In the contingent-reward condition, norepinephrine as an index of sympathetic activity was positively correlated with entropy indicating exploration in decision-making. Norepinephrine was negatively correlated with neural activity in the right posterior insula, rostral anterior cingulate cortex, and dorsal pons, suggesting neural bases for detecting changes of bodily states. Furthermore, right anterior insular activity was negatively correlated with entropy, suggesting influences on exploration in decision-making. By contrast, in the random-reward condition, entropy correlated with activity in the dorsolateral prefrontal and parietal cortices but not with sympathetic activity. These findings suggest that influences of sympathetic activity on exploration in decision-making and its underlying neural mechanisms might be dependent on the degree of uncertainty of situations.We previously reported that sympathetic activity was associated with exploration in decision-making indexed by entropy, which is a concept in information theory and indexes randomness of choices or the degree of deviation from sticking to recent experiences of gains and losses, and that activation of the anterior insula mediated this association. The current study aims to replicate and to expand these findings in a situation where contingency between options and outcomes is manipulated. Sixteen participants performed a stochastic decision-making task in which we manipulated a condition with low uncertainty of gain/loss (contingent-reward condition) and a condition with high uncertainty of gain/loss (random-reward condition). Regional cerebral blood flow was measured by  Electrophysiological , EEG, cardiovascular, neuroendocrine, and immune parameters were measured during a stochastic decision-making task. In that task, we manipulated the degree of contingency between options and outcomes (monetary gains and losses) to examine variations of association between the brain and autonomic activities during decision-making corresponding to uncertainty of situations. In a condition with lower uncertainty called the contingent-reward condition, an advantageous option, compared to a disadvantageous option, is associated with monetary gains at a higher probability and with monetary losses at a lower probability. On the other hand, in another condition with higher uncertainty called the random-reward condition, the gains and losses were delivered randomly for both stimuli. Thus, the situation was substantially stochastic and participants could not learn the contingency. One merit of utilization of this dataset is that involvement of brain regions which are well known to relate to decision-making, including the anterior cingulate, orbitofrontal, and dorsolateral cortices  and dorsal striatum, during the stochastic decision-making task has been clarified and published elsewhere  in peripheral blood, and cardiovascular indices (heart rate (HR), mean blood pressure (MBP), total peripheral resistance (TPR), and heart rate variability (HRV) representing vagal (parasympathetic) activity  measured by  Sayers, . Because Sayers, , 2010, wSixteen healthy right-handed Japanese male undergraduate and graduate students who had no past history of psychiatric and neurological illness were recruited . They gave written informed consent in accordance with the Declaration of Helsinki. The present study was approved by the Ethics Committee of Kizawa Memorial Hospital.The timeline of a trial of the stochastic decision-making task which participants performed is shown in Figure Participants performed eight blocks of the decision-making task. Three blocks were for the contingent-reward condition, three blocks were for the random-reward condition, and two blocks were for the control condition. Each block lasted for 4 min, with an 11-min interval from the previous block, and contained 40 trials. Each condition was consisted of three continuous blocks, and the order of the contingent-reward condition and the random-reward condition was counterbalanced between the participants. Both in the contingent-reward condition and in the random-reward condition, the advantageous and disadvantageous stimuli were counter-balanced between participants, and the same stimulus was delivered as an advantageous stimulus in all blocks for a participant. Blocks for the control condition were placed in the 1st and 5th block, such that a control condition was followed by either blocks of the contingent-reward condition or blocks of the random-reward condition. The contingency between stimuli and outcomes in each control block was matched to the in the following experimental blocks; i.e., 70:30% gain/loss mapping to stimuli in one control block and 50:50% gain/loss mapping to stimuli in the other control block. Participants were told that gain and loss in the control conditions would also influence the money paid for participation.PET scanning to collect rCBF data was conducted in each block. Cardiovascular parameters  were measured for 2 min before each block as baseline and for 4 min during the task. For measurement of plasma catecholamine (epinephrine and norepinephrine), blood samples were taken using a heparinized 22-gage butterfly catheter placed in the antecubital vein of the right forearm, for 1 min just before the baseline period of measurement of cardiovascular parameters and for the last 1 min of each block. Finally, participants were remunerated. Although participants were told that their payment would depend on their performance, all participants were paid 15,000 JPY (140 USD) for participation.a) under a state (S). Here, the action is a choice of the same stimulus that was chosen in the previous trial or that of another stimulus that was not chosen in the previous trial (Stay or Shift). The state is an outcome (gain or loss) in the previous trial. Thus, the conditional probability P(a|S) is calculated as follows:Task performance was evaluated in two behavioral indexes: response bias and reward acquisition. Response bias means the rate of choice of the advantageous stimulus. Reward acquisition was defined as the rate of getting gain regardless of choice of advantageous or disadvantageous stimulus. Following our previous study  is the number of Stay or Shift (a) under a state S, and Num(k|S) is the number of total choices k under a state S. The constant c was introduced to stabilize the calculated probability, and was fixed to 1 here. Therefore, four conditional probabilities were calculated: (1) Stay  when gain was given in the previous trial, (2) Stay when loss was given in the previous trial, (3) Shift  when gain was given in the previous trial, and (4) Shift when loss was given in the previous trial. Then, entropy H was estimated as follows:where N is a number of states S. The value of entropy H was standardized from 0 to 1 by dividing by N . Thus, entropy calculated by this formula reflects the degree of deviation from dependence of a choice on the outcome of the previous trial. If a participant chooses the same stimulus regardless of whether it is advantageous or disadvantageous in all trials, H will be a minimum . If a participant always chooses the same stimulus as the previous trial when gain was given in the previous trial and shifts the choice when loss was given in the previous trial , H will also be the minimum. These patterns of decision-making can be regarded as fixed strategies, independently from task performance reflected by response bias and reward acquisition. Conversely, if a participant chooses a stimulus totally independently from the outcome in the previous trial in all trials (random choice), entropy H will be a maximum (approaching to 1). Response bias, reward acquisition, and entropy were determined at each block of the contingent-reward and random-reward conditions, respectively.where We recorded MBP and HR by using the finger cuff of a Portapres Model 2  which was attached to the third finger of the dominant arm of each participant. HR was also measured and analyzed by using photoplethysmography using the Portapres at a sampling rate of 200 Hz, and the Beatfast software using a model flow. TPR was obtained by analyzing the sampled arterial pressure waveforms with the Beatfast software. Mean values of MBP, HR, and TPR were calculated for 2 min just before the task as baseline and during 4 min of the task in each block, for analyses.We further measured components of HRV on the basis of HR data as indices of sympathetic and parasympathetic activity. Similar to other cardiovascular indexes, HRV was analyzed for 2 min just before the task for baseline and 4 min during the task in each block. First, the tachogram data on interbeat-intervals were re-sampled at 4 Hz to obtain equidistant time-series values. Then, a power spectral density was obtained by a fast Fourier transformation. The data were linearly detrended and filtered through a rectangular window. The integral of the power spectrum was measured in a low-frequency band  and a high-frequency band . Herein, we report the absolute value of HF power as an index of parasympathetic activity. For statistical analyses, we examined the LF and HF component expressed as natural logarithm values of the percentages of LF power and HF power of the total power in the spectrum  were performed to detect combinations of data points which showed significant differences.We performed two-way  repeated-measures analyses of variance (ANOVAs) for data of response bias, reward acquisition, and entropy, separately. Three-way  repeated-measures ANOVAs were performed for autonomic data . The Greenhouse-Geisser epsilon correction factor, ε  and change scores of autonomic indices  were examined. Furthermore, we performed step-wise regression analyses by using change scores of autonomic indices  as independent variables in the contingent-reward condition and random-reward condition, separately, to examine the effects of sympathetic and parasympathetic parameters on entropy. To calculate the change scores of the autonomic indices, subtractions of the autonomic indices at baseline from values during the task in each block were conducted first. Mean scores of the subtracted values within three blocks were then calculated for each indices both in the contingent-reward condition and in the random-reward condition, and used for the regression analyses.68germanium pin source was completed for 10 min. 370-MBq bolus injection was started 60 s after initiation of each block. Scanning was started 30 s after initiation of bolus injection and continued for 60 s. The integrated radioactivity accumulated during the scanning was measured as the index of rCBF. Eight scans were performed for each participant, and the 15 min interval between successive scans was placed for clearance of radioactive levels. A Hanning filter was used to reconstruct images into 35 planes with 4.5 mm thickness and a resolution of 2 × 2 mm .The distribution of rCBF was measured by using a PET scanner  in a high-sensitivity three-dimensional mode at each block. A venous catheter for administering the tracer was inserted in an antecubital fossa vein in the left forearm of each participant. The participant's head was fixed in an inflatable plastic head-holder that prevented head movement. Then, a transmission scan using a rotating We used SPM 99  for the correlation analyses, we focused on the prefrontal, limbic, and striatum areas for interpretations, as we had found neural activity and neuro-autonomic associations in such regions during similar tasks of decision-making  and K > 10. This threshold is relatively liberal in the current standard. However, it was chosen considering the balance between risks of the type-1 error and type-2 error  and standard errors (SEs) of response bias, reward acquisition, and entropy are shown in Table F = 7.10, p < 0.05, η2p = 0.32] was significant, but neither a main effect of Block nor an interaction of Condition and Block was significant (F < 1.78), for response bias. Naturally, reward acquisition showed similar results as response bias, namely, a significant main effect of Condition . No significant effect was obtained on entropy .Means  < 0.17, ns.], indicating that entropy is independent of performance of the decision-making task.Entropy was not correlated with response bias and reward acquisition in either condition , suggesting that MBP in the contingent-reward condition was higher than that in the random-reward condition, and that MBP elevated during the task compared to the baseline. For HR, a significant main effect of Condition was shown , indicating that HR was higher in the contingent-reward condition compared with that in the random-reward condition. TPR showed a significant interaction of Condition and Block , indicating that TPR was markedly increased in the random-reward condition but not in the contingent-reward condition, during the third block. A significant main effect of Condition in the HF component of HRV  was also observed, suggesting that parasympathetic activity was more enhanced in the random-reward condition compared with that in the contingent-reward condition. The LF/HF ratio of HRV showed no significant effects in either condition.F = 6.10, p < 0.05, η2p = 0.29], indicating that overall concentration of epinephrine was higher in the contingent-reward condition compared with that in the random-reward condition. For norepinephrine, a significant interaction of Condition and Period was observed . Further it was indicated that norepinephrine concentration did not change between baseline and task periods in the contingent-reward condition, while it was reduced during the task period in the random-reward condition.For catecholamine, epinephrine showed a significant main effect of Condition , including norepinephrine and the LF/HF ratio of HRV as independent variables. The analysis also revealed that the change of norepinephrine as an index of sympathetic activity , but not the LF/HF ratio of HRV, significantly and positively contributed to entropy  = 0.53, ns.].In the contingent-reward condition, a hierarchical regression analysis on entropy adopted a significant model [adjusted The change of norepinephrine showed significant negative correlations with rCBF in brain regions including the parahippocampal gyrus, cerebellum, rostral ACC, right posterior insula, prefrontal cortex, globus pallidus, thalamus, putamen, and postcentral gyrus in the contingent-reward condition Figure , while rx = 60, y = 22, z = −4, Z = 4.11), lateral prefrontal cortex , putamen , posterior insula , and rostral ACC   predicted entropy in the contingent-reward condition. This seemed that signals of peripheral sympathetic activity affecting exploration are conveyed to the brain mainly via the neurochemical route including the afferent vagus nerve expressing β-adrenergic receptors, NTS, LC-norepinephrine system, and basal forebrain cholinergic system, as proposed by several researchers  and a Grant-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Research Area) 2010 (No. 4102-21119003) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Carrying load alters normal walking, imposes additional stress to the musculoskeletal system, and results in an increase in energy consumption and a consequent earlier onset of fatigue. This phenomenon is largely due to increased work requirements in lower extremity joints, in turn requiring higher muscle activation. The aim of this work was to assess the biomechanical and physiological effects of a multi-joint soft exosuit that applies assistive torques to the biological hip and ankle joints during loaded walking.EXO_ON), unpowered (EXO_OFF) and unpowered removing the equivalent mass of the device (EXO_OFF_EMR). Seven participants walked on an instrumented split-belt treadmill and carried a load equivalent to 30 % their body mass. We assessed their metabolic cost of walking, kinetics, kinematics, and lower limb muscle activation using a portable gas analysis system, motion capture system, and surface electromyography.The exosuit was evaluated under three conditions: powered (EXO_ON condition (7.5 ± 0.6 W kg−1) was 7.3 ± 5.0 % and 14.2 ± 6.1 % lower than in the EXO_OFF_EMR condition  and in the EXO_OFF condition , respectively. The exosuit also reduced the total joint positive biological work  when comparing the EXO_ON condition (1.06 ± 0.16 J kg−1) with respect to the EXO_OFF condition  and to the EXO_OFF_EMR condition .Our results showed that the exosuit could deliver controlled forces to a wearer. Net metabolic power in the The results of the present work demonstrate for the first time that a soft wearable robot can improve walking economy. These findings pave the way for future assistive devices that may enhance or restore gait in other applications.The online version of this article (doi:10.1186/s12984-016-0150-9) contains supplementary material, which is available to authorized users. Carrying heavy loads alters the biomechanics of walking, leading to an increased metabolic burden. This negative consequence of load carriage has been reported in soldiers, first responders, and recreational athletes who are required to execute physically demanding tasks during walking , 2. SeveLower-limb exoskeletons have been proposed as a means to augment or assist human locomotion for many applications . Some exAs an alternative to rigid exoskeletons, we have developed a multi-joint soft exosuit –22 that Our research group has demonstrated reductions in metabolic cost during load carriage with a tethered soft exosuit , 24. OneEXO_ON), with the device unpowered (EXO_OFF) and with the device unpowered with equivalent mass removed (EXO_OFF_EMR). The second condition (EXO_OFF) was evaluated to assess the penalty associated with carrying the additional mass represented by the device itself, an important consideration in the design of such systems. To obtain additional insights on the benefit of wearing the soft exosuit and to extend the knowledge on the biomechanical and physiological effects of this device, we evaluated metabolic cost, muscle activation and joint mechanics which have been shown to be relevant for regulating metabolic energy cost during gait [Therefore, the aim of this work was to perform the first study with an autonomous (fully portable) multi-joint  soft exing gait .A lower extremity soft exosuit Fig.  and the The load-transferring elements in the exosuit followed two distinct paths in each leg as described in Fig. Two actuation units Fig.  connecteThe ankle and hip contribute together approximately 80 % of the positive mechanical power produced by the lower limb joints during walking , 27. PowThe actuation system we built for the exosuit enabled two motors to actuate all four of the load paths (two load paths per leg). Each of the two actuation units Fig.  consisteWe recruited seven load carriers from the local community . All participants reported no musculoskeletal injuries or diseases and provided written informed consent. The participants whose images appear in the manuscript have provided written consent for the publication of their images according to the policies of the Journal of NeuroEngineering and Rehabilitation. The study was approved by the Harvard Medical School Committee on Human Studies.EXO_ON, EXO_OFF and EXO_OFF_EMR. Participants walked on an instrumented split-belt treadmill measuring three-dimensional ground reaction forces  at a constant speed of 1.5 m s−1 while carrying a load equivalent to 30 % of their body mass , and unpowered (EXO_OFF and EXO_OFF_EMR). In the EXO_OFF_EMR condition, used as baseline comparison, the equivalent weight of the multi-joint soft exosuit (6.6 kg) was removed from the load in the backpack, further details on this procedure are provided in Additional file EXO_OFF_EMR and in the EXO_OFF condition and for nine minutes in the EXO_ON condition. The first three minutes of the EXO_ON condition allowed the device’s controller to ramp up forces to the desired level   Fig. . Carbon th order low pass Butterworth filter with a 5–9 Hz optimal cut-off frequency selected using a custom residual analysis algorithm . Joint angles, net joint moments and powers were calculated in the sagittal plane using filtered markers and forces by means of an inverse kinematic and dynamic approach . Net joint moments and powers were then normalized by each participant’s body mass. An automatic gait event detection algorithm  was used to determine heel strikes that defined gait cycles. Ten strides per condition were used for generating mean kinematic and kinetic data for each participant, which were subsequently combined to calculate condition mean data.Three-dimensional (3D) gait analysis was performed during treadmill walking. The marker set used for 3D motion capture  was composed of 50 markers placed on specific anatomical bony landmarks Fig. . Single EXO_OFF_EMR condition. Ten strides per condition were used to compute mean muscle activation across each stride.During all trials, surface electromyography (EMG) signals from eight lower limb muscles were measured by means of a wired system  simultaneously with the motion data measured by the VICON system. Muscles investigated were: rectus femoris (RF), vastus medialis (VM), vastus lateralis (VL), gluteus maximus (GM), biceps femoris (BF), soleus (SOL), medial gastrocnemius (MG) and tibialis anterior (TA) Fig. . EMG sigEXO_ON condition, the actuation units were synchronized to the Vicon system using a 5 V signal generated at the beginning of the motion capture data collection. Forces measured by load cells at the ankle and the hip during the EXO_ON condition  from the net joint moment at the ankle (or hip) as per [EXO_ON condition were calculated for the ankle and the hip joints of the right leg by integrating over time the positive and negative corresponding biological powers. For the EXO_OFF_EMR and the EXO_OFF conditions, positive and negative biological work were calculated by inverse dynamics.To compute the biological components of net joint moment and power during the ion Fig.  were segion Fig.  were calion Fig. . To asseion Fig. . In this) as per . BiologiEXO_OFF_EMR, EXO_OFF and EXO_ON) were used to verify the effect of the device on metabolic power and on the average muscle activation across the stride. The biomechanical variables of interest included: peak flexion and extension joint angles and moments, as well as absorbed and generated joint power (positive and negative area of the power trace curves) at the ankle, knee and hip. Additional one-way repeated measures ANOVA were conducted on positive and negative biological joint work and power . Bonferroni post hoc tests were performed to identify differences between conditions when a statistically significant main effect was identified by the ANOVA. The significance level was set at p < 0.05 for all analyses. Effect sizes were calculated using Cohen’s d method.Statistical analysis was conducted in SPSS . One-way repeated measures analyses of variance (ANOVA) with three modes  and 14.2 ± 6.1 %  lower than in the EXO_OFF_EMR and for the EXO_OFF conditions, respectively. This resulted in a significant metabolic power reduction during the EXO_ON condition of 35.2 ± 23.0 W  and 74.8 ± 33.6 W  with respect to the EXO_OFF_EMR and to the EXO_OFF condition, respectively . Significantly lower average muscle activation (8.4 ± 9.8 %) was also reported in the soleus for the EXO_ON condition with respect to the EXO_OFF condition . No main effect of muscle activation was reported for the six other lower limb muscles investigated. Muscle activations during the three conditions of testing are presented in Fig. Significantly lower average muscle activation (4.7 ± 7.0 %) was reported in the vastus lateralis for the Stride length, stride frequency, duty factor and stance and swing times were not significantly different between the three testing conditions. A complete overview of these parameters is presented in Additional file EXO_ON condition with respect to the EXO_OFF_EMR  and the EXO_OFF  conditions. Additionally, a significantly lower knee flexion angle peak was reported in the beginning of the stance phase in the EXO_ON condition with respect to the EXO_OFF_EMR  and to the EXO_OFF  conditions. No other significant differences were reported in kinematics  and a higher knee flexion moment peak in the EXO_ON condition with respect to the EXO_OFF condition . Significantly higher ankle power generation was reported for the EXO_ON condition with respect to the EXO_OFF_EMR  and to the EXO_OFF  conditions. Additionally, significantly lower knee power absorption was reported in the EXO_ON condition with respect to the EXO_OFF condition  and between the EXO_OFF_EMR condition and the EXO_OFF condition . A complete overview of the joint kinetics data is presented in Additional file The only statistically significant differences in joint moments Fig. , were a EXO_ON condition with respect to the EXO_OFF condition  and to the EXO_OFF_EMR condition . Further, hip biological positive work was significantly reduced in the EXO_ON condition with respect to the EXO_OFF condition  and to the EXO_OFF_EMR condition , and ankle biological positive work was significantly reduced in the EXO_ON condition with respect to the EXO_OFF_EMR condition . Total joint biological negative work was not significantly different between the three conditions; nevertheless, knee biological negative work was significantly reduced in the EXO_ON condition with respect to the EXO_OFF condition .Average peak forces generated by the exosuit across the seven participants were 272 ± 43 N of ankle plantarflexion, 204 ± 32 N of hip flexion, and 68 ± 24 N of hip extension. These external forces caused a significant reduction of total joint biological positive work  when comparing the EXO_ON (1.02 ± 0.10 W kg−1) with respect to the EXO_OFF condition  and to the EXO_OFF_EMR condition . Hip biological positive power was significantly reduced in the EXO_ON condition with respect to the EXO_OFF condition  and to the EXO_OFF_EMR condition , and ankle biological positive power was significantly reduced in the EXO_ON condition with respect to the EXO_OFF condition . Total joint biological negative power was not significantly different between the three conditions. Knee biological negative power was significantly reduced in the EXO_ON condition with respect to the EXO_OFF condition  and in the EXO_OFF condition with respect to the EXO_OFF_EMR condition   was significantly reduced when comparing the 85) Fig. .The aim of this study was to evaluate the effects of an autonomous (fully portable) multi-joint soft exosuit on the metabolic cost of loaded walking. A net reduction in the metabolic cost was reported while walking with the exosuit compared to wearing the exoskeleton unpowered with effective mass removed. This finding Fig.  represenRecent work from two different research groups , 31 alsoEXO_ON condition produced a larger reduction in metabolic cost when compared to the EXO_OFF condition than to the EXO_OFF_EMR condition. Although the relationship between muscle-tendon behavior and whole body energy consumption is complex [EXO_ON vs EXO_OFF comparison and concealed, at least in part, by the additional load imposed by the system in the EXO_OFF_EMR condition.As expected, the  complex , we hypoEXO_ON condition suggests that the exosuit facilitated a return to gait patterns that resemble unloaded walking [Walking with the powered device did not alter participants’ spatio-temporal parameters, indicating that the exosuit’s assistive forces were not disruptive to participants’ freely-selected step frequencies and step lengths. Nevertheless, some alterations in participants’ kinematics and kinetics were present. Given the aforementioned finding of reduced metabolic cost during walking, we posit that these changes may have permitted the musculoskeletal system to operate more efficiently. Indeed, considering that ankle dorsiflexion and knee flexion have been shown to increase with an increase of load , our obs walking .Walking with the soft exosuit reduced the total biological joint work produced by the lower limbs, and the most marked reduction in biological work production was at the hip joint. This reduction is of particular interest especially considering that the hip joint seems to have lower efficiency compared with the other lower limb joints . This hyEXO_ON condition during push-off corresponded to a lower knee moment on the contralateral limb during weight acceptance (Fig. EXO_ON condition with respect to the EXO_OFF condition. It can be assumed that the ankle joint also contributed to the metabolic reduction and this rationale can be supported by the reduced soleus activation in the EXO_ON condition with respect to the EXO_OFF condition. The reason this was not found when comparing the EXO_ON and EXO_OFF_EMR conditions may have been that the effect was masked by the wearer having to carry the increased mass of the device.Although the main reduction in the biological work was reported at the hip, the knee and ankle may also have contributed to lowering the metabolic burden. Interestingly, the assistance provided by the soft exosuit reduced knee extension moments Fig.  with an nce Fig. , similarSurprisingly, despite the fact that biological work was significantly reduced, only small differences were found in muscle activation between conditions Fig. . ReporteThe observed variability in the reduction of metabolic cost between participants may be due to several contributing factors. The fixed external assistance across participants coupled with small variations in how the exosuit fit to different body sizes and shapes resulted in variations in the percentage of the delivered assistance relative to nominal biological joint torques at the hip and ankle. In addition, small variations in how the multi-articular load path crossed the knee may have resulted in small torques at the knee joint for some participants. Additionally, neuromuscular adaptation associated with the use of wearable robots remains an active research area , with liBased on the results of this study, we believe that there is potential for further enhancing the exosuit’s performance. First, it should be noted that the joint torques applied by the exosuit to the wearer were relatively low compared to the joint moments experienced by load carriers . This waOur results demonstrate that an autonomous soft exosuit can reduce the metabolic burden experienced by load carriers, possibly augmenting their overall gait performance. Although many basic fundamental research and development challenges remain in actuator development, textile innovation, sensing and control, this proof of concept study provides the first demonstration of a soft wearable robot to augment gait. This work also presented the first demonstration that an autonomous multi-joint wearable robot can achieve a metabolic reduction. Future studies will be necessary to explore the effects of assisting walking with a soft exosuit and explore if, for a given level of mechanical work, the most effective approach to providing assistance is by augmenting the function of a single joint or multiple joints. Moreover, the realization of devices that assist joints other than the ankle can increase the knowledge on the determinants of energy cost, potentially revealing different adaptive mechanisms of the musculoskeletal system. Finally, apart from assisting load carriers, we are exploring how the soft exosuit can be used as a platform to assist individuals with compromised ability to produce adequate forces during locomotion , paving


This phenotype shared features with that produced when the Wnt/β-catenin pathway co-receptor Lrp6 is mutated or when retinoic acid (RA) signaling in the eye is compromised. Consistent with this, microarray and cell fate marker analysis identified a series of expression changes in genes known to be regulated by RA or by the Wnt/β-catenin pathway. Using pathway reporters, we showed that Wnt ligands from the surface ectoderm directly or indirectly elicit a Wnt/β-catenin response in retinal pigment epithelium (RPE) progenitors near the optic cup rim. In fl/flLe-cre; Wls mice, the numbers of RPE cells are reduced and this can explain, using the principle of the bimetallic strip, the curvature of the optic cup. These data thus establish a novel hypothesis to explain how differential cell numbers in a bilayered epithelium can lead to shape change.The Wnt/β-catenin response pathway is central to many developmental processes. Here, we assessed the role of Wnt signaling in early eye development using the mouse as a model system. We showed that the surface ectoderm region that includes the lens placode expressed 12 out of 19 possible Wnt ligands. When these activities were suppressed by conditional deletion of wntless ( Summary: During optic cup morphogenesis, Wnt ligands expressed in the surface ectoderm control cell proliferation in the retinal pigmented epithelium, and thus influence bending of the neural retina. Eye development begins with bilateral evagination of the diencephalon to form the optic vesicles  . We show+ cells from Z/EG mice  . Thus, tl allele  with thefl/flLe-cre; Wls mice, Wls immunoreactivity is lost from the lens placode and periocular ectoderm at E9.5 , TCF/LEFlacZ is, like TOPGAL, expressed in RPE adjacent to the retinal rim at E11.5 , mapped positive cells from representative sections (n=11 for each genotype) onto a schematic of eye sections (+ cells in each of ten sectors that represented different optic cup regions (+ cells was significantly reduced in the two sectors (1 and 10) that represented the rim of the optic cup . However, deletion of fl/flWls with the mesenchymal Wnt1-cre  and no change in TOPGAL expression  despite evidence of Wls protein loss . In addition, deletion of Wls from RPE progenitors using DctCre . These data suggest that surface ectoderm Wnt ligands either signal to the optic cup directly or signal using a Wnt ligand-independent relay.Analysis of Wnt1-cre  producedg DctCre  also profl/flWls mice with the experimental genotype fl/flLe-cre; Wls. RNA for the analysis was generated from E10.5 whole eye tissue containing eye cup, lens pit and POM. A comparison of transcript levels in the two genotypes revealed that 44 transcripts showed a greater than 1.5-fold change. A 2-fold reduction in the Wls transcript helped verify conditional deletion . When we examined the immunoreactivity for Raldh1 (Aldh1a1), Raldh3 (Aldh1a3) and Pax2 in dorsoventral sections of control and fl/flLe-cre; Wls mice, we observed a very similar distribution , even though the altered shape of the retinal rim in fl/flLe-cre; Wls mice gave the impression of higher expression ventrally. This observation is consistent with the suggestion that RA signaling does not alter dorsoventral patterning in the optic cup  in the optic vesicle results in early eye defects including anophthalmia   . Similarcoloboma  or defeccoloboma .The RA receptor RXR and RAR families are required for eye development, as are the RA synthesis enzymes Raldh1 and Raldh3. Although there are many nuances in an interpretation of their function, there is strong evidence to indicate that RA synthesized by Raldh1 and Raldh3 in the optic cup signals to the surrounding ocular mesenchyme via Rarα-Rarβ and Rarα-Rarγ receptor heterodimers  placodal and periocular ectoderm expresses 12 out of 19 possible Wnt ligands and this raises the very simple question of what each of them does. The expressed Wnt ligands fall into both Wnt/β-catenin and non-canonical signaling ‘classes’ [but see Although the current data provide strong evidence that the surface ectoderm is a crucial source of Wnt ligands required for optic cup morphogenesis, many questions remain unanswered. The relative roles of different Wnt ligands is unclear. Our data suggest that Pax6-expressing  and TCF/LEFlacZ . RNA was isolated using the RNeasy Kit (Qiagen). End-point PCR was performed using primer sequences for Wnts 1-11 from the qPrimerDepot database.Developing eye regions at E10.5 were isolated from mouse litters of the following crosses: N=3) and the data were averaged during the analysis and grouping of genes. Microarray data are available in the GEO repository under accession number GSE65154.E10.5 embryos were dissected into cold PBS. Eye regions were removed using the MC-2010 Micro-Cautery Instrument  and flash frozen in 30 µl RLT buffer from the Qiagen Micro RNeasy Kit. Samples from two to four embryos of the same genotype were combined to create a single sample containing four to eight eye regions. Tissue was then processed for RNA isolation using a standard protocol from the Micro RNeasy Kit to produce 20 ng total RNA. Affymetrix GeneChip Mouse Genome 430 2.0 gene expression arrays were analyzed using an Agilent Bioanalyzer. Microarrays for each genotype were performed in triplicate (


Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL). This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5) on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i) in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment. Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus that infects an estimated 15–20 million people worldwide ,4. HTLV-As a complex retrovirus, HTLV-1 has a genome that encodes structural and enzymatic proteins , regulatory proteins (Tax and Rex), and several accessory proteins p30, p12/p8, HBZ). Studies have shown that at least two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process ,10. Tax /p8, HBZ.in vivo and transforms CD4+ T-cells are not completely understood. The requirement for Tax and other viral proteins in vivo suggests that expression of viral proteins early in infection plays a major role in viral replication, infected cell survival, and disease development. A favored theory within the field is that the virus is critically dependent on Tax early in infection to initiate transformation, but then Tax expression is highly regulated and often times silenced to prevent immune detection. HBZ is hypothesized to provide the maintenance or cell survival signal necessary for the transformation process. Over time, the combination of genetic and epigenetic changes in an HTLV-1-infected cell can lead to transformation and potentially, disease development -labeled dTTP . A volume of 10 µL was spotted onto a DEAE filtermat (Perkin Elmer), air dried at room temperature, then washed 5× with 1× saline-sodium citrate buffer (SSC) and 2× with 85% ethanol. Filtermats were air dried and exposed to a phosphorimaging screen for 2.5 h at room temperature. Density, counts/mm2, was determined using the Typhoon Scanner  and Quantity One software (Bio-Rad).ACH-2 , 100 ng LTR-1-luciferase, 1 μg ACHneo, 500 ng Flag-PRMT5, 500 ng pcDNA3.1-p30, 100 ng S-tag-Tax, or 500 ng S-tag-HBZ, where indicated. HEK293T cells were harvested 48 h post-transfection in Passive Lysis Buffer (Promega). Relative firefly and Renilla luciferase units were measured in a FilterMax F5 Multi-Mode Microplate Reader using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer’s instructions. Each condition was performed in duplicate. Extracts also were subjected to immunoblotting to verify equivalent protein levels. Cell supernatants (48 h) were used for p19 ELISA (Zeptometrix).HEK293T cells were transfected using Lipofectamine6 cells/mL. Indicated concentrations of the PRMT5i were added to the appropriate wells. Cells were incubated at 37 °C for 24 h. After incubation, cells were collected by slow centrifugation  for apoptosis analysis via flow cytometry. Collected cells were stained using the FITC Annexin V Apoptosis Detection Kit I  according to the manufacturer’s instructions.Lymphoid cells were seeded into a 6-well plate at 1 × 10®2000 according to the manufacturer’s instructions. Cells were cross-linked in fresh 1% paraformaldehyde for 10 min at room temperature. The cross-linking reaction was quenched using 125 mM glycine. Following cell lysis and DNA fragmentation by sonication, DNA-protein complexes were immunoprecipitated with anti-PRMT5 (Santa Cruz) and control anti-IgG (Santa Cruz) antibodies. Immunoprecipitated DNA-protein complexes were washed using sequential low-salt, high-salt, lithium chloride, and Tris-EDTA (TE) buffers. DNA was purified using the Qiagen Gel Extraction Kit (Qiagen). The presence of specific DNA fragments in each precipitate was detected using PCR. Primers used for amplifying the HTLV-1 LTR were 5'-CCACAGGCGGGAGGCGGCAGAA-3' and 5'-TCATAAGCTCAGACCTCCGGGAAG-3' and LacZ-coding region were 5'-AAAATGGTCTGCTGCTG-3' and 5'-TGGCTTCATCCACCACA-3'. Quantification of each ChIP experiment was performed using ImageJ software.pA-18G-BHK-21 cells are a Syrian Hamster kidney cell line stably transfected with a plasmid vector containing the lacZ bacterial gene under the control of a HTLV-1-LTR promoter as previously described . pA-18G-Recently, PRMT5 over-expression was identified to be involved in the pathogenesis of hematologic (lymphoma) and solid tumors  ,30,31,32in vitro transformation assay. Protein and RNA were isolated from two independent wells of cells at weekly time points. Our data revealed that PRMT5 protein and RNA were upregulated at each time point, to varying degrees, throughout the transformation assay.We next determined whether PRMT5 becomes dysregulated and over-expressed during HTLV-1-driven T-cell transformation. Freshly isolated human PBMCs co-cultured with lethally irradiated HTLV-1 producer cells (729.ACHi) in the presence of 10 U/mL of human IL-2 showed progressive growth consistent with HTLV-1 immortalization B and RNATo determine whether PRMT5 over-expression is a marker for T-cell transformation and/or contributes to the process of HTLV-1 transformation, we utilized shRNA vectors to knockdown PRMT5 expression in three different HTLV-1-transformed cell lines. As shown in LAV) were also treated with titrating amounts of PRMT5i  was developed . This nof PRMT5i H to examIn a recent report, PRMT5 was linked to proliferation in B-cell lines and MCL because knockdown of PRMT5 expression reduced cell proliferation . TreatmeA recent report identified PRMT5 as a binding partner of the HTLV-1 accessory protein p30, a known negative regulator of HTLV-1 gene expression . To invein vivo, we performed ChIP assays using pA-18G-BHK-21 cells. The Syrian Hamster kidney cell line was stably transfected with a plasmid vector containing the HTLV-1-LTR promoter that drives expression of lacZ [We next asked what effect PRMT5 had on Tax and HBZ transcriptional activity. HEK293T cells were transfected with LTR-1-luc, TK-renilla control, the ACHneo proviral clone, flag-PRMT5 expression vector, S-tag-Tax expression vector, and S-tag-HBZ expression vector as indicated A. Tax ac of lacZ . pA-18G-HTLV-1 is a tumorigenic retrovirus and the causative infectious agent of ATLL, an extremely aggressive and fatal disease of CD4+ T-cells ,3,4. In et al. found decreased PRMT5 mRNA levels in mantle cell lymphoma cell lines despite abundant PRMT5 protein over-expression [et al. found both PRMT5 protein and mRNA were upregulated in lung tumors [in vitro, we also examined the level of PRMT5 protein and RNA in total PBMCs isolated from ATLL patients  . Using stax/rex mRNA in the nucleus. Taken together, these results suggested that other viral proteins were required for the repressive effects of PRMT5, and/or PRMT5 affected a cellular transcription factor responsible for activating viral transcription. Although not required for the repressive effects of p30 on viral gene expression, we did find PRMT5 and p30 had additive repressive effects on viral transcription, which adds yet another level of regulation to HTLV-1 gene expression. The roles of additional PRMT5 interacting partners, such as MEP50, in PRMT5-mediated HTLV-1 gene regulation are also a possibility to explore in the future. MEP50 is a WD-40 repeat protein and a common PRMT5 cofactor, likely present in most PRMT5-containing complexes in vivo [Using reporter gene assays, we found PRMT5 inhibited HTLV-1 gene transcription, but not Tax protein specifically A,B. We a in vivo . Phospho in vivo .Importantly, we found PRMT5i treatment or shRNA-mediated knockdown of PRMT5 in HTLV-1-positive cell lines caused a decrease in cell proliferation compared to HTLV-1-negative cell lines A,B. Furt


Motor neurons (MNs) are neuronal cells located in the central nervous system (CNS) controlling a variety of downstream targets. This function infers the existence of MN subtypes matching the identity of the targets they innervate. To illustrate the mechanism involved in the generation of cellular diversity and the acquisition of specific identity, this review will focus on spinal MNs (SpMNs) that have been the core of significant work and discoveries during the last decades. SpMNs are responsible for the contraction of effector muscles in the periphery. Humans possess more than 500 different skeletal muscles capable to work in a precise time and space coordination to generate complex movements such as walking or grasping. To ensure such refined coordination, SpMNs must retain the identity of the muscle they innervate. Within the last two decades, scientists around the world have produced considerable efforts to elucidate several critical steps of SpMNs differentiation. During development, SpMNs emerge from dividing progenitor cells located in the medial portion of the ventral neural tube. MN identities are established by patterning cues working in cooperation with intrinsic sets of transcription factors. As the embryo develop, MNs further differentiate in a stepwise manner to form compact anatomical groups termed pools connecting to a unique muscle target. MN pools are not homogeneous and comprise subtypes according to the muscle fibers they innervate. This article aims to provide a global view of MN classification as well as an up-to-date review of the molecular mechanisms involved in the generation of SpMN diversity. Remaining conundrums will be discussed since a complete understanding of those mechanisms constitutes the foundation required for the elaboration of prospective MN regeneration therapies. Motor neurons (MNs) are neuronal cells located in the central nervous system (CNS) controlling a variety of downstream targets. There are two main types of MNs, (i) upper MNs that originate from the cerebral cortex and (ii) lower MNs that are located in the brainstem and spinal cord. Among the latest, spinal MNs (SpMNs) have been intensively studied during the last decades and therefore provide an interesting framework for further molecular characterization. SpMNs are located in the ventral horn of the spinal cord and control effector muscles in the periphery. They form the ultimate and irreplaceable component of the neuronal circuitry since there is no alternative route to convey the commands from the processing centers located in the CNS to the effector muscles in the periphery. Their axon extending through several meters in mammals constitute an exceptional and unique anatomical feature. SpMNs are therefore the longest known cell type.Complex movements such as walking or grasping require the cooperation of several dozens of muscles. Additionally, sensory-motor feedback loops are essential for the real-time tuning of gestures. To ensure such refined coordination, SpMNs must acquire and retain the identity of muscles they innervate as well as be integrated in a coherent and functional neuronal circuitry. Hollyday et al.  and LandMNs are exceptional cell types that can be divided into two main categories according to the location of their cell body: (i) upper and (ii) lower MNs. Upper and lower MNs must be considered as distinct entities despite of their shared nomenclature. Table Upper MN cell bodies are located in the pre-motor and primary motor region of the cerebral cortex also known as the “motor strip.” Since upper MNs make glutamatergic connections with lower MNs located in the CNS, they are exclusively confined to the latter. Typical clinical symptoms of upper MN lesion include uncontrolled movement, decreased sensitivity to superficial reflex stimulation and spasticity  as well as from interneurons (INs). Paralysis is a typical clinical symptom of lower MN lesions since once damaged there is no alternative route to convey the information to the muscle targets in the periphery. Lower MNs are classified into three groups according to the type of target they innervate: (i) branchial, (ii) visceral, and (iii) somatic MNs.Branchial MNs are located in the brainstem and form, together with SNs, the cranial nuclei. They innervate branchial arch derived muscles of the face and neck through 5 cranial nuclei: the trigeminal (V), facial (VII), glossopharyngeal (IX), vagus (X) and accessory (XI) nerves. Despite their similar function, muscles of the neck and the face differ from other skeletal muscles in their embryological origin since they do not derive from the somites, but instead from the branchial arches. Such developmental difference is mirrored by specific characteristics reviewed in depth by Chandrasekhar .Visceral MNs belong to the autonomic nervous system (ANS) responsible for the control of smooth muscles  and glands. The ANS can be described as the association of two components: (i) preganglionic MNs located in the CNS connected to ganglionic neurons belonging to the peripheral nervous system (PNS). In turn, peripheral ganglionic neurons target to the final effector organ. Additionally, the ANS is anatomically and functionally divided into two structures: (i) the sympathetic system and (ii) the parasympathetic system.Motor neurons of the sympathetic system. The sympathetic nervous system is involved in the traditional “fight or flight” responses, recruiting energy storage, increasing awareness, and leading to a global activation of the body metabolism. Central MNs of the sympathetic system are located in the spinal cord from the thoracic segment 1 (T1) to the lumbar segment 2 (L2). These MNs have an intermedio-lateral position and constitute the preganglionic column (PGC) that will be described below. They connect to 3 different targets: two chains of ganglia adjacent to the spinal cord named (i) paravertebral and (ii) prevertebral as well as directly to (iii) the chromaffin cells of the adrenal medulla responsible for the release of the catecholamines  in the circulation, in response to stress stimuli. On the other hand, paravertebral and prevertebral ganglia connect to a wide variety of targets including the heart, lungs, kidneys, intestines and the colon.Motor neurons of the parasympathetic system. The parasympathetic system controls glands secretion and activates the gastrointestinal tract as well as sexual behavior, which are summarized as “rest and digest” functions. Central MNs of the parasympathetic system are located in the brainstem and contribute to the formation of the cranial nerves . Parasympathetic MNs are also found in sacral segments 2 to 4 (S2–S4) of the spinal cord. They innervate ganglia located in the proximity of the peripheral targets such as the heart, bladder, lungs, kidneys, and pancreas.In summary, visceral central MNs from the sympathetic and parasympathetic systems relay information from the CNS to ganglionic neurons of the PNS. In turn those ganglia antagonistically control a large number of various visceral targets. In contrast to branchial mentioned previously and somatic MNs described below, visceral MNs do not directly connect to the final effector. As a result, they constitute an anatomical and functional exception among lower MNs.Somatic MNs are located in the Rexed lamina IX in the brainstem and the spinal cord and innervate skeletal muscles responsible for movements Rexed, . MNs forThree classes of muscles can be anatomically and functionally distinguished: (i) cardiac muscles, (ii) smooth muscles and (iii) skeletal muscles. Cardiac muscles are responsible for the rhythmic contraction of the heart while smooth muscles control the diameter of blood vessels and the internal digestive and secretion organs. Both smooth and cardiac muscles are innervated by the ANS (described above). In contrast, somatic MNs exclusively innervate skeletal muscles that are the most abundant muscle class, with around 639 different muscles in the human body  alpha, (ii) beta and (iii) gamma that will be further described below.Alpha motor neurons. Alpha MNs exclusively innervate extrafusal muscle fibers and are the key of muscle contraction  static, innervating nuclear chain fibers and static nuclear bag fibers and (ii) dynamic, innervating the dynamic nuclear bag fibers  receive input information from the periphery and transmit it either directly to alpha MNs located in the ventral horn (monosynaptic connections) or to association neurons  that, in turn, process and convey the information toward the MNs. MNs then stimulate their respective effector that will generate the appropriate output response  the endoderm, (ii) the mesoderm, and (iii) the ectoderm. Individual layers generate progenies restricted to a limited number of distinct fates. The ectoderm undergoes a process called neurulation in which it folds inward and leads to the formation of three ectodermic masses: (i) the neural tube, (ii) the neural crest cells, and (iii) the external ectoderm. The external ectoderm generates the epidermis whereas the neural crest cells form the peripheral ganglion, the pigments of the skin as well as the dorsal root ganglia. Finally, the neural tube gives rise to the CNS, composed of the brain and the spinal cord     protein OLIG2  , the growth differentiation factor 11 (GDF11 or BMP-11), members of transforming growth factor beta (TGFB) family as well as RA    has been shown to be required for proper SAC MN generation  Figure  where thIsl1 gene to maintain its expression . However, these cells belong to the parasympathetic system (rest and digest) while thoracic PGC MNs belong to the sympathetic autonomic system (fight or flight). In addition to these functional differences, thoracic and sacral PGC MNs also differ in terms of axonal projections. While thoracic PGC MNs connect to the sympathetic chain ganglia located in the proximity of the spine, sacral PGC MNs connect to ganglia in the vicinity of the effector targets . Therefore, the molecular properties of sacral PGC MNs that remain largely unknown are presumably substantially different from thoracic PGC MNs.Initially the MMC had been separated in two divisions: (i) a medial MMC (MMCm), described above as MMC, targeting to axial musculature and present all along the rostro-caudal axis and (ii) a lateral MMC (MMCl) targeting to the body wall and present only in the thoracic segments  and Latissimus dorsi (LD) muscles and induces the expression of ETV4 in the corresponding MN pools  is secreted by both Together these results illustrate the coordination between intrinsic and extrinsically-induced cues. While the first group allows MN development independently of the environment, the second ensures the completion of essential steps. Together these mechanisms create a flexible process allowing MNs to adapt to environment variability. By definition, MN pool specification is intimately linked to axonal targeting. Intensive works have identified various molecules involves in SpMN axonal targeting. We will dedicate the next section to the review the known molecular mechanisms controlling SpMN axonal targeting.Axonal targeting is a critical process of MN development. MN axons emerge within the CNS and transit through different tissues to reach and connect to their specific muscle target in the periphery. Axonal targeting not only provides to MNs their unique anatomical characteristic and therefore their irreplaceable function but also ensures their persistence through the action of trophic signals. In order to complete such critical process, MNs combine several mechanisms in a stepwise manner Figure . SeveralThe very first choice SpMN axons make occurs within the spinal cord . MNs are intrinsically competent to recognize and connect to either fast or slow muscle fibers . Subsequently, while SpMNs strengthen their motor identity, they segregate into anatomical columns termed motor columns. Combinations of LIM homeodomain proteins provide a unique molecular profile for each motor column. In parallel, the LIM code induces the initial steps of a crucial process: MN axonal targeting. SpMN axonal targeting and further differentiation occurs in a step-wise manner. Checkpoints are established along the route to ensure the completion of critical steps. Furthermore, these checkpoints are informative and instruct developing SpMNs of the environment at the growth cone. The SpMN target can be seen as the last checkpoint of the chain. Upon reaching their final destination, SpMNs are required to complete their differentiation process and form functional connection with their target. SpMN identity echoes muscle fiber type properties. Finally, as a mechanism controlling the integrity of SpMN development, naturally programmed cell death induces the elimination of inadequate MNs and ensure the formation of a coherent circuitry.Olig2 mRNA  oriented investigation of downstream targets of known intrinsic regulators such as LIM and/or HOX proteins for example and (ii) unbiased screenings combining, viral retrograde tracing (Stepien et al., rhomboideus MN pool. These neurons constitute, in fact, the only known exception to the MN columnar organization described earlier. Although innervating an axial muscle, this MN pool is located in the lateral component of the ventral horn at caudal brachial segments; a position typical of LMC MNs (Straznicky and Tay, SpMNs are anatomically well organized. This morphological arrangement correlates with the position of their respective target in the periphery as reviewed by Kania . Thus, SFinally, this review deliberately focused on SpMN development from a motor perspective. However, SpMNs are “only” one constituent of a larger coherent circuitry. Complex movements require the control of individual muscles in a collaborating manner. This coordination relies on a highly organized circuitry between SNs, association neurons, and SpMNs as reviewed by Ladle et al. . In a peNicolas Stifani reviewed the relevant literature, wrote the manuscript and generated the figures.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aberrant activation of the Wnt/β-catenin pathway is a major and frequent event in liver cancer, but inhibition of oncogenic β-catenin signaling has proven challenging. The identification of genes that are synthetically lethal in β-catenin-activated cancer cells would provide new targets for therapeutic drug design.CTNNB1 (gene coding for β-catenin) to obtain an isogenic cell line pair with or without aberrant β-catenin signaling. Using this hepatoblastoma isogenic cell line pair, we performed a human kinome-wide siRNA screen to identify synthetic lethal interactions with oncogenic CTNNB1. The phenotypic readouts of the screen were cell proliferation, cell cycle arrest and apoptosis, which were assessed by image-based analysis. In addition, apoptosis was assessed by flow cytometric experiments and immunoblotting. The potential synthetic lethal relationship between candidates genes identified in the screen and oncogenic CTNNB1 was also investigated in a different cellular context, a colorectal HCT116 isogenic cell line pair.We transfected the parental HuH6 hepatoblastoma cell line with a doxycycline-inducible shRNA against CTNNB1 and maximal reduction of β-catenin signaling activity in response to doxycycline treatment. Following high throughput screening in which 687 genes coding for kinases and proteins related to kinases (such as pseudokinases and phosphatases) were targeted, we identified 52 genes required for HuH6 survival. The silencing of five of these genes selectively impaired the viability of HuH6 cells with high β-catenin signaling: HGS, STRADA, FES, BRAF and PKMYT1. Among these candidates, HGS depletion had the strongest inhibitory effect on cell growth and led to apoptosis specifically in HuH6 with high β-catenin activity, while HuH6 with low β-catenin activity were spared. In addition, HGS was identified as a potential synthetic lethal partner of oncogenic CTNNB1 in the HCT116 colorectal isogenic cell line pair.We first determined the experimental conditions that led to the efficient expression of shRNA against HGS. Importantly, HGS depletion specifically affected cells with uncontrolled β-catenin signaling activity in two different types of cancer , and thus may represent a new potential target for novel therapeutic strategies in liver and colorectal cancer.These results demonstrate the existence of crosstalk between β-catenin signaling and The online version of this article (doi:10.1186/s12885-015-2037-8) contains supplementary material, which is available to authorized users. BRCA and PARP1. Indeed, PARP1 inhibitors show promising activity in clinical trials of breast, ovarian and other cancers associated with BRCA mutations [Genetic alterations and modifications of the tumor environment often lead to the apparition of weaknesses specific to the tumor that could be exploited therapeutically . For insutations .nd and 4th leading causes of death by cancer worldwide in 2012, respectively (WHO). Indeed, more that 50 % of hepatoblastoma (HB) and a third of hepatocellular carcinoma (HCC) display aberrant activation of Wnt/β-catenin signaling caused by stabilizing mutations of β-catenin in the CTNNB1 gene [APC, which lead to the ectopic activation of Wnt/β-catenin signaling, are considered the major initiating event in colorectal cancer (CRC) [Deregulation of the Wnt/β-catenin pathway, which is a key developmental biology signaling pathway, is a major event in liver cancer and colorectal tumorigenesis , 5, whicNB1 gene , while mer (CRC) . Thus, ter (CRC) . Hence, CTNNB1 activating mutation. One of these genes (HGS) is an SL partner of oncogenic β-catenin in colorectal HCT116 cancer cells, suggesting that the LS interaction between HGS and CTNNB1 is not limited to liver cancer.Here, we used an siRNA kinome library to perform a high throughput (HT) SL screen of HuH6 isogenic HB cell lines, and we examined the effect of knockdown on cell proliferation, the frequency of mitotic events and induction of apoptosis. The depletion of transcripts of five genes by siRNA led to lethality only in a cellular context characterized by the aberrant activation of the Wnt/β − catenin signaling pathway due to a 2. Parental HuH6 cells were transfected with pTER-β-catenin plasmid using Lipofectamine 2000 (Life Technologies) to generate HuH6CTNNB1sh cells [Human hepatoblastoma HuH6 cells were grown in Dulbelcco’s modified Eagle’s medium  with 10 % fetal bovine serum and 100 U/ml penicillin/streptomycin. Colorectal carcinoma HCT116 cells were cultivated in McCoy’s medium, with 10 % fetal bovine serum, at 37 °C in 5 % COB1 cells . PositivCTNNB1sh were cultivated in the presence or absence of 2 μg/ml of doxycycline for 72 h and transfected with reporter plasmids using Lipofectamine2000 in triplicate in accordance with the manufacturer’s instructions. The pRL-TK plasmid  was co-transfected to control for transfection efficiency. Forty-eight hours after transfection, Luciferase activity was measured with the Dual-Luciferase reporter assay system (Promega).The TOPflash/FOPflash reporter plasmids  were used to determine β-catenin-induced TCF/LEF transcriptional activity. TOPflash is a reporter plasmid containing two sets of three copies of wild-type TCF binding sites driven by the thymidine kinase minimal promoter located upstream from a luciferase reporter gene. FOPflash contains mutated TCF binding sites and is used as a negative control for TOPflash activity. HUH6 and HUH6Total RNA was isolated with TriZol reagent according to the manufacturer’s instructions (Life Technologies). Reverse transcription was performed from 1 μg of total RNA with the Transcriptor First Strand cDNA Synthesis Kit  and random hexamer primers. PCR amplification was performed on the LightCycler 480 system with SYBRGreen PCR mix (Roche Diagnostic) and the following primers: HGS forward 5’- CTCCTGTTGGAGACAGATTGGG -3’ and HGS reverse 5’- GTGTGGGTTCTTGTCGTTGAC -3’, 18S forward 5’-GTAACCCGTTGAACCCCATT-3’ and 18S reverse 5’-CCATCCAATCGGTAGTAGCG-3’, CTNNB1 forward 5’- GCTTTCAGTTGAGCTGACCA-3’ and CTNNB1 reverse 5’-GCTTTCAGTTGAGCTGACCA-3’ or Axin2 forward 5’- TGTCTTAAAGGTCTTGAGGGTTGAC-3’ and Axin 2 reverse 5’- CAACAGATCATCCCATCCAACA-3’.After validating RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA was reverse transcribed with the Ovation PicoSL WTA System V2 . Briefly, the resulting double-stranded cDNA was used for amplification based on SPIA technology. After purification according to the manufacturer’s protocol, 2.5 μg of single-stranded DNA was fragmented and labeled with biotin using the Encore Biotin Module (NuGEN). Fragment size was verified with the Bioanalyzer 2100, cDNA was then hybridized to GeneChip® human Gene 1.0 ST (Affymetrix) at 45 °C for 17 h. After overnight hybridization, the chips were washed on the fluidic station FS450 according to the manufacturer’s protocol  and scanned with the GCS3000 7G. The image was then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for quality control. The evaluation of some of these metrics and the distribution of raw data showed no experimental outliers. RMA normalization was performed with R.4Cl and permeabilized first with a PBS solution containing 0.2 % BSA and 0.05 % saponin, and subsequently with 0.5 % Triton-X100. Cells were then incubated for 60 min with rabbit primary antibody anti-cleaved Caspase 3  and mouse anti-Ki67 . Next, cells were washed twice in PBS and incubated with Alexa Fluor 488- or Cy3 coupled secondary antibodies (Jackson ImmunoResearch). Nuclei were stained with 0.2 μg/ml DAPI. Images were acquired with an INCell2000 automated wide-field system  at 10x magnification . Images were analyzed with the INCell Analyzer workstation software  and the mean fluorescence intensity of nuclear Ki67 (a proliferation marker) and cleaved-caspase3 antigens was quantified in each cell. Three independent experiments were performed for each treatment.A kinome siRNA library of 2748 siRNAs targeting 687 genes (four siRNAs per gene) was obtained from Qiagen. We also included a negative siRNA control  and a positive lethal siRNA (anti-KIF11) on each plate. A total of 750 cells were seeded on a 384-well plate  in 40 μL of DMEM medium supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin. Cells were transfected the day after with 10 nM siRNA using INTERFERin, . Transfection efficiency was estimated by comparing 4,6-diamidino-2-phenylindole (DAPI) staining between cells transfected with negative control siGL2 and those transfected with siKIF11. Cells were fixed 72 h after transfection with 4 % (w/v) formaldehyde for 15 min and washed with phosphate buffered saline (PBS). Cells were next quenched with 0.05 M NHZ-scores were calculated for the comparison of each siRNA against the GL2 negative control population. A gene was identified as a ‘hit’, if the RZ-score for at least two of four siRNAs was > 2 or < -2 in at least two of three replicates. Selected hits were further reordered and validated with four siRNAs per gene (Qiagen), for HUH6 and HUH6CTNNB1sh cell lines treated or not treated with 2 μg/ml doxycycline.Positive hits for each gene were identified as follows. Data were first transformed with log or logit functions. B-score normalization was then applied to each replicate, separately, and included corrections for plates, rows and columns , 11. MedCTNNB1sh cells were cultured with 2 μg/ml of doxycycline for 48 h prior to siRNA transfection and were kept in the presence of doxycycline for the following 72 h. HCTT116, HuH6 and HuH6CTNNB1sh cells were plated at 0.1 × 106 cells per well on 6-well plates and transfected with siRNA as indicated. Seventy-two hours after transfection, cells were washed once in PBS and incubated in a crystal violet solution  for 10 min at room temperature. Plates were washed once with PBS and twice by immersion in water. Cells were then incubated in 1 % SDS solution until the dye was completely solubilized and absorbance was read at 570 nm.HuH6(3)) and 7.5 μM propidium iodide (PI), (Life Technologies). Cells were incubated for 20 min at 37 °C and samples were analysed using a BD LSR Fortessa flow cytometer .Cells were harvested and centrifuged at 200 g for 5 min and the pellet was resuspended in 500 μl of culture medium containing 200nM 3,3'-Dihexyloxacarbocyanine iodide  containing 1X of complete protease inhibitor cocktail (Roche Diagnostics) and centrifuged at 13,000 g for 10 min at 4 °C. Total proteins were resolved by SDS-PAGE, transferred to nitrocellulose and blocked with 5 % BSA. Blots were separately incubated overnight at 4 °C with specific primary antibodies, including 1:1000 anti-HGS/HRS , 1:5000 anti-β-catenin , 1:5000 anti-γ tubulin and 1:5000 anti-β-Actin (Sigma-Aldrich). After further washing and incubating with corresponding secondary antibodies, the blots were developed by enhanced chemiluminescence .CTNNB1 , and a cell line with a WT CTNNB1 gene (HCC HuH7 cells). HeLa and Human Embryonic Kidney 293 T cells (HEK293T) were used as negative controls. Only background activity was detected in HeLa and HEK293T cells, whereas HuH7 cells showed modest but significant β-catenin co-transcriptional activity, despite the WT status of β-catenin , whereas activity was 8- and 36-fold higher than background levels in HepG2 and HuH6 cells, respectively -regulated shRNA against CTNNB1 , to obtaRNA Fig. . Additio complex , 15) mRNB1 Table , confirmCTNNB1sh. Thus, parental Huh6 and HuH6CTNNB1sh (without Dox) were used as reference cells with a high β-catenin signaling activity and Dox-treated HuH6CTNNB1sh cells were used as reference cells with low β-catenin signaling activity.Surprisingly, the almost complete inhibition of β-catenin signaling activity did not appear to correlate with β-catenin protein levels (which were only 40 % lower in Dox-treated cells than in control cells). This may be explained by the dual role of β-catenin at the plasma membrane, where it has a structural function in cadherin‐based adherens junctions . Indeed,CTNNB1sh cells was associated with a substantial cell growth defect when cells were seeded at very low density  and PI co-staining. DiOC6(3) is a membrane-permeable lipophilic cationic fluorochrome that is used as a probe for mitochondrial transmembrane potential (Δψm). In living cells, DiOC6(3) accumulates in mitochondria and low DiOC6(3) staining reflects a collapse of Δψm. Dissipation of mitochondrial transmembrane potential is a sensitive marker of early apoptotic events. In addition, PI stains only cells whose plasma membrane integrity is compromised. Thus, cells that showed low staining for both DiOC6(3) (loss of Δψm) and PI (loss of plasma membrane integrity) were considered as apoptotic cells. Interestingly, we observed a significant increased in number of apoptotic cells (8.7 % Vs 17.3 %) only in Dox-treated HuH6shCTNNB1 when seeded at low density  ; therefoZ-scores for each siRNA. All siRNAs with a median Z-score >2 or < −2 were considered significant hits, and genes with at least two (out of four) ‘hit’ siRNAs were selected as candidate genes  and one with low β-catenin activity (HuH6CTNNB1sh + Dox cells). Proliferation index was slightly lower in HuH6CTNNB1sh + Dox cells than in the other three conditions and was strongly influenced by cell density , and for at least two siRNAs; and (ii) knockdown had no significant effect (|median robust Z-score| < 2) in cells with low β-catenin signaling (HuH6CTNNB1sh + Dox) for all phenotypes analyzed. This counter-screen identified five genes that are potential SL partners of oncogenic β-catenin (Table HGS (hepatocyte growth factor-regulated tyrosine kinase substrate), STRADA , FES (feline sarcoma oncogene), BRAF  and PKMYT1 .We then carried out an SL counter-screen with siRNA against the 52 survival kinases to identify kinases whose inhibition would affect the viability of HuH6 cells with oncogenic β-catenin activity, but not that of HuH6 cells with low β-catenin activity (sh-induced knock-down of β-catenin). A scheme of the counter-screen is illustrated in Fig. HGS, STRADA, FES, BRAF and PKMYT1 by RNAi experiments in HuH6CTNNB1sh  or in Dox-treated HuH6CTNNB1sh  and assessed cell survival by crystal violet staining /PI double negative cells measured by flow cytometry  and has one WT allele and one mutant allele with a 3-bp deletion that eliminates the serine residue at codon 45, leading to synthesis of mutant β-catenin which is constitutively active [CTNNB1WT/- cells with low β-catenin activity in which the CTNNB1del45 allele is invalidated [WT/del45 and HCT116WT/- cells with the most efficient siRNA against one of the five potential SL partners of oncogenic β-catenin. Depletion of STRADA, FES, BRAF and PKMYT1 affected cell survival in both HCT116WT/del45 and HCT116WT/-, whereas HGS knockdown significantly impaired cell survival only in HCT116WT/del45  , suggesting that HGS expression is required for HCT116WT/del45 survival . However, HGS protein levels were not significantly different between HCT116WT/del45 and HCT116WT/- cells  is an essential protein that belongs to multi-vesicular bodies (MVB)/Lysosome complex, which is required to sort membrane proteins and direct them toward either degradation in lysosomes or recycling at the plasma membrane [HGS in fibroblasts led to a defect in E-cadherin degradation and accumulation of E-cadherin at the plasma membrane, which in turn trapped β-catenin at the membrane, preventing its translocation to the nucleus. In addition, HGS depletion attenuates the cell growth, tumorigenesis and metastatic potential of Hela cells [HGS depletion in HuH6 cells leads to the accumulation of E-cadherin at the plasma membrane, which may trap the stabilized form of β-catenin, thus impairing signaling through this pathway and attenuating its oncogenic effect. However, HGS is also a key regulator of the molecular machinery involved in receptor sorting and recycling; therefore, HGS depletion may affect several signaling pathways. Hence, the exact nature of the SL relationship between oncogenic CTNNB1 and HGS requires further investigation.membrane , 21. Intla cells . TherefoHGS as a potential SL partner of oncogenic CTNNB1. To ensure high confidence in the hits selected with our approach, we measured three fitness variables  [HGS and oncogenic CTNNB1 in HuH6 and HCT116 cells; nonetheless, further investigation is required to validate HGS as a new therapeutic target for liver cancers with CTNNB1 mutations.In conclusion, we performed a siRNA screen that led to the identification of  events)  in two dThe data sets supporting the results of this article are included within the article and its additional files.


Discosoma sp. red fluorescent protein (DsRed)-tagged cadherin cytoplasmic domain in cells inhibited the cell surface localization of endogenous E-cadherin, leading to morphological changes, the inhibition of junctional assembly and cell dissociation. These changes were associated with increased cell migration, but were not accompanied by the down-regulation of epithelial markers and up-regulation of mesenchymal markers. Thus, these changes cannot be classified as EMT. The cadherin cytoplasmic domain interacted with β-catenin or plakoglobin, reducing the levels of β-catenin or plakoglobin associated with E-cadherin, and raising the possibility that β-catenin and plakoglobin sequestration by these constructs induced E-cadherin intracellular localization. Accordingly, a cytoplasmic domain construct bearing mutations that weakened the interactions with β-catenin or plakoglobin did not impair junction formation and adhesion, indicating that the interaction with β-catenin or plakoglobin was essential to the potential of the constructs. E-cadherin–α-catenin chimeras that did not require β-catenin or plakoglobin for their cell surface transport restored cell–cell adhesion and junction formation.The downregulation of E-cadherin function has fundamental consequences with respect to cancer progression, and occurs as part of the epithelial–mesenchymal transition (EMT). In this study, we show that the expression of the  E-Cadherin, a prototypical member of this family, is a transmembrane protein that forms the adherens junction between epithelial cells. The cytoplasmic domain of E-cadherin interacts directly with β-catenin or plakoglobin. α-Catenin interacts with the cadherins indirectly via interactions with β-catenin or plakoglobin, and links the cadherin–catenin complex to the actin cytoskeleton through interactions with α-actinin, vinculin, formin, EPLIN , and actin filaments Cadherins comprise a large family of CaThe loss of epithelial characteristics and the gain of a mesenchymal phenotype–a process referred to as the epithelial-to-mesenchymal transition (EMT)–is considered to be a hallmark of neoplastic transformation. A key initial step in EMT is the downregulation of E-cadherin, which at the transcriptional level is repressed by several factors: namely, ZEB1, ZEB2, Snail, Slug, and Twist In vitro studies using function-perturbing antibodies have indicated that E-cadherin-mediated adhesion is a necessary prerequisite for the formation of other cell junctions, including desmosomes and tight junctions in vivo study employing the conditional inactivation of E-cadherin in stratifying epithelia showed that E-cadherin is required for tight junction, but not desmosome, formation in vivo. Since a single cell type can express several different cadherins, the specific knockdown of E-cadherin may be accompanied by the increased expression of other cadherins, which compensate for the loss of E-cadherin Several cadherin functions have been elucidated using dominant-negative mutants. Pioneering work by Kintner It was recently shown that the expression of the dominant-negative protein induces EMT Discosoma sp. red fluorescent protein (DsRed)-tagged cadherin cytoplasmic domain in MDCK cells inhibited the cell surface localization of endogenous E-cadherin, leading to morphological changes, the inhibition of assembly of desmosome and tight junction components, and a reduction in the mechanical integrity of the epithelial cell sheets. Thus, contrary to previous reports that the soluble cadherin cytoplasmic domains do not affect cadherin function, we showed that the cytoplasmic constructs exhibited dominant-negative activities. The observed morphological changes were not accompanied by the down-regulation of epithelial markers and the up-regulation of mesenchymal markers. Thus, these changes could not be classified as EMT. The constructs associated with β-catenin and plakoglobin, and reduced the level of β-catenin or plakoglobin associated with endogenous E-cadherin, raising the possibility that sequestration of β-catenin and plakoglobin by the constructs induced the intracellular localization of E-cadherin. The introduction of E-cadherin–α-catenin chimeras that did not require β-catenin or plakoglobin for their cell surface transport restored cell–cell adhesion and junction formation.Here, we show that the expression of a Experiments with recombinant DNA technology were performed in agreement with the guidelines of Kagoshima University Committee on recombinant DNA security.CCTCGAGGGAGAACGGTGGTCAAAGA and ATCGTCGTCCTCGCCACCG or CCTCGAGGCCGGGATAAAGAACG and ATCGTCATCACCTCCACCATACA, respectively. The products were digested with Xho I and cloned into the Xho I and EcoR V sites of the pC-DsRedFLAG vector GAGTTTATCGATGCTTCCCGC and ATCAATGCTGTCCATAGCTTTGAA or CCATCGATGTGGAAGATGGTATCTTGAA and ATCCTGTCTACGCAAGTCCC, respectively. The PCR products were digested with Cla I, and then cloned into the Cla I and EcoR V site of the pC-ELAHA vector, yielding pC-ELAαC and pCELAαM. An expression vector for DNCT under the control of the Tet-repressible transactivator (pU-DNCT) was constructed as follows: cDNA encoding DsRed, the N-cadherin cytoplasmic domain, and the FLAG tag in a pC-DNCT vector was amplified by PCR using the following primer pairs CCGGTCGCCACCATGGACAA and GCTCTAGACGCCCTTGTCGTC, digested with Xba I, and cloned into the EcoR I and Xba I site of the pUHD10-3 vector The mammalian expression vectors containing hemagglutinin (HA)-tagged E-cadherin cDNA encoding either the wild-type (pC-EcadHA), or modified proteins , or HA-tagged N-cadherin (pC-NcadHA) were previously described Cell culture conditions for the Type I Madin-Darby canine kidney (MDCK) cells have been described The following monoclonal antibodies were used to detect E-cadherin: DECMA-1, raised against the extracellular domain of E-cadherin (provided by Rolf Kemler of the Max-Planck Institute for Immunobiology); ECCD-2, recognizing the distinct extracellular domain of E-cadherin ; and C20820, a mAb detecting the cytoplasmic domain of E-cadherin . A rat mAb against HA (3F10) was purchased from Roche Molecular Biochemicals . A mouse mAb against FLAG (DYKDDDDK) was purchased from Wako Pure Chemical Industries, Ltd. . Mouse mAbs recognizing N-cadherin, β-catenin, plakoglobin, and p120 were purchased from BD Biosciences, and a mAb detecting vinculin was obtained from Sigma-Aldrich Japan . A mouse mAb against vimentin, and rabbit antibodies targeting ZO-1, claudin 1, and occludin were purchased from Zymed Laboratories . A mouse mAb against desmoplakin was purchased from Progen Biotechnik GmbH . All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. .6) were lysed in a buffer . The proteins were collected with mAbs that had been preabsorbed to protein G–Sepharose.Immunoprecipitation and immunoblot analyses were carried out as described Immunofluorescence labeling of cells was performed as described Cells were washed with PBS and then incubated for 2 h in DMEM supplemented with 10% FCS containing 2.4 U/ml of dispase (Gibco). Detached cells were subjected to mechanical stress by pipetting with a 1 ml pipette. The extent of cell dissociation was represented by the index Np/Nc, where Np and Nc are the total numbers of particles and cells per dish, respectively. To obtain Nc, the detached cells were incubated for 10 min in the presence of 5 mM EGTA, and then subjected to the mechanical stress.Cells were plated on 35 mm dishes and grown to confluency. Then, the cell monolayer was manually scratched with a pipette tip, washed with PBS, and incubated for 24 h. A phase contrast microscope was used to photograph the cells at 0 h and 24 h after performing the scratch.4 cells in serum-free DME medium. After incubation for 22 h at 37°C, the cells that penetrated through the Matrigel to the lower surface of the filters were stained, and then quantified by microscopy.Invasion was measured using BioCoat MatriGel Invasion Chambers (BD Biosciences) according to the manufacturer’s instructions. The lower chambers were filled with DME medium containing 5% FBS as a chemoattractant. The upper insert chambers were seeded with 2.5×10Total RNA was prepared from DsRed+, DECT+, Snail+ MDCK cells using TRIzol Reagent (Invitrogen) and purified using SV Total RNA Isolation System (Promega) according to the manufacturer’s instructions. cRNA was amplified and labelled using a Quick Amp Labelling Kit (Agilent Technologies) and hybridized to a 44 K Agilent 60-mer oligomicroarray  according to the manufacturer’s instructions. The hybridized microarray slides were scanned using an Agilent scanner. The relative hybridization intensities and background hybridization values were calculated using Agilent Feature Extraction Software (version 9.5.1.1). Microarray data analysis was supported by Cell Innovator .We made the chimeric construct (DECT) comprising a fluorescent protein, DsRed, the cytoplasmic domain of E-cadherin (ECT), and a C-terminal FLAG tag , and expThe disruption of epithelial sheets containing DECT+ cells was confirmed by cell dissociation assays. DECT+ cells, but not DsRed cells, were dissociated into single cells after detachment from the dishes by dispase and subsequent mechanical dissociation by pipetting . Thus, DThe acquisition of these fibroblastic characteristics by the DECT+ cells suggested that the cells had undergone EMT. To determine whether the molecular alterations associated with EMT had occurred upon expression of DECT, we assessed the status of EMT markers in DECT+ cells using Snail+ cells as a positive control. Upon Snail-induced EMT, the expression of mesenchymal proteins, e.g., fibronectin, N-cadherin, and vimentin, was markedly upregulated . By cont2+, but not in the absence of Ca2+. Cadherins localized in the intracellular compartments resists tryptic digestion even in the absence of Ca2+2+ (TC) or 1 mM EGTA (TE), followed by immunoblot analysis with an anti-E-cadherin mAb has been used to quantify E-cadherin on the cell surface or inside the cells Immunofluorescence staining using the E-cadherin monoclonal antibody, DECMA-1, which recognizes the extracellular part of the protein, revealed that endogenous E-cadherin did not exist at the surface of DECT+ cells . This waImmunofluorescence staining of DECT+ cells revealed that β-catenin did not exist on the cell surface membrane, but rather that it co-localized with DECT in intracellular compartments . AlthougAlanine substitution of the conserved eight serine residues in the catenin-binding site of E-cadherin has been shown to weaken the interaction with catenins Stable transfectants expressing these proteins were isolated and confirmed by immunoblot analysis . Like DEPrevious experiments using E-cadherin deletion proteins revealed that the C-terminal half of the cytoplasmic domain is enough for binding of catenins To show that the disruption of cell–cell junction biogenesis was due to a depletion in β-catenin and plakoglobin, which is required for the correct localization of E-cadherin, we performed rescue experiments using E-cadherin–α-catenin chimeric molecules . The chiExpression vectors for these constructs, ELA, ELAαM, and ELAαC, were introduced into DECT+ cells, established by selection with hygromycin, and stable double transfectants were isolated after selection with G418, followed by immunostaining and immunoblotting with anti-HA . ELA expThe easy mechanical dissociation of DECT+ cells suggested that the expression of DECT affected the assembly of adherens junctions and other junctional complexes, e.g., desmosomes and tight junctions. Consistent with this idea, the expression of DECT in MDCK cells induced the intracellular localization of desmoplakin, a desmoglein-associated desmosomal protein, and ZO-1, a tight junction component .The expression of ELAαM and ELAαC chimeras in DECT+ cells restored the mechanical integrity of cell sheets because cells expressing these proteins were resistant to mechanical dissociation . ConsistTo explore the reversibility of the cadherin cytoplasmic domain activity, we expressed DNCT under the control of the Tet-repressible transactivator. In the presence of either Tet or Dox, the expression of exogenous protein is repressed 2+. Therefore, the Ca2+-dependency of the sheet integrity was assessed as follows. Cells were first detached from the dishes by dispase in the presence of Ca2+. Then, the detached cells were incubated in the presence of EGTA for an additional 10 min, and subjected to mechanical dissociation. When the detached cells were incubated with EGTA to remove Ca2+ before mechanical dissociation, the cells were completely dissociated  in MDCK cells resulted in partial disruption of adherens junctions and the accumulation of β-catenin in the cytoplasm and the nuclei. Expression of the shorter (30 amino acid) cadherin tail fragment, which effectively inhibited β-catenin-mediated signaling, in MDCK cells resulted in the accumulation and diffuse distribution of β-catenin but without a detectable effect on its organization in adherens junctions. These authors, however, did not examine the interaction of the fragments with plakoglobin. As shown in the present study, the shorter E-cadherin cytoplasmic domain (DECTC) having β-catenin-binding ability showed weaker binding to plakoglobin than the entire cytoplasmic domain and loses the ability to disrupt junctions. Another E-cadherin cytoplasmic domain chimera fused to βTrCP ubiquitin–protein ligase was expressed in DLD1 cells Since the amino acid substitutions introduced into the cytoplasmic domain of E-cadherin weakened its interaction with β-catenin and plakoglobin and abrogated its activities as a dominant-negative protein, the amount of the cytoplasmic domain produced may be critical to the experimental outcome. In the previous experiments, 1) the cytoplasmic domains of E-cadherin or N-cadherin It has been shown that the expression of a dominant-negative mutant of E-cadherin  in A431 cells caused EMT The cell–cell adhesion molecule E-cadherin is localized to the basolateral membrane of polarized epithelial cells. Little is known, however, about mechanisms regulating the intracellular trafficking of E-cadherin The defects in the cell junctions of DECT+ cells were rescued by the expression of E-cadherin–α-catenin chimeras (ELAαM and ELAαC). Immunofluorescence staining of the rescued cells revealed that the tight junction and desmosomal components were properly assembled. Furthermore, the cell sheets resisted the mechanical force inflicted in the dissociation assay. We do not know how these chimeric proteins rescued the junctional assembly defect in DECT+ cells. Although ELAαM and ELAαC could not interact with β-catenin and plakoglobin, they were efficiently transported to the sites of cell–cell contact. α-Catenin is a protein with multiple domains that interacts with a number of proteins, including actin and actin-binding proteins–actin (α-catenin residues 685–883) Previous studies have suggested that the actin cytoskeleton is involved in desmosomal and tight junction assembly, which are sensitive to various actin-disrupting drugs, e.g., cytochalasin D Figure S1Confocal imaging of MDCK cells expressing DsRed or DECT. Cells were stained with anti-β-catenin (A) or anti-plakoglobin (B) antibodies together with DAPI to visualize nuclei. Although a significant part of DECT was detected in cytoplasm together with β-catenin or plakoglobin, a small amount of these proteins was present in the nucleus. In contrast, a significant part of RsRed was detected in nucleus. Bars, 25 µm.(TIF)Click here for additional data file.