, it is possible that congenital one-handers have typical sensorimotor representation of their missing hand, but they did not access it due to unnatural task demands  hand in the missing hand cortex. In two-handed controls, finger movements lead to individuated digit representation in specific cortical patches in ipsilateral M1 and SI, which tightly correspond to the activity patches engaged in the movement of the mirror-symmetric contralateral finger . Importa(28)=1.42, p=0.166, BF = 0.209; typicality: t(28)=.69, p=0.498, BF = 0.244). In contrast, ipsilateral representation in congenital one-handers was significantly lower than in amputees (dissimilarity: t(29)=3.81, p<0.001; typicality: t(29)=3.05, p=0.005) and showed similar trends versus controls (dissimilarity: t(23)=2.20, p=0.038; typicality: t(23)=2.19, p=0.039). Together, these findings provide additional support for the reduced existence of inter-digit representational difference in the missing hand cortex of congenital one-handers versus amputees, independent of missing hand motor skill.Mean (intact hand) ipsilateral digit dissimilarity and typicality were not significantly different between amputees and controls =2.51, p=0.027), indicating that there were significant differences between the activity patterns associated with each finger. We therefore wished to determine whether the dissimilarity measures reflect meaningful (though reduced) sensorimotor digit information, or rather the increased sensitivity of RSA to other (not sensorimotor) inter-digit differences . For example, a recent study demonstrated that visual information about touch on the hand is sufficient to induce some residual digit-selective activity patterns in SI . We therHere, we demonstrated long-term stability of SI hand representation in a group of acquired amputees with diverse phantom sensations, including those experiencing limited phantom sensations. Using RSA, we find that amputees show individuated digit representation for their missing hand, as exemplified by significantly greater inter-digit dissimilarity values in amputees versus congenital one-handers. The inter-digit pattern comprising the missing hand representation was typical to SI hand representation in amputees and was not significantly different from controls . Importantly, by studying individuals with a varying range of phantom sensations, we were able to confirm stable hand representation even after phantom sensations have diminished. This result confirms the persistence of hand representation as a general principle in amputees, contrary to recent reports . Using tWe also explored whether we could activate the representation of the missing hand indirectly by movements of the fingers of the intact hand. Previous studies in two-handers have demonstrated that contralateral and ipsilateral hand movements produce identical representational patterns . Since tIt has previously been shown that restored peripheral input, for example via hand transplantation  or targeIt is important to consider how the finding of robust persistence of hand representation, despite the physical absence of a hand, conceptually aligns with other reports of brain reorganisation. Since the pioneering work of Hubel and Wiesel, demonstrating that input loss to visual cortex in early development leads to profound physiological changes , it has As mentioned above, the missing hand cortex in amputees, but not in congenital one-handers, has been previously shown to respond to inputs from the intact hand , presumaTo conclude, here we show that once sensorimotor hand-representation is formed, it is generally immutable to change: We identified stable hand representation in amputees’ sensorimotor cortex using representational similarity analysis, despite years (and even decades) of amputation and irrespective of their phantom sensations vividness. In contrast, individuals born with a missing hand  did not show normal representation of their nonexisting hand. We therefore suggest that consistent sensory representation despite input loss may be a common organising principle . How canWe tested 18 acquired amputees with an average of 18 years since amputation , 13 congenital one-handers , and 12 two-handed control participants . All amputees reported experiencing phantom sensations after amputation, but vividness of these sensations varied across participants at the time of the study . Informed consent and consent to publish was obtained in accordance with ethical standards set out by the Declaration of Helsinki. Control participants were recruited as to match the other two groups in term of age, gender and handedness (with respect to the intact hand). When possible, control participants were friends and family of the one-handed participants. All participants were compatible with local magnetic resonance imaging (MRI) safety guidelines.https://osf.io/gmvua/). Here we focus on procedures related to the representation of the missing hand in amputees and congenital one-handers.The experimental procedures described in this manuscript were run as part of a larger study  of either hand, as well as no movement (rest) blocks. Each condition was repeated three times in a semi-counterbalanced order within each run. Each run comprised a different block order.To probe somatosensory digit representation, we used a visually cued active (motor) task. In an intact sensorimotor system, movement recruits a combination of peripheral receptors, encoding a range of somatosensory modalities , as well as efferent information from the motor system. Using an active task, we have previously shown high consistency of SI digit topography across multiple scanning sessions  with their nondominant (controls), phantom (amputees), or missing hand . Handless individuals were instructed to attempt performing actual movements with the digits of their missing hand, even when not being able to feel their digits, rather than using motor imagery. Controls moved their nondominant hand digits in mid-air. To ensure good understanding of these instructions, outside the scanner, the amputees were asked to demonstrate to the experimenter the extent of volitional movement they felt they were able to carry out in each of their phantom digits, by mirroring each movement onto their intact hand.On ‘intact hand blocks’, all participants performed a comparable task with their intact/dominant hand by exerting force on a button box. Participants received real-time visual feedback of how much force each digit exerted by means of moving vertical bars on ‘intact hand blocks’, but not on ‘missing hand blocks’. The dominant hand of controls was paired up with the intact hand because, through intensive use, amputees’ and congenital one-handers’ intact hand becomes their de facto dominant hand . All groMRI images were acquired using a 3T MAGNETON Prisma MRI scanner  with a 32-channel head coil. Functional images were collected using a multiband T2*-weighted pulse sequence with a between-slice acceleration factor of 4 and no in-slice acceleration. This provided the opportunity to acquire data with high spatial (2 mm isotropic) and temporal (TR: 1500 ms) resolution, covering the entire brain. The following acquisition parameters were used: TE: 32.40 ms; flip angle: 75°, 72 transversal slices. Field maps were acquired for field unwarping. A T1-weighted sequence was used to acquire an anatomical image .https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/, https://www.fil.ion.ucl.ac.uk/spm/, humanconnectome.org) in combination with other Matlab scripts (version R2016a), both developed in-house  analysis was carried out, as implemented in SPM12. In brief, each of the experimental conditions was modelled for each run separately against rest. Regressors were created by convolving stimulus presentation (as a boxcar function) with a double-gamma hemodynamic response function (HRF). In the GLM estimation, the functional data was weighted using the robust Weighted Least Squares approach , which eThe dissimilarity between activity patterns within each ROI was measured for each digit pair using the cross-validated squared Mahalanobis distance, or ‘crossnobis’ distance . We calcWe extracted two measures from the resulting inter-digit representational dissimilarity matrix (RDM). As a measure of strength of the representation, we used the mean dissimilarity, the average dissimilarity between the ten unique digit pairs . The typicality of the representational structure was assessed by calculating the Spearman’s rho correlation between the measured RDM and the average RDM of the dominant hand of two-handed controls . Because the representational structure can be related to behavioural aspects of hand use and is highly invariant in controls . MDS projects the higher-dimensional RDM into a lower-dimensional space, while preserving the inter-digit dissimilarity values as well as possible . MDS wasAs mentioned above, to determine typicality we correlated RDM from the current study with the average representational structure of the dominant hand of two-handed controls, in an independently acquired cohort of participants. The full details of the acquisition parameters are described in Statistics were calculated using Matlab R2016a. Subsequent to normality validation (using the Shapiro-Wilk test), we used paired/independent-sample two-tailed Student’s t-tests to compare activity levels and distance measures within/between groups, and one-sample t-tests to compare group measures to zero. Correlations were calculated as Spearman’s rho. Partial correlation effects were calculated using linear regression. For the main analysis concerning RSA, within each ROI, group t-tests were adjusted to three comparisons, using the Bonferroni correction (α = 0.05/3), to account for the three inter-group comparisons. Post-hoc correlations between SI typicality and key clinical measurements  were also corrected for three comparisons (α = 0.05/3). One post-hoc comparison involving a small subset of amputees was done using a Mann-Whitney U test (see Results - section 2). For a control analysis, involving visual and somatosensory ROIs, we also used a mixed-design analysis of variance (ANOVA) to identify interactions across groups and ROIs.In order to assess whether any aspect of the representational structure in the amputees was not different from that in controls, we used Bayesian statistics as implemented in Javascript . Our alt2 >0.1. As a large number of predictor variables were included in the model and stepwise regression is generally only recommended for exploratory analysis, we aimed to establish internal replicability using bootstrap resampling (e.g. 2. We interpreted high proportion of inclusion (p>0.75) as evidence for internal replicability  in healthy controls, in amputees and in congenitally one-handed participants. Two approaches are taken. The first is to instruct participants to "move" their (absent) digits. Then a representational similarity analysis is conducted to test for the representational structure across (digit-) conditions. BOLD activity, average representational dissimilarity and "typicality" (a comparison to controls) are similar between controls and amputees, but virtually absent in congenital one-handers. Second, as a control to ensure that this is not simply due to an inability of congenital one-handers in performing the task, a comparison is made using the ipsilateral representation of the other hand, confirming the initial findings.eLife. The difference is that the current paper includes congenital one-handed participants, which allows one to tell whether the representational structure is experience-dependent. The reviewers thought that this is a valuable addition to the previous study. However, there were a number of residual concerns that need to be addressed further.The experiment builds upon and extends a previous paper by the group published in Essential revisions:1) For the first finding, the only support is a correlation between one imaging measure  and one questionnaire response (# of independently controllable parts of the hand). Given this, one would expect very large differences between those who can and cannot move their fingers independently – not only in representational typicality, but also in dissimilarity. Surprisingly, the correlation between kinaesthesia and dissimilarity is not reported in the manuscript. As stated in the Introduction, "greater individuation of inputs will induce greater representational dissimilarity." Therefore, if amputees vary on self-reported individuation, then there should be a correlation between these two variables. This needs to be reported. If there isn't a relationship between the two variables, then it makes it quite difficult to interpret the significant correlation between typicality and "kinaesthesia". Second, it is difficult to be confident about correlations with questionnaire data with a small sample size (e.g. the 95% CI for the reported correlation ranges from.33 to.89). Therefore, the relationship between typicality and kinaesthesia would be more convincing if there were other, related correlations that are also significant.  Further, the authors stated in the introduction that they would examine "whether persistent representation of a missing hand…will also be observed in amputees with little phantom sensations." Based on this, we were expecting an analysis of the specific individuals with little/no phantom sensations  have a phantom representation similar to intact controls). This should be included in a future submission.2) The authors show that congenital amelics demonstrate reduced activity and digit discriminability in S1 for phantom movements compared to controls. We share some of the concerns of an earlier reviewer. Given that the authors suggest here and previously  that the S1 activity observed when moving the phantom is due to efference copy, it is unlikely that congenital amelics can generate efference copy for a limb that never existed. However, the lack of efference copy activation in S1 of congenital amelics is not the same as demonstrating that the hand representation has not developed without sensory experience – one of the goals of this paper (see Introduction). That is, there could be a hand representation that exists, but simply hasn't been accessed with the movement task. The manuscript, at times, seems to conflate a lack of activation in S1 for moving a phantom/non-existent hand as evidence for the lack of any hand representation. Although we are sympathetic to this view, we don't think the evidence is there given this single task – and so the claims should be softened.3) The authors find that controls and amputees did not significantly differ across a number of variables. One example is that typicality did not differ between the two groups. The authors used Bayes factors, using a BF<1/3 as positive evidence in support of the null. Given the editor's concerns regarding exactly what "substantial" evidence means in the context of BFs, one suggestion would be to use frequentist tests designed specifically to examine whether two groups are equivalent , Lakens, 2017). This provides a standard p-value that is more straightforward to interpret and is consistent with the (primarily) frequentist tests used throughout the manuscript.4) The argument for using the ipsilateral hand representation to probe digit structure in the missing hand (Results section) was quite unclear. We understand that Diedrichsen showed a relationship between ipsilateral and contralateral digit representation. But this was in individuals with intact hands. Why would the fact that the "ipsilateral digit representation is a reliable predictor of contralateral hand representation" in two-handers mean that this would also be true for amputees or congenital one-handers? This logic should be laid out.5) We suggest to briefly explain that the absence of information for the congenital one-handed participants means that the effect is not driven by the visual cue.6) Is the MDS-analysis cross-validated?7) To which degree is the typicality analysis influenced by SNR? Does the low dissimilarity of the congenital group  mean that the responses were reproducibly very similar across conditions ?8) Is this a completely separate dataset than in the original paper? This should be clarified.[Editors’ note: further revisions were requested before acceptance.]eLife. Your revised article has been favorably evaluated by Eve Marder (Senior Editor) and two reviewers.Thank you for resubmitting your work entitled "Obtaining and maintaining cortical hand representation as evidenced from acquired and congenital handlessness" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below: We assume that you will be able to address these issues by editorial revision of the text. Reviewer #1 is now satisfied, and Reviewer 2 agrees that modest textural revisions will be sufficient, and I see no reason why these would need to go back to the reviewers as long as you address the issues.From Reviewer 2 "With regards to the comment starting with "Further, the authors stated in the Introduction that they would examine.…", my question was regarding whether those with little/no phantom sensations were SIMILAR TO INTACT CONTROLS. However, the author's comments seem to be related to whether these individuals were different from congenital one-handers. First, they say that the intercept for the regression line between phantom vividness and typicality was greater than zero. While this provides evidence for some representational similarity in those with low vividness, it does not mean that they are similar to intact controls. For their second point, they state that the 3 participants with little/no phantom vividness have higher typicality than congenital one-handers. While true, higher typicality than congenital one-handers is not the same as "similar to intact controls". This is not a major point, and doesn't need to be addressed, but could improve the manuscript.More importantly, with regards to point #4 , we do understand the evidence showing a relationship between ipsilateral and contralateral digit representations in neurologically intact individuals, and the logic that the authors use here. However, no mechanistic argument is presented as to why such a relationship would occur in congenital one-handers, even if they had an intact "missing hand" representations. As it stands, the reader is left with the following implied argument: Since there is a precise correlation between the representational patterns for moving the contralateral hand and ipsilateral hand in the same is, then it must be that representational patterns for the ipsilateral hand assess the representation of the missing hand.This argument seems to be based on the idea that a strong correlation = causal evidence. Just because this correlation was observed does not mean that moving the ipsilateral hand is necessarily a measure of the contralateral hand representation. For example, one possibility is that in individuals that have had hands at some point in their life, there is a strong relationship between the two representations such that the activity for moving the ipsilateral and contralateral hand in one hemisphere map perfectly. However, there is no mechanistic reason presented to think that this relationship would necessarily hold in those who have never had a hand….it could be that moving the ipsilateral hand does not result in any kind of organized activity in the "missing hand" hemisphere in this population.Note that I think that this analysis is clever and creative, and I am sympathetic to it. However, as it stands, I don't see the clear mechanistic account for a) why activity for moving the ipsilateral hand is an index of the contralateral hand representation  and b) why your argument would necessarily hold in congenital one-handers. I believe this could be improved with some mechanistic arguments to support the claim. There is some work on what ipsilateral activation indexes …maybe there is something in this literature that could strengthen the claims?" The experiment builds upon and extends a previous paper by the group published in eLife. The difference is that the current paper includes congenital one-handed participants, which allows one to tell whether the representational structure is experience-dependent. The reviewers thought that this is a valuable addition to the previous study. However, there were a number of residual concerns that need to be addressed further.We agree with the reviewers’ summary but would like to add that the key innovation extends beyond the study of congenital one-handers. Primarily, whereas all previous studies (both from our own group and beyond) tested people with highly vivid phantom sensations, we have tested a large group of amputees with varying phantom sensations, allowing us to determine whether the experience of having a hand is necessary for the maintenance of the digit maps. Also, our M1 results further complement our original report and are of interest to many people in the field, particularly those working on brain-machine interfaces.Essential revisions:1) For the first finding, the only support is a correlation between one imaging measure  and one questionnaire response (# of independently controllable parts of the hand). Given this, one would expect very large differences between those who can and cannot move their fingers independently – not only in representational typicality, but also in dissimilarity. Surprisingly, the correlation between kinaesthesia and dissimilarity is not reported in the manuscript. As stated in the Introduction, "greater individuation of inputs will induce greater representational dissimilarity." Therefore, if amputees vary on self-reported individuation, then there should be a correlation between these two variables. This needs to be reported. If there isn't a relationship between the two variables, then it makes it quite difficult to interpret the significant correlation between typicality and "kinaesthesia".We agree with the reviewers’ reasoning. Since our main prediction concerned typicality and not dissimilarity , we wished to limit the number of comparisons. Yet, based on the reviewers’ suggestion, we have now tested this correlation and added it to the manuscript. As stated below, we found a (trending) positive correlation between kinaesthesia and dissimilarity .Subsection “Missing hand representation in acquired amputees is persistent even after phantom sensations have diminished”: “The correlation between kinaesthesia and dissimilarity in SI approached significance .”Second, it is difficult to be confident about correlations with questionnaire data with a small sample size (e.g. the 95% CI for the reported correlation ranges from.33 to.89). Therefore, the relationship between typicality and kinaesthesia would be more convincing if there were other, related correlations that are also significant. We agree that, where possible, questionnaires should be validated with other empirical evidence. Since kinaesthesia was one of our two key predictors  we actually asked participants to show us the range of their missing hand movement by mimicking it with their intact hand (see the full study protocol on https://osf.io/gmvua/). This was recorded on video immediately after the scan. Unfortunately, due to a human error, we lost a substantial amount of this data and only have a sub-set of the first 8 acquired amputees tested. These existing videos strongly confirm the self-reports: rates estimated from the videos are strongly correlated with those estimated in the questionnaires (rho=.840). Considering the small sample size, we felt that this extended evidence does not substantially benefit the paper. However, should the editor or reviewers feel differently, we will be happy to include this supporting information.Regardless, the reviewers make a strong point that we had multiple measures that could potentially be relevant to phantom finger representation. In our first submission, we attempted to deal with this by using a stepwise regression analysis as an exploratory tool. We had to be careful not to overfit the data, so we chose 7 measures that we felt were most appropriate for the missing hand’s typicality measure – time since amputation, age at amputation, kinaesthesia, non-painful phantom vividness (both acute and chronic), and phantom limb pain (both acute and chronic) – as well as typicality of the intact hand’s contralateral activity patterns. The analysis confirmed that kinaesthesia was most relevant to our fMRI measure, allowing us to focus on kinaesthesia in our Results section. We strongly agree that without our regression analysis this focus is not sufficiently justified. Yet, we were also attuned to the original reviewers’ concern that our regression model is not sufficiently reliable for its exploratory purpose.2 of.645 . Out of 1000 iterations, our measures were included in the final model 96% of the times. We believe that this analysis adds clarity to our Results section without reducing the rigidity of the overall findings. Nevertheless, as in our original finding, we suggest this analysis as an exploratory means only, and do not interpret it further. We have updated the manuscript as follows:In the revised manuscript we implemented a bootstrapping approach to provide a measure of confidence for the output of the regression analysis. The final model included an intercept and kinaesthesia as the only regressor (p<.001), with an adjusted RSubsection “Missing hand representation in acquired amputees is persistent even after phantom sensations have diminished”: “Next, we evaluated whether the consistency of hand representation in SI during missing hand movements correlates with amputees’ subjective reports of phantom sensations. We first carried out an exploratory forward stepwise regression with typicality as the dependent variable. The following factors were tested as independent variables: kinaesthesia of phantom sensations . The final model  and the intercept . This regression was submitted to a bootstrapping analysis, allowing us to estimate the consistency of the final model . This bootstrapping analysis returned kinaesthesia as the final variable in.963% of the iterations . The proportion of the other included factors in the final model was: typicality of the intact hand (7.0%); time since amputation (9.5%); age at amputation (9.6%); vividness of nonpainful phantom sensations ; phantom limb pain, acute: 22.7%; chronic: 10.1%).”2 > 0.1. As a large number of predictor variables were included in the model and stepwise regression is generally only recommended for exploratory analysis, we aimed to establish internal replicability using bootstrap resampling . In particular, we randomly sampled (with replacement) the full data matrix and repeated the stepwise regression 1000 times. We subsequently computed the proportion of bootstrap samples in which each factor which included as well as confidence bounds on the model’s adjusted R2. We interpreted high proportion of inclusion (p >.75) as evidence for internal replicability.”Subsection “Statistical Analysis”: “In order to gauge which aspects of phantom sensation and key demographics may relate to the amputees’ representational structure’s typicality, we performed an exploratory forward stepwise regression. The dependent variable was SI typicality in amputees. The following factors were used as independent variables : typicality of the intact hand ; age at amputation (in years); vividness of nonpainful phantom sensations, as experienced during the study  and chronically ; intensity of phantom limb pain, as experienced acutely during the study, and chronically (as detailed for nonpainful sensations). Only linear factors were considered, i.e. no interaction terms, and the criterion for inclusion was an increase in RFurther, the authors stated in the introduction that they would examine "whether persistent representation of a missing hand…will also be observed in amputees with little phantom sensations." Based on this, we were expecting an analysis of the specific individuals with little/no phantom sensations  have a phantom representation similar to intact controls). This should be included in a future submission.Indeed, this question was of interest to us. We explored this in our previous submission in two different ways (see Results section of our previous submission). First, we showed that the intercept for the regression line between phantom vividness and typicality was significantly greater than 0 (.75). This means that, based on our population of n=18 amputees, individuals with no phantom sensation are still predicted to show some hand typicality. Secondly, we show that the 3 participants experiencing little to no phantom vividness (below 10/100) showed significantly higher typicality than congenital one-handers. In the revised manuscript, we have reordered the presentation of results and elaborated these results so that they are not easily missed by the readers.intercept=.52). This was also the case when phantom vividness was the (non-significant) dependent variable . These results predict that even amputees who do not experience any phantom sensations will retain some typical missing hand representation. To test this prediction directly, we examined the 3 amputees in our dataset showing weak to no chronic phantom vividness (below 10/100). Despite not being able to experience clearly their phantom hand when performing the phantom movements task, these individuals showed high typicality  =.83). Moreover, when comparing their typicality to that found in the congenital one-handers (who were arguably better matched to this sub-group in terms of task demands), the amputees with diminished phantom sensations showed significantly stronger correlations with the canonical hand structure . Together, these additional analyses confirm that the representational structures’ typicality in SI of amputees is still present in those with little to no phantom or kinaesthetic sensations.”Subsection “Missing hand representation in acquired amputees is persistent even after phantom sensations have diminished”: “Regardless of the positive relationship between kinaesthesia and typicality, amputees with little to no kinaesthetic sensations still showed missing hand representation. As stated above, the regression line between kinaesthesia and typicality included an intercept (rho2) The authors show that congenital amelics demonstrate reduced activity and digit discriminability in S1 for phantom movements compared to controls. We share some of the concerns of an earlier reviewer. Given that the authors suggest here and previously  that the S1 activity observed when moving the phantom is due to efference copy, it is unlikely that congenital amelics can generate efference copy for a limb that never existed. However, the lack of efference copy activation in S of congenital amelics is not the same as demonstrating that the hand representation has not developed without sensory experience – one of the goals of this paper (see Introduction). That is, there could be a hand representation that exists, but simply hasn't been accessed with the movement task. The manuscript, at times, seems to conflate a lack of activation in SI for moving a phantom/non-existent hand as evidence for the lack of any hand representation. Although we are sympathetic to this view, we don't think the evidence is there given this single task – and so the claims should be softened.We are grateful for this comment, as this is a point that we were very keen to clearly address in our previous submission, and we therefore appreciate the opportunity to improve our delivery. The task constraints imposed on congenital one-handers are similar to those encountered by the few acquired amputees that no longer experience phantom sensations. As highlighted above, these individuals were still able to show higher typicality then congenital one-handers (see above). Regardless, as we highlighted in the original manuscript (and emphasise further in the revised manuscript), we recognise that the task we used might have induced performance differences across the one-handed groups. For this reason, we designed two further analyses that are based on intact hand movements, which all participants could perform equally.The representational structure of contralateral hand movement can be studied indirectly by examining activity in the same brain region induced by the ipsilateral hand . Just to reiterate this point, as an example – left hand movements would evoke strong activity patterns in the contralateral hand area (in the right hemisphere). When participants are moving their ipsilateral (right) hand, that same brain area would show activity patterns, that although smaller, crucially reflect the same representational inter-digit relationships . Because of this, the deprived hand area can be studied based on the ipsilateral intact hand, without imposing unnatural task demands on the congenital one-handers. If congenital one-handers truly have diminished hand representation for their missing hand, their ipsilateral movement should not induce typically structured activity patterns, whereas such activity patterns are expected for the acquired amputees and controls. To test this very specific prediction, we recorded individuated finger movements, which all groups were able to perform equally well. We find that ipsilateral activity following intact hand movement is structured similarly in acquired amputees and controls, but it is reduced in congenital one-handers. In fact, missing hand representation in congenital one-handers was found to be diminished to the point where it is not different from a control visual area.As such, we are basing our interpretation on multiple converging pieces of evidence: diminished missing hand representation in congenital one-handers using our missing fingers movement task; persistent missing hand representation in acquired amputees with diminished-to-nonexisting phantom sensations; diminished ipsilateral intact hand representation in congneital one-handers; similar ipsilateral digit information in SI and V5 in congenital one-handers, but not amputees/controls.Nevertheless, we have done our best to frame these results cautiously, e.g. in the abstract and elsewhere we refer to the congenital one-handers missing hand representation as “reduced” or “diminished” rather than non-existent. In the discussion we acknowledge the limitations of our results and call for further research to conclusively resolve this question. We also refrain from inferring that the missing hand representation never developed (as indicated by the reviewers), and instead suggest that the missing hand maps could have originally formed (e.g. based on a genetic blue-print) but later diminished due to insufficient relevant inputs.In the revised manuscript we have re-written substantial portions of the text (including the abstract), and better structured the Results section (by grouping the findings into sub-sections) to communicate these ideas more clearly. Here are a few examples of revised text addressing this point:Abstract:“We used representational similarity analysis in primary somatosensory and motor cortex during missing and intact hand movements. We found that key aspects of acquired amputees’ missing hand representation persisted, despite varying vividness of phantom sensations. In contrast, missing hand representation of congenital one-handers, who do not experience phantom sensations, was significantly reduced. […] We conclude that once cortical organisation is formed, it is remarkably persistent, despite long-term attenuation of peripheral signals.”Discussion section:“Still, it is possible that rudimentary missing hand representation, e.g. determined by genetic factors , has originally formed in congenital one-handers but later diminished due to lack of consistent sensorimotor input. Our results were insufficiently conclusive to address this important question. Bearing this caveat in mind, our findings suggest that early-life experience is necessary to create typical functional sensory organisation, but not to maintain it.”3) The authors find that controls and amputees did not significantly differ across a number of variables. One example is that typicality did not differ between the two groups. The authors used Bayes factors, using a BF<1/3 as positive evidence in support of the null. Given the editor's concerns regarding exactly what "substantial" evidence means in the context of BFs, one suggestion would be to use frequentist tests designed specifically to examine whether two groups are equivalent , Lakens, 2017). This provides a standard p-value that is more straightforward to interpret and is consistent with the (primarily) frequentist tests used throughout the manuscript.We appreciate the suggestion to substitute our Bayesian analysis with the frequentist equivalence test TOSTER. After implementing this analysis, we conclude that while the resulting p value might be more intuitive to interpret, the effect size estimate required for conducting the test is conceptually quite tricky. The goal in the TOSTER approach is to specify lower and upper bounds, such that group differences falling within this range are deemed non-meaningful. But how do we choose the a priori equivalence bounds for the existence of a hand representation? One option was using the effect size of the group difference between the controls (who have a hand) and the congenital one-handers (who do not have a hand). Using this criterion, the typicality of the amputees was significantly equivalent to that of the controls . But although appropriate for the Bayesian analysis, this boundary might be not suitable for TOSTER. This is because the lower bound should represent the smallest reasonable effect of having a hand, whereas we believe the congenitals do not have hand representation altogether. In other words, the congenital one-handers fall outside this lower boundary. Alternative measures of the lower hand representation boundary can come from the control participants: e.g. contralateral vs. ipsilateral univariate activity  or the comparison between SI and V5 typicality . But the fact that these tests produce stronger evidence in favour of the null than our experimental results with the congenital one-handers lead us to doubt this boundary as meaningful as well.While the TOSTER method clearly supports our main interpretation, in our view, there are two main reason why the Bayesian analysis is better suited to our paper. First, the inclusion of the congenital one-handers in our study was specifically designed to allow us to interpret the persistence of missing hand representation in the acquired amputees. Indeed, the Bayesian analysis allows us to compare the group difference ‘amputees vs controls’ as a measure of having a hand representation. Secondly, the TOSTER method is not quite standard in the field and much less widespread than the Bayesian statistics we have used. Therefore, we prefer implementing the current gold standard for interpreting null results . Should the editor or reviewers feel that the TOSTER provides substantial further evidence, we will be happy to include it as well.4) The argument for using the ipsilateral hand representation to probe digit structure in the missing hand (Results section) was quite unclear. We understand that Diedrichsen showed a relationship between ipsilateral and contralateral digit representation. But this was in individuals with intact hands. Why would the fact that the "ipsilateral digit representation is a reliable predictor of contralateral hand representation" in two-handers mean that this would also be true for amputees or congenital one-handers? This logic should be laid out.We apologise for being unclear. We have adapted the text more explicitly address this crucial point.Subsection “Diminished missing hand representation in congenital one-handers even when task performance is matched”: “To probe digit structure in the missing hand cortex using an alternative task, we examined ipsilateral hand representation of the intact hand (i.e. in the missing hand cortex). Ipsilateral digit representation in two-handed controls was previously shown to precisely predict contralateral hand representation . In other words, individuated digit movements with the ipsilateral hand in sensorimotor cortex produce an identical representational pattern to that of the contralateral hand (though this pattern can be masked by noise). As such, ipsilateral representation of the intact hand provides an indirect assay into the representation of the missing hand, while controlling for task demands across groups. Importantly, all three groups were able to perform the individuated digit movement task, with similar success . We compared the intact/dominant inter-digit representational structure in the missing/nondominant hand area of one-handers/controls (respectively). We predicted that persistent missing hand representation in amputees should result in similar ipsilateral representation in their missing hand cortex as controls. If missing hand representation is diminished in congenital one-handers, then ipsilateral representation of their intact hand (in the missing hand area) should show reduced representational features as those found in amputees.”Discussion section:“We further show that ipsilateral digit-representation in the missing hand cortex of the intact hand supports the existence of persistent missing hand representation in amputees and diminished representation in congenital one-handers. Previous studies demonstrate that contralateral and ipsilateral hand movements produce identical representational patterns . As such, the ipsilateral digit representational pattern is a reliable predictor of contralateral hand representation. This discovery provides us with a unique and novel opportunity to interrogate the information content underlying the “deprived” hand cortex despite the physical absence of the hand. This approach provided converging evidence, but using different task demands, for similar (missing) hand representation for amputees and controls, but not for congenital one-handers.”5) We suggest to briefly explain that the absence of information for the congenital one-handed participants means that the effect is not driven by the visual cue.That is a good point. We have added it to our revised manuscript.Discussion section:“Using the same task, we were unable to identify similar digit representation for the missing hand of congenital one-handers, demonstrated by significantly reduced pattern typicality compared to both amputees (and even in comparison to those few amputees with little phantom sensations), as well as controls. This result confirms that the persistent hand representation observed in amputees does not reflect mere cognitive task demands .”6) Is the MDS-analysis cross-validated?We believe that MDS does not easily lend itself to quantitative analysis, therefore in the manuscript we have relied on cross-validated Mahalanobis distances for all analyses and only used the MDS for visualisation purposes. This is now clarified in the Introduction, in both the Materials and methods section and the Figure legend.Subsection “fMRI analysis”:Note that MDS is presented for intuitive visualisation purposes only, and was not used for statistical analysis.Figure 1:E) Two-dimensional projection of the representational structure (D) .7) To which degree is the typicality analysis influenced by SNR? Does the low dissimilarity of the congenital group  mean that the responses were reproducibly very similar across conditions ?1H-AMP=.001) and controls . We note that by splitting the data we are reducing the effectiveness of our analysis . Nevertheless, the relative difference in split-half consistency between the congenital one-handers and the other two groups indicates that there is no strongly consistent digit information in the missing hand area of the congenital one-handers during our task. We have added the following text to the manuscript to reflect this point:This is a good point that we didn’t address so far. When the SNR is low, dissimilarity will tend to 0. Typicality will therefore also be indirectly affected by SNR. In our view, this is not a design flaw, but, in the case of the congenital group, an indication that although the missing hand area of congenital one-handers was activated, it was not activated consistently across scans. To directly address the reviewers’ suggestion, we tested whether the representational patterns evoked by the congenital one-handers are reproducible, by calculating split-half consistency. For each participant, we calculated an RDM for the missing/non-dominant hand using the two odd runs, and one using the two even runs. The correlation between the odd and even RDMs was significantly lower in the congenital one-handers (rho=-.02) compared to both amputees  and controls . We note that by splitting the data we are reducing the effectiveness of our analysis. Nevertheless, the relative reduction in split-half consistency indicates that there is no strongly consistent digit information in the missing hand area of congenital one-handers during this task.”Subsection “Phantom hand movements elicit typical hand representation in the missing hand area of acquired amputees”: “Although the inter-digit representational structure of congenital one-handers is atypical with respect to canonical hand representation, it is possible that it is still consistent within participants. To explore this idea, we split individual participants’ data to odd and even scans. For each participant, we calculated an RDM in the missing/nondominant hand area using the odd and even runs, and correlated the two RDMs. The correlation between odd and even RDMs was significantly lower in congenital one-handers (rho=-.02) compared to both amputees  Is this a completely separate dataset than in the original paper? This should be clarified.We apologise that we did not mention this in our original submission. All three participants from the original paper were scanned again in the current study, namely participants A4, A7, and A2 . After excluding these participants, our two main results still hold: typicality is not significantly reduced in the amputees compared to controls , and is significantly correlated with kinaesthesia . We have added this information to the text.Subsection “Participants”:“3 participants in the amputees group tested here also took part in our previous study .”[Editors’ note: further revisions were requested before acceptance.]From reviewer 2 "With regards to the comment starting with "Further, the authors stated in the Introduction that they would examine…", my question was regarding whether those with little/no phantom sensations were SIMILAR TO INTACT CONTROLS. However, the author's comments seem to be related to whether these individuals were different from congenital one-handers. First, they say that the intercept for the regression line between phantom vividness and typicality was greater than zero. While this provides evidence for some representational similarity in those with low vividness, it does not mean that they are similar to intact controls. For their second point, they state that the 3 participants with little/no phantom vividness have higher typicality than congenital one-handers. While true, higher typicality than congenital one-handers is not the same as "similar to intact controls". This is not a major point, and doesn't need to be addressed, but could improve the manuscript.We agree that adding the direct comparison between controls and amputees with little no phantom vividness improves the manuscript and apologise for not having included this initially. The difference between these groups is strongly non-significant . We’ve appended the following sentence to the manuscript:Results section:“These results predict that even amputees who do not experience any phantom sensations will retain some typical missing hand representation. To test this prediction directly, we examined the 3 amputees in our dataset showing weak to no chronic phantom vividness (below 10/100). Despite not being able to experience clearly their phantom hand when performing the phantom movements task, these individuals showed high typicality  =.83). Moreover, when comparing their typicality to that found in the congenital one-handers (who were arguably better matched to this sub-group in terms of task demands), the amputees with diminished phantom sensations showed significantly stronger correlations with the canonical hand structure . Typicality was not different between these 3 amputees and controls . Together, these additional analyses confirm that the representational structures’ typicality in SI of amputees is still present in those with little to no phantom or kinaesthetic sensations.”More importantly, with regards to point #4 , we do understand the evidence showing a relationship between ipsilateral and contralateral digit representations in neurologically intact individuals, and the logic that the authors use here. However, no mechanistic argument is presented as to why such a relationship would occur in congenital one-handers, even if they had an intact "missing hand" representations. As it stands, the reader is left with the following implied argument: Since there is a precise correlation between the representational patterns for moving the contralateral hand and ipsilateral hand in the same is, then it must be that representational patterns for the ipsilateral hand assess the representation of the missing hand.This argument seems to be based on the idea that a strong correlation = causal evidence. Just because this correlation was observed does not mean that moving the ipsilateral hand is necessarily a measure of the contralateral hand representation. For example, one possibility is that in individuals that have had hands at some point in their life, there is a strong relationship between the two representations such that the activity for moving the ipsilateral and contralateral hand in one hemisphere map perfectly. However, there is no mechanistic reason presented to think that this relationship would necessarily hold in those who have never had a hand….it could be that moving the ipsilateral hand does not result in any kind of organized activity in the "missing hand" hemisphere in this population.Note that I think that this analysis is clever and creative, and I am sympathetic to it. However, as it stands, I don't see the clear mechanistic account for a) why activity for moving the ipsilateral hand is an index of the contralateral hand representation  and b) why your argument would necessarily hold in congenital one-handers. I believe this could be improved with some mechanistic arguments to support the claim. There is some work on what ipsilateral activation indexes ….maybe there is something in this literature that could strengthen the claims?"We apologise for misunderstanding the concerns of reviewer 2 in our previous round of revisions. We agree that the reviewer raises an important issue that requires further consideration.Recent evidence showing correspondence between ipsilateral and contralateral representations (across the two hands) sheds some light on the potential mechanism giving rise to ipsilateral hand representation in SI/M1. As we emphasised before, past studies have shown that the activity patterns underlying the ipsi- and contralateral representational structure in one hemisphere are spatially overlapping . This is generally interpreted as both hands’ representation being facilitated by the same architecture rather than two distinct representations with a correlating structure. In other words, the ipsilateral hand reactivates the patches of cortex associated with the contralateral hand. Strong evidence for this is indeed provided by Diedrichsen et al., : During bimanual asymmetric movement, SI activity patterns are dominated by contralateral actions only and ipsilateral representation disappears. Most recently, we also showed that ipsilateral representation is only present during active movement (as opposed to passive touch) and not related to uncrossed sensory inflow . Taking this evidence together, we have good reason to suggest that in two-handers the motor command to move one hand activates (through the back door) the corresponding representations of the other hand and not a distinct (correlated) representation of the ipsilateral hand. We realise that some of the language used in our manuscript, e.g. emphasising that one representation predicts another, has not reflected this interpretation well and we have made some edits to elaborate on this suggested mechanism.How does this shared architecture across hands emerge? As pointed out by the reviewers, the two key mechanisms are: Bilateral hand experience, emerging over life, and innate structural connections (e.g. relating to mirroring).What does this mean for the congenital one-handers? As we see it, there are three possibilities: (1) The deprived cortex develops separate representations for both contralateral (missing) hand and ipsilateral (intact) hand. Here we assume that the overlap and functional interaction seen in controls require coordinated bimanual behaviour, so in one-handers both ipsi- and contralateral hand remain represented distinctly and independently within the deprived cortex. (2) The deprived cortex develops contralateral hand representation (of the missing hand), but intact hand movements do not recruit it, e.g. due to impaired connectivity across hemispheres. (3) The deprived cortex does not develop structured hand representation .If option 1 were true (though unlikely in light of the research in controls discussed above), then one would expect to find strong ipsilateral representation of the intact hand. Yet, our data does not reflect this hypothesis. Despite typical contralateral dissimilarity for the intact hand , average ipsilateral dissimilarity (in the deprived cortex) is not higher than our control area, which does not represent hands .Option 2 cannot be disproven by our current data, but our previous findings suggest that callosal connections are functionally preserved in congenital one-handers, at least to some extent. We showed that the strength of functional connectivity across the two sensorimotor hand areas correlates with the extent to which congenital one-handers engage their stumps  in performing “bi-manual” tasks . Moreover, congenital one-handers who strongly rely on stump usage show normal inter-hemispheric connectivity in comparison to two-handed controls. Since the stump has been shown to strongly activate the missing hand cortex in congenital one-handers , it appears that given the right input, the two hemispheres can engage. Please also note that in Makin et al. (2013) we explored structural connectivity changes (using DTI) and did not identify any striking callosal differences between congenital one-handers and controls.This leaves us with option 3 (no hand representation in the deprived cortex). Taken together, it appears that sensorimotor experience is necessary for developing a hand area, including its connectivity to other areas in the sensorimotor network. We have clarified this both in the Results section and Discussion section, as follows:Results section:“To probe digit structure in the missing hand cortex using an alternative task, we examined whether we could observe a representation of the ipsilateral (intact) hand in the missing hand cortex. In two-handed controls, finger movements lead to activity in specific cortical patches in ipsilateral M1 and SI, which tightly correspond to the activity patches engaged in the movement of the mirror-symmetric contralateral finger . Indeed, this ipsilateral representation fully overlaps with the representation of the contralateral hand . Furthermore, ipsilateral representation disappears completely during asymmetric bimanual finger movements, during which activity in M1 and SI is fully determined by the contralateral hand . As such, the ipsilateral representation of one hand is likely elicited due to recruitment of the representation of the contralateral hand . Ipsilateral representation of the intact hand can therefore provide an indirect assay into the representation of the missing hand, while controlling for task demands across groups.”Results section:“If missing hand representation is diminished in congenital one-handers, then ipsilateral representation of their intact hand (in the missing hand area) should show reduced representational features compared to those found in amputees .”Discussion section:“We also explored whether we could activate the representation of the missing hand indirectly by movements of the fingers of the intact hand. Previous studies in two-handers have demonstrated that contralateral and ipsilateral hand movements produce identical representational patterns . Since this ipsilateral representation is completely overwritten by the contralateral hand if the two hands are engaged in dissociated movements , it has been proposed that the ipsilateral hand reactivates the cortical resources associated with the contralateral hand. Based on this evidence, the ipsilateral digit representation can serve as an indirect measure of the contralateral hand representation.”Discussion section:“It might be worth considering whether congenital one-hander’s deprived cortex could have developed separate representations for both the contralateral (missing) and ipsilateral (intact) hand, which are uncorrelated due to anatomical or behavioural differences from two-handers. If this were possible, we would predict maintained, or even stronger, ipsilateral representation of the intact hand. Yet, our data does not reflect this hypothesis. Moreover, poor ipsilateral representation in the deprived cortex does not seem to stem from reduced inter-hemispheric connectivity, which appears to be functionally and structurally preserved in congenital one-handers, depending on lateralisation strategies in daily behaviour .”


Hepatocellular adenoma (HCA) is a rare liver tumor that has the potential for rupture and malignant transformation. Here, we report a case of multiple hepatocellular adenomas (HCAs) that were treated by surgical resection.2. A liver tumor was incidentally found on abdominal ultrasonography. Contrast-enhanced computed tomography and gadoxetic acid-enhanced magnetic resonance imaging revealed three hepatic tumors that were 68 mm, 16 mm, and 9 mm in segments 3/4, 8, and 1, respectively. A percutaneous needle biopsy of the largest tumor was performed, the diagnosis of unclassified type HCA was made, and laparoscopic partial liver resection was performed of all three. The postoperative course was uneventful, and the patient was discharged 12 days later. An immunohistochemical examination revealed positivity for serum amyloid A protein, no decrease in fatty acid-binding protein, and negativity for β-catenin, glutamine synthetase, and cytokeratin 7. Therefore, these tumors were diagnosed as inflammatory type HCAs.An 18-year-old man was admitted to our hospital with proteinuria. His height was 176.5 cm, weight was 126 kg, and body mass index was 40 kg/mWe reported an extremely rare case of multiple resected HCAs in a young, obese Japanese man. Our findings suggest that HCA should be considered in the differential diagnosis of liver tumor in obese patients. Further studies that consider clinical and molecular risk factors are required to establish individualized treatment plans for HCA in obese patients. Hepatocellular adenoma (HCA) is a rare benign liver tumor, most frequently affecting young women with a history of oral contraceptive use . HCA is Here, we report a rare case of multiple hepatocellular adenomas (HCAs) in a young Japanese man with severe obesity that was treated with laparoscopic liver resection (LLR).2. He was taking no medication and had never consumed alcohol. A physical examination demonstrated no findings. Laboratory investigation results are shown in Table An 18-year-old man was admitted to our hospital with proteinuria identified in a medical checkup. He had no personal or familial medical history. His height was 176.5 cm, weight was 126 kg, and body mass index (BMI) was 40 kg/mHCAs are rare in children, men, and the elderly. The male-to-female ratio in the incidence of HCA is approximately 1:10 . The domIn the diagnosis of HCA, non-invasive tools such as imaging play an important role, in addition to biopsies. Among the imaging tools, CECT and MRI are useful for diagnosing HCA. HCA is typically hypervascular and heterogenous on the arterial phase but isoattenuating or hypoattenuating on the portal venous phase of CECT . The diaAccording to the World Health Organization classification edited in 2010, HCA can be categorized into four subgroups: (i) HNF-1α-inactivated HCA , (ii) β-catenin-activated HCA , (iii) inflammatory HCA , and (iv) unclassified HCA (tumors without any mutation/activation and no inflammatory infiltrate) . In our The management of HCA includes multidisciplinary treatments due to the nature of the associated complications that may include bleeding and malignant transformation. In a systematic review of 1176 patients with HCA, van Aalten et al. reported an overall frequency of 27.2% for hemorrhage and 17.5% for rupture and intraperitoneal bleeding . The risp < 0.001), a need for transfusion , and a shorter hospital stay  [2), LLR of the three separate lesions was successfully performed without excessive blood loss.LLR is also considered a feasible option with comparable efficacy and safety relative to the open liver resection for HCAs. A multi-institutional study showed that laparoscopic surgery can reportedly achieve short-term outcomes similar to those of open surgery for HCAs and has the additional benefits of a reduced blood loss  . LLR als< 0.001) , 22 and < 0.001) , 24. In Recent studies on obese female patients with HCA have reported successful treatment by weight loss or bariatric surgery , 9, 25. We reported a case of successful laparoscopic management of multiple HCAs in a young, obese, Japanese man. Our findings suggest that HCA should be considered in the differential diagnosis of liver tumors in the obese patients. Further studies are required to establish individualized HCA treatments in obese patients, in considering clinical and molecular risk factors.


A digital-coding programmable metasurface (DCPM) is a type of functional system that is composed of subwavelength-scale digital coding elements with opposite phase responses. By configuring the digital coding elements, a DCPM can construct dynamic near-field image patterns in which the intensity of each pixel of the image can be dynamically and independently modulated. Thus, a DCPM can perform both spatial and temporal modulations. Here, this advantage is used to realize multichannel direct transmissions of near-field information. Three points are selected in the near-field region to form three independent channels. By applying various digital phase codes on the DCPM, independent binary digital symbols defined by amplitude codes  are transmitted through the three channels. The measured near-field distributions and temporal transmissions of the system agree with numerical calculations. Compared with the conventional multichannel transmission, the proposed mechanism achieves simultaneous spatial and temporal modulations by treating DCPM as an energy radiator and information modulator, thereby enduing DCPM with high potential in near-field information processing and communications. Scientists have developed a programmable metasurface capable of controlling both the spatial and temporal modulations of light, opening the door for tunable optical components that are flat and easily integrated into other devices. Metasurfaces are ultrathin materials made from metals or semiconductors that can modulate the amplitude, phase, and polarization of light waves. However, most metasurfaces currently available are static in nature, with their optical properties fixed during fabrication. Now, Xiang Wan and colleagues from Southeast University in China have developed a digital-coding programmable metasurface (DCPM) that can dynamically control light in real time. Made from subwavelength-scale digital coding elements with opposite phase responses, the proposed DCPM is capable of multi-channel direct transmissions of near-field information and could be used in a range of applications, including wearable devices, autonomous vehicles, and sensing, imaging, and display technologies. With the rapid growth in the number of devices that are in use, the fifth generation (5G) of communication will involve communications not only between humans but also between objects and between humans and objects. Hence, in addition to improving the network capacity and the spectrum efficiency, establishing additional communication modes is an evolution direction of 5G. Device-to-device (D2D) communication is a type of communication technology that can directly transmit and receive information between terminals; hence, it can reduce the burden on the base stations and decrease the end-to-end delay, improve the spectrum efficiency and reduce the transmission power of the terminals. However, the communication systems that are based on the classical superheterodyne architecture can no longer satisfy the requirements of modern communications due to the high complexities, high cost, and large volumes. Furthermore, the digitization of modern communication systems has evolved from baseband to radio frequency links and antennas. Therefore, more flexible hardware architectures, new types of information, and innovative breakthroughs in the theories of information and communication are urgently needed for satisfying the substantial demands of 5G for “huge capacity, huge connections, and extensive applications”.2, which is a type of artificial material, has been demonstrated to control macroscopic phenomena of electromagnetic (EM) waves, such as negative reflections4, perfect lenses5, and invisible cloaks10. Microscopic phenomena, such as molecular spontaneous emission11 and bound states in the continuum12, can also be manipulated by metamaterials. After years of development, metamaterials have been fully developed in various morphologies, from acoustic waves13 to light14. A metasurface is a two-dimensional morphology of metamaterials. According to a generalized version of Snell’s law15, the manipulation of EM waves does not necessarily require bulky gradient metamaterials, which typically have a size of several wavelengths according to effective medium theory16; instead, the manipulations can be realized within the subwavelength scale. Many types of metasurfaces have been proposed for controlling the phases19, amplitudes23, and polarizations29 of EM wave fronts and for novel applications such as holographic imaging32, vortex beams35 and conversions of propagation waves, and surface waves37. Active metasurfaces have also been proposed for manipulating the EM waves in various frequency regimes41.Metamaterial45 were proposed for designing more functional devices and/or systems, thereby introducing metasurfaces into informatic fields47, in which the digital coding metasurface can manipulate EM waves by arranging the states of digital elements instead of designing effective medium parameters. Moreover, digital coding programmable metasurfaces (DCPMs) were proposed for performing dynamic spatial modulations50. A DCPM is a type of artificial encoded aperture that is constructed of digital elements and digital coding information is written into the aperture by independently applying a driven voltage on each digital element. Hence, a DCPM can control EM waves dynamically in a programmable way. However, only far-field EM features  of DCMs and DCPMs have been explored in the literature50. According to Fresnel diffraction theory, the aperture field is related to the diffracted field via the Fourier transform. Therefore, spatial modulations of near fields can be realized by configuring the digital coding schemes of DCPMs, which have higher information capacities.With further research, digital coding metasurfaces (DCMs)Since the aperture codes of a DCPM change over time, the digital coding mechanism naturally involves temporal operations. Hence, for each position on the near-field plane, the amplitudes and phases of near fields can be modulated in the time domain by sequentially changing the aperture codes. Therefore, DCPMs can perform near-field modulations in both the space and time domains, which can be used to directly transmit near-field information through multiple channels. To interpret the mechanism, a near-field communication system with multiple channels is established using a DCPM. Three points are selected in the near-field region to form three independent channels. By applying space-time digital codes on a DCPM, independent binary symbols are transmitted through the three channels. The measured near-field distributions and temporal transmissions of the system accord with the numerical calculation results. As the spatial and temporal modulations are directly realized by a DCPM, the presented mechanism of multichannel transmission of near-field information will enrich the modes of 5G communication and improve the mechanisms of near-field information processing and communications.A conceptual diagram of the multichannel transmission system is presented in Fig. By combining spatial and temporal modulations, a multichannel transmission system can be realized by using a DCPM. Photos of the fabricated DCPM are shown in Fig. Because the presented multiple channels are realized by rearranging the near-field energies at three channels, the amplitudes of the symbols may fluctuate in the space and time domains. In the space domain, the imbalanced energy of the channels is caused by factors such as the asymmetry of the coding scheme and the fabrication errors. The symbol “1” may differ in amplitude among the three channels due to this imbalanced energy. In the time domain, there is an inherent imbalance among the channels. If only one channel transmits the symbol “1”, the signal intensity of this channel is stronger than that of each channel if all three channels transmit the symbol “1”. Because the energy is focused on a single channel rather than being distributed among three channels, the amplitude of a symbol in the same channel will depend on the combination of symbols in the three channels.To evaluate the fluctuations of the channel strengths, the near fields are integrated around the focal point within an integral area, in which the field values are higher than 10% of the value at the focal point. All the channel strengths are normalized to a fixed value, which is obtained by integrating the values of near-field pattern “010”. Figure The results of the following transmission experiments demonstrate that the DCPM can transmit different near-field symbols among channels. To receive the symbols, a general signal processing board was used to sample the signal at each channel. For convenience of sampling, the carrier waves of the transmitted signals were converted from 10 GHz to 3 MHz. The period of each symbol is 2 μs and the sampling frequency is 31.44 MHz. Hence, each period of the signal will be sampled by 63 times. The digital receiver can record 400 sampling points. The left column of Fig. For spatial modulations, the fabricated DCPM was measured in a near-field microwave anechoic chamber. Figure From both calculations and measurements, we observe the fluctuations in the space and time domains. However, they are not sufficiently violent for hindering the identification of symbols. Typically, the receivers of communication systems have sufficient dynamic ranges for covering the fluctuations. In the presented experiments, the period of the transmitted symbols is selected as 2 μs for convenience of sampling. The minimum period of the transmitted symbols is determined by the fastest switching time of the DCPM. The main limiting factor of the speed of the DCPM is the switching time of the Positive-Intrinsic-Negative (PIN) diode and the DC feeding method. In the present work, all units of the DCPM are controlled by a digital circuit board  in parallel; hence, the switching time of the whole aperture is very close to the switching time of a single PIN diode. The fastest switching time of the DCPM is 25 ns according to the test. However, short periods may result in larger errors in the transmitted symbols. Hence, the period of the transmitted symbols must balance the rate and accuracy of the symbols.In conclusion, the dynamic property of DCPMs is used to realize multichannel direct transmission of near-field information. Spatial modulations are performed on the near fields to form three transmission channels and independent binary amplitude modulations are performed on each channel to transmit distinct digital information. Both spatial and temporal modulations are achieved by directly configuring a DCPM. Comparing with the conventional multichannel transmissions, the new transmission mechanism does not require phase shifters for the spatial modulations or baseband modulators for the temporal modulations. These advantages endow DCPMs with high potential in near-field information processing and high-capacity communications.First, the capacity of a DCPM for performing near-field spatial modulations is investigated. As an example, six focal points are defined arbitrarily on a near-field plane that is 600 mm away from the DCPM. Figure Based on the spatial modulations, multiple channels for the near-field transmission can be physically established. For convenience of testing, three points are specified as three transmission channels. When the DCPM focus the energy at one point, the digital symbol “1” will be transmitted through the corresponding channel; otherwise, the digital symbol “0” will be transmitted. To independently transmit binary digital symbols through the three channels, eight types of coding schemes for the DCPM are needed. The correspondences between the coding schemes and the transmitted symbols in the three channels are presented in Fig. For temporal modulations, one can specify a series of coding schemes for the DCPM for transmitting a series of binary symbols. As an example of independent temporal modulations, periodic bytes that each contain four binary symbols are specified in the three channels: “1010”, “0110”, and “0001” in channel 1, channel 2, and channel 3, respectively. Figure Multichannel direct transmission of near-field information_SM


A novel ultra‐low‐cost biochemical analysis platform to quantify protein dissociation binding constants and kinetics using paper microfluidics is reported. This approach marries video imaging with one of humankind's oldest materials: paper, requiring no large, expensive laboratory equipment, complex microfluidics or external power. Temporal measurements of nanoparticle–antibody conjugates binding on paper is found to follow the Langmuir Adsorption Model. This is exploited to measure a series of antibody–antigen dissociation constants on paper, showing excellent agreement with a gold‐standard benchtop interferometer. The concept is demonstrated with a camera and low‐end smartphone, 500‐fold cheaper than the reference method, and can be multiplexed to measure ten reactions in parallel. These findings will help to widen access to quantitative analytical biochemistry, for diverse applications spanning disease diagnostics, drug discovery, and environmental analysis in resource‐limited settings. Today, a new generation of low cost consumer electronic‐based biosensors is emergingE. coli detection using quantum dots.To date, the use of smartphone cameras to interpret lateral flow tests has focused on individual still image end‐point readings to interpret paper‐based tests for diagnostics,KD and kinetic kon and koff rates. We overcome potential barriers associated with quantitative analysis on lateral flow tests cited in previous work, including sample volume limitations,Here, for the first time in the literature, we progress beyond still images of lateral flow tests to video analysis in order to investigate whether dynamic ligand–receptor binding on μPADS follows the Langmuir Adsorption Isotherm Model,The ability to measure such fundamental chemical binding constants and kinetic reaction rates lies at the heart of chemistry and traditionally relies on access to sophisticated laboratory instrumentation, such as surface plasmon resonanceI. The pixel values are extracted and averaged across the width of the μPAD to reduce noise, creating a line profile along the strip. The peak height is then outputted as a function of time for each μPAD. Here, the green channel of the RGB color‐space is used to match the absorption peak of 20 nm AuNPs, but this can be tailored to the type of nanoparticles employed. Due to the timescales considered here, a sampling rate of 1 Hz is used; however, this could be increased up to 30 Hz to measure faster biochemical reactions.Our low‐cost technique uses a simple set‐up consisting of a consumer camera or smartphone, a series of direct‐detection μPADs, and a 96‐well plate. Lines of antigen are immobilized on nitrocellulose paper strips. When the antibody‐functionalized gold nanoparticles (Ab‐AuNP) flow along the membrane, they bind to the test line Scheme  a, generm). The number of antibodies per AuNP, and antigen‐on‐strip concentration, are held constant. Time‐intensity plots are fitted to the exponential equation, shown below as Equation Figure I is intensity (test line peak height), I∞is the equilibrium intensity value, kobs is the fitted observed binding rate, and t is time. As t→∞, I→I∞. The I∞values are determined for each concentration of analyte, and fitted to a Langmuir model shown below as Equation where a is the saturation intensity when all available binding sites are occupied, and C is Ab‐AuNP concentration.where kon and koff rates, the values of kobs are extracted from the fits of Equation (1) and plotted against C. They are then fitted to following the relationship in order to extract kon and koff, shown below as Equation To determine the An example of this is shown in Figure Our results show strong agreement between the binding kinetics measured by video analysis and the Langmuir model.KD fits shown in Figure KD values measured by video‐μPADs and interferometry show a linear relationship with a gradient of 1.1 (standard error 0.032), and an adjusted R2 value of 0.996. For all raw data and fits, see SI, Figures S6, S7, and Table S3. The estimated concentration of antibodies in video‐μPAD assays assumes that all antibodies bind to AuNPs in an active, available conformation, equating to ≈15 antibodies per AuNP. We note that the measured kon and koff values differ from those measured by interferometry, although the relationship KD=koff/kon still holds . This interesting result highlights the value of quantifying antibody‐antigen reaction kinetics on μPADs in order to optimize diagnostic test performance to achieve fast kinetics and a strong KD, giving a sensitive, rapid test with low‐sample volume.We then apply video analysis to five different antibody‐antigen pairs . In parallel, benchmarking studies are performed with the same proteins using a FortéBio Octet RED96 benchtop interferometer. The KD values , confirming the LG Nexus 5 smartphone can be used for KD measurements.A proof‐of‐principle of video‐μPADs using a smartphone is shown with an LG Nexus 5 Figure  a. The pKD measured from singleplex and multiplex video‐μPADs are not significantly different , validating this reversed orientation .Building on our work with single antibody–antigen pairs, we then sought to investigate whether video‐μPADs could measure multiple antibody–antigen interactions simultaneously. This could be useful for antibody and drug screening to quantify multiple antibodies’ binding affinities to a single target. Therefore, in contrast to the above, the AuNPs are functionalized with the antigen, and the antibody is spotted on the μPADs. The proof‐of‐concept is shown in Figure KD values for five antibody–antigen pairs show excellent agreement with a reference benchtop interferometer . The assumptions and justifications for using the Langmuir model are discussed in SI Discussion and Table S4. We demonstrate that a smartphone can measure KD values, and create a multiplex platform to detect multiple ligand–receptor interactions in parallel. Although AuNP labeling is used here, future assays could employ a label‐free competitive inhibition format.Herein we harness consumer electronic video imaging for low‐cost μPADs, creating an accurate platform for measurement of antibody–antigen dissociation constants. Our approach does not require expensive, complex laboratory‐based equipment, simply using a camera or smartphone. The measured video‐μPAD The low‐cost of video‐μPADs is a major advantage over expensive benchtop instrumentation. SI Table S1 lists the cost, size, and weight of video‐μPAD instrumentation compared to the FortéBio Octet RED96, showing that video‐μPAD equipment costs are around 250–580 times cheaper, making it much more accessible to academic laboratories and resource‐limited settings. Low‐end smartphones are amenable to this application, since neither high camera resolutions nor large processing power are needed. Moreover, video‐μPADs require 64‐fold lower amounts of capture reagents (SI Table S1), advantageous for early stage discovery projects requiring biophysical characterization of reagents, where the quantities of material may be limited.In our study, video analysis is performed off‐device; however, this could be performed in real‐time, even on a low‐cost smartphone, with a capture rate of 1 Hz. In future, automatic strip detection by traditional image processing or machine learning would make the method more user‐friendly.To close, this technique allows low‐cost, quantitative, multiplexed analysis and is generalizable to a wide range of biological and chemical ligand–receptor interactions, with many potential applications in analytical chemistry, biomedicine, forensics, and environmental analysis.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Impaired activity of the Na+‐Cl−‐cotransporter (NCC) has been associated with hypermagnesiuria and hypomagnesemia, while increased activity of NCC, as observed in patients with Gordon syndrome, is not associated with alterations in Mg2+ balance. To further elucidate the possible interrelationship between NCC activity and renal Mg2+ handling, plasma Mg2+ levels and urinary excretion of sodium (Na+) and Mg2+ were measured in a mouse model of Gordon syndrome. In this model, DCT1‐specific expression of a constitutively active mutant form of the NCC‐phosphorylating kinase, SPAK (CA‐SPAK), increases NCC activity and hydrochlorothiazide (HCTZ)‐sensitive Na+ reabsorption. These mice were normomagnesemic and HCTZ administration comparably reduced plasma Mg2+ levels in CA‐SPAK mice and control littermates. As inferred by the initial response to HCTZ, CA‐SPAK mice exhibited greater NCC‐dependent Na+ reabsorption together with decreased Mg2+ reabsorption, compared to controls. Following prolonged HCTZ administration (4 days), CA‐SPAK mice exhibited higher urinary Mg2+ excretion, while urinary Na+ excretion decreased to levels observed in control animals. Surprisingly, CA‐SPAK mice had unaltered renal expression of Trpm6, encoding the Mg2+‐permeable channel TRPM6, or other magnesiotropic genes. In conclusion, CA‐SPAK mice exhibit normomagnesemia, despite increased NCC activity and Na+ reabsorption. Thus, Mg2+ reabsorption is not coupled to increased thiazide‐sensitive Na+ reabsorption, suggesting a similar process explains normomagnesemia in Gordon syndrome. Further research is required to unravel the molecular underpinnings of this phenomenon and the more pronounced Mg2+ excretion after prolonged HCTZ administration.Active reabsorption of magnesium (Mg Defects in either one of these components may result in hypomagnesemia  or hypermagnesemia   , which is regulated at the level of transcription, plasma membrane abundance and activity   reabsorption. Here, Na+ is reabsorbed through the apically located Na+‐Cl−‐cotransporter (NCC). The activity of this transporter, which facilitates electroneutral transport of Na+ and chloride (Cl−), is regulated through phosphorylation by the with‐no‐lysine kinase ‐Ste20p‐related proline‐ and alanine‐rich kinase (SPAK) pathway  characterized by hypokalemia and hypomagnesemia  through phosphomimetic mutations at the key activation sites (T243E and S383D) Arbr/J). DCT1‐specific CA‐SPAK (Parv‐Cre/CA‐SPAK) (n = 14) and control mice, expressing the parvalbumin Cre‐driver alone (n = 14), had ad libitum access to water and food. Mice were treated with an intraperitoneal (i.p.) injection of either hydrochlorothiazide  or vehicle, once daily for 4 days. Subsequently, all mice were anesthetized by an i.p. injection consisting of 100 mg/kg ketamine and 10 mg/kg xylazine and their kidneys were collected. The cortex was subsequently isolated and stored in RNAlater . Twenty‐four hour urine was collected at baseline and during each day of treatment. Blood samples were drawn from the carotid artery on day 4 after administration and immediately spun down to isolate the plasma. All animal experiments were performed in adherence to the NIH Guide for the Care and Use of Laboratory Animals and approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee.The generation of the CA‐SPAK mice has been described previously . RNA integrity was determined at the Core Facility of the University of Maryland School of Medicine using the RNA Integrity Number (RIN) method  for analysis of plasma and urinary MgP < 0.05 was considered statistically significant. All statistical analyses were performed using Graphpad Prism 5.0 (Graphpad Software). All experiments were performed in a blinded manner and unblinding occurred immediately before statistical analysis.All data are presented as mean ± SEM. Student's independent t‐test was used for statistical comparison; a 2+ balance, plasma Mg2+ levels were determined. The CA‐SPAK mice had similar plasma Mg2+ levels as control mice , did the CA‐SPAK mice display a significantly higher urinary Mg2+ excretion than control mice . This indicates that NCC‐dependent Na+ reabsorption is required for Mg2+ reabsorption in the DCT. Indeed, this notion is supported by the observation that SPAK−/− mice, which lack the kinase responsible for phosphorylating NCC, also develop hypomagnesemia and renal Mg2+ wasting (Yang et al. + reabsorption is not coupled to Mg2+ reabsorption. As estimated by the initial urinary response to HCTZ, we found NCC‐dependent Mg2+ reabsorption is actually suppressed in the CA‐SPAK mice, despite increased NCC activity. We speculate that enhanced Mg2+ reabsorption in other segments offsets the decrease in DCT‐specific Mg2+ reabsorption to maintain normal Mg2+ balance.It is well established that decreased NCC activity is associated with urinary Mg+ and Mg2+ reabsorption in CA‐SPAK mice. It is possible, for example, that constitutive SPAK signaling directly inhibits TRPM6, the apical Mg2+ channel responsible for Mg2+ reabsorption in the DCT. Technically demanding patch‐clamp experiments, in vivo or in vitro, on TRPM6 will be required to test this idea. In addition, the activities of other magnesiotropic proteins might also be contributory.Since we did not observe any significant differences in the mRNA expression of magnesiotropic genes between control mice and CA‐SPAK mice under control conditions, we speculate that post‐transcriptional modifications are responsible for opposite responses on Na2+ homeostasis is altered as a direct result of CA‐SPAK and whether a WNK4 mouse model of Gordon syndrome exhibits the same phenotype (Wilson et al. Wnk1, reported normal plasma Mg2+ levels and urinary Mg2+ excretion (Vidal‐Petiot et al. 2+ reabsorption (de Baaij et al. 2+ handling in TAL and DCT may explain the normomagnesemia and normomagnesiuria in patients with Gordon syndrome.Because SPAK is the terminal kinase in the WNK signaling pathway that is altered in Gordon syndrome, our results should be broadly applicable to the disease. Nevertheless, it will be important to determine whether Mg2+ wasting. The specific genetic background of CA‐SPAK mice could also have implications for renal Mg2+ handling in the thick ascending limb of Henle's loop given the data suggesting a role for SPAK in NKCC2 function (Moriguchi et al. 2+ than control animals after prolonged HCTZ treatment. However, in contrast to NCC knockout mice and chronic HCTZ administration in wild‐type mice, which exhibit downregulation of Trpm6 (Nijenhuis et al. Trpm6 RNA levels are not affected in CA‐SPAK mice by prolonged HCTZ treatment. Thus, high levels of urinary Mg2+ excretion in the thiazide‐treated CA‐SPAK mice cannot be attributed to changes in Trpm6 expression. We speculate that HCTZ‐induced atrophy of the DCT might be more pronounced in CA‐SPAK mice. This warrants additional experiments into alterations in tubular morphology following prolonged HCTZ treatment and their association with Mg2+ reabsorption.It is also important to consider that the DCT1‐specific CA‐SPAK mice are engineered on a SPAK null background. Thus, when NCC is inhibited, these mice revert to a SPAK null phenotype. We (Grimm et al. + and Mg2+ reabsorption are not coupled in CA‐SPAK mice. These mice display an impaired NCC‐dependent Mg2+ reabsorption, despite constitutive hyperactivation of NCC. Like human subjects with Gordon syndrome, CA‐SPAK mice do not exhibit an altered Mg2+ balance. We speculate that enhanced Mg2+ reabsorption in other segments offsets the decrease in DCT to maintain normal Mg2+ balance. Further research is required to define the mechanisms.In summary, thiazide‐sensitive NaThe authors declare that there is no conflict of interest.