For decades, the emergence and progression of infant reaching was assumed to be largely under the control of vision. More recently, however, the guiding role of vision in the emergence of reaching has been downplayed. Studies found that young infants can reach in the dark without seeing their hand and that corrections in infants' initial hand trajectories are not the result of visual guidance of the hand, but rather the product of poor movement speed calibration to the goal. As a result, it has been proposed that learning to reach is an embodied process requiring infants to explore proprioceptively different movement solutions, before they can accurately map their actions onto the intended goal. Such an account, however, could still assume a preponderant (or prospective) role of vision, where the movement is being monitored with the scope of approximating a future goal-location defined visually. At reach onset, it is unknown if infants map their action onto their vision, vision onto their action, or both. To examine how infants learn to map the feel of their hand with the sight of the object, we tracked the object-directed looking behavior (via eye-tracking) of three infants followed weekly over an 11-week period throughout the transition to reaching. We also examined where they contacted the object. We find that with some objects, infants do not learn to align their reach to where they look, but rather learn to align their look to where they reach. We propose that the emergence of reaching is the product of a deeply embodied process, in which infants first learn how to direct their movement in space using proprioceptive and haptic feedback from self-produced movement contingencies with the environment. As they do so, they learn to map visual attention onto these bodily centered experiences, not the reverse. We suggest that this early visuo-motor mapping is critical for the formation of visually-elicited, prospective movement control. Reaching for objects is a fundamental skill that emerges in infancy around 3–5 months of age. Understanding how this skill forms and develops has been a core area of study in developmental psychology since the 1930s  and reaching in three infants that we followed weekly throughout the transition to reaching, to examine how the above scenarios play out. We attempt to gain insights on the process underlying the formation of visuo-motor mapping at reach onset (1) by identifying whether looking patterns at the target objects prior to the onset of reaching can help predict the formation of early goal-directed movement, (2) by tracking whether these looking patterns at the object change in the weeks following reach onset, and (3) by examining if there is some spatial correspondence between the history of looking patterns at the object and the history of point of hand-object contact after reach onset that could support one of the suggested scenarios. As we will show, these preliminary data further extend previous embodied accounts of infants learning to reach. They suggest the possibility that mapping the feel of the hand with the sight of the object occurs by learning to align visual attention to the point of first hand-object contact, and not the reverse, as previously thought. We discuss the implication of these findings for the development of prospective control from an embodied perspective.The goal of this paper is to examine anew the role of vision in relation to the emergence of goal-directed reaching in infancy, particularly in light of the more recent embodied accounts on learning to reach. We ask how do infants figure out how to map the feel of their arm to a specific location identified visually if infants' first reaching attempts are mainly controlled proprioceptively? Does vision provide any specific information in this process Traditional accounts on the development of infant reaching greatly emphasized the role of vision in the process of guiding the hand toward the target. Piaget  was the White et al.  describeSubsequent studies recorded the kinematics of infants' reaching trajectories. They found that infants' early reaching trajectories were poorly controlled and contained many corrections and changes in direction before the hand attained the target  and bringing the hand in that specific location come together in the first place?The embodied accounts reviewed above have somewhat downplayed the critical role of vision for learning to reach despite abundant evidence indicating that visual input matters for reaching. As mentioned earlier, when infants are approaching reach onset, they fixate the target object intensely . This scenario assumes that vision dictates where the hand needs to go . This would imply that infants learn to map the feel of their arm onto the sight of the object by learning to control their arm increasingly accurately to bring it where visual attention on the object is directed. Predictions from this scenario would imply that a visual selection process or particular pattern of fixations at the object could possibly form and become more observable in the weeks preceding reach onset. At reach onset and in subsequent weeks, as infants increase proprioceptive control of their arm, they would gradually figure out how to better align the transport of their hand to the vicinity of the object area visually attended. The developmental origins for such a visuo-motor mapping process can be found in the initial eye-hand coordination and pre-reaching skills of newborns . This scenario would assume that infants can take advantage of their proprioceptive experience prior to reach onset to discover how to direct their arm to a specific location. Vision would become calibrated to the action as the result of accidental events. Early in life, infants move their arms around in seemingly unintentional ways. Doing so, they can hit objects inadvertently, without seeing them, but receive immediate haptic and proprioceptive feedback about the posture and location of their arms and hands in space. Such accidental events could eventually cause the infants to direct their regard toward the location where hand-object contact occurred. Hence, vision could begin to be associated to the arm movement experience of the child as a consequence of the haptic and proprioceptive feedback received. After many repetitions of such events, a basic form of movement intentionality could emerge when the feel of an arm movement in a given direction would happen to match the sight of an object in that same direction, but the experience would still be proprioceptively-guided, not visually-elicited. As vision would continue to map onto these emergent, successful, proprioceptively controlled goal-directed movements, a more accurate selective or prospective role of vision could develop, mainly as a consequence of connecting what is seen to what is felt, not the reverse. Predictions from this scenario would entail that infants can demonstrate a relatively accurate sense of their arm projection in space at reach onset. We would expect little or no specific pre-selective looking patterns at the object in the weeks preceding the emergence of reaching. Over time, we should see increased visual attention directed toward where the hand is reaching as a result of infants discovering how to align vision to their actions and beginning to anticipate where to bring their arm in space. This sensory-motor scenario can be rooted in infants' early sense of their own body and movements . This third scenario would correspond to a mix of the two described above and would not assume any dominance of vision over proprioception (scenario 1), or proprioception over vision (scenario 2), but would rather cast the emergence of reaching as the product of a continuous process where both prospective vision and proprioceptive feel of the arm experienced in the month before reach onset become progressively integrated. Predictions should show that both sight of the object and feel of the arm are increasingly mapped onto each other, but are not related to particular visual looking trends prior to reaching onset, nor to any movement tendency after reach onset.To examine the plausibility of these scenarios, we documented the looking (captured via eye-tracking) and reaching patterns of three infants that we began to see from the age of 2–2.5 months old (that is prior to the emergence of reaching). We followed them until they were 12 months of age, but for the purpose of this report we focus only on an 11-week period around the transition to reaching. Each week, infants were presented with 3D objects that they could visually scrutinize for up to 5 s before they would be allowed to reach for them. Infants were presented with five kinds of objects. Here we describe in detail the results related to a drumstick-shaped object (a sphere attached to the end of a rod) and contrast them with those of a plain rod with no distinct features. For each week, we report how looking patterns were distributed on the objects. When infants began to reach, we documented where they brought their hand to make the first contact with the object and related it to the looking patterns. We also compared their performance to a group of 9-month-old infants tested in the same conditions. Because 9-month-olds have more reaching experience and demonstrate decent prospective control in reaching  were 9 months old ±1 week) at the time of testing. They were seen only once and their data were used in this report to provide a developmental reference norm. The other three infants  were followed longitudinally from about 2 months of age, and up to the end of their first year of life. This report presents the 11-week period around the transition to reaching . Table  week at Testing sessions were completed in a well-lit room. A custom-designed infant seat reclined 10 degrees from vertical was used for infant seating. It provided full trunk support via a 15-cm-wide padded foam strap wrapped around the infants' torso and allowed free-range arm and leg movements. A small pillow was used for the head. Before infants could support the weight of their own heads, infants were seated in their caregivers' lap. When transitioned to the infant seat, caregivers sat nearby in another chair. Both MC and ME had already transitioned to the infant seat for collection of the data reported. AC transitioned to the seat at week 19, thus was the only infant who provided data while on her mother's lap.To minimize ambient distractions, a custom-designed, black, tri-fold, wooden theater was positioned directly in front of the infants  was located at the bottom of the presentation window, directly under the flat-screen monitor to capture infants' eye movements during calibration and object presentations. The eye-tracker was positioned at a 60 cm distance from the infants' eyes and its angle was adjusted to accommodate the height of the infants' eyes . The eye-tracker, operated through Tobii software (Studio v. 2.0.8), used an infrared light source on the cornea relative to the center of the pupil. Estimated directions of visual fixation and saccade gaze were recorded at a rate of 50 Hz and then were superimposed onto a live video recording of the infants' visual scene, which was captured by a digital camera located directly behind the infant.Reaching behavior was recorded with three cameras. A small, black webcam facing toward the infant and secured on top of the presentation opening recorded the infants' faces, arms, and hands. This webcam view was merged and saved with the live scene recording containing the infants' looking behaviors. Two additional video cameras were situated on the right and left sides of the infants. They were connected to a Digital Video Switcher , which merged the left and right side camera views into one split-screen arrangement and then recorded with an added image frame counter  on a VCR. All camera views,  were synchronized to each other using a small custom-made diodes system  playing on the flat-screen monitor positioned in the theater window. When the infant's attention focused on the monitor, the angle of the eye-tracker and the distance between the infant eyes and the eye-tracker were adjusted. Once the capture of the infant's pupils displayed a clear and stable signal, eye calibration using five points began. Calibration points were located at the four corners and center of the monitor. Colorful pictures of objects moving and sounding in concert were displayed consecutively in each of the five areas until the infant had looked at each location for 3–5 s. If any calibration points were missing or inaccurate for either eye, those points were repeated until eye calibration was accurate on at least four out of five points for both eyes. Occasionally, three points were used. When sounds and pictures on the monitor were not sufficient at holding infants' attention to the calibration areas, the experimenter shook small rattles in front of the target areas. Calibration typically lasted between 3 and 10 min.While seated, infants were shown a Sesame Street video . Coding was performed by trained independent observers who identified the onset/offset of fixation points according to predefined regions or areas of interest on the objects and also coded the point of first hand/object contact according to these same predefined object areas , ME = 5.076 (SD = 1.80), and AC = 8.21 (SD = 3.09)]. However, looking behavior was not solely directed at the object, it could be directed at the experimenter's hand holding the object or at the surrounding scene, and, in some trials, infants never looked at the object. We eliminated trials with no or minimal looking at the object, which constituted 13.25% of our data sample, and did not consider looking times that were not directed at the object . Our final data samples and average looking durations at the objects for the longitudinal infants over the 11 weeks used in this report corresponded to: MC = 209 trials, object-directed average looking time = 2.51 s (SD = 1.45), ME = 105 trials, object-directed average looking time = 2.42 s (SD = 1.53), and AC = 145 trials, object-directed average looking time = 2.76 s (SD = 1.57). The drumsticks and rods used for this report constituted 47% of this overall sample. ME and AC produced less object-directed useable data for some weeks preceding reaching onset, which resulted in missing data for those weeks , we report significance at the 0.05 level, but also Figure p-values of the statistical analyses performed on these longitudinal data following the strategy outlined above are presented in Tables pre-reaching looking period, both MC and AC divided their object-directed visual attention mainly between the sphere and the middle of the rod. Their amounts of looking at those two areas were significantly greater than at the end of the rod. No test was ran on ME's pre-reaching looking period due to only 2 weeks of useable data up to reach onset. AC's p-values for that period were nearing significance. For the post-reaching looking period, visual attention to the drumstick was still mainly directed toward the sphere area, middle rod, or both depending on the child. MC's and AC's looking patterns were still mainly distributed between sphere and middle rod, while ME's visual attention was mainly directed to the sphere. Wilcoxon tests performed on the reaching patterns indicated a significant bias toward more frequent first touches at the sphere area. All three babies directed their hand and made first contact more frequently with the sphere than the middle rod (significant trend), and end rod (nearing trend). There were no differences in frequency of first touches between middle and end rod areas.The looking behavior assessed with linear curve estimations (Table p < 0.0001). The linear curve estimations performed on the reaching data did not reveal consistent significant developmental trends across babies, except for AC who increased her object contacts at the middle. For all three babies, the predominant tendency to touch the sphere more frequently remained about the same over the 6 weeks post-reaching period. The 9-month-old infants also displayed significantly more first touches at the sphere .Developmental trends in ns Table  only revlooking behavior, the developmental trends described above were confirmed. The Mann–Whitney tests revealed significant differences between longitudinal and 9-month-old infants for looking at the middle and the sphere areas of the drumstick in the weeks preceding and just following reach onset . However, those group differences were no longer significant for the last 3 weeks post-reaching . There were no significant differences between groups for looking at the rod end of the drumstick . Thus, the longitudinal infants looking patterns at the drumstick, initially different from those of the 9-month-old infants prior to and right around reach onset, became increasingly more similar to those of the 9-month-olds by week 4–6 after reach onset. For reaching behavior, there were no significant differences between groups , verifying that distribution of the point of first hand/object contact of the longitudinal infants did not differ from those of the 9-month-old group.To compare the looking and reaching trends of the longitudinal infants with those of the 9-month-old group, we collapsed the 9-week period (3 weeks before and 6 weeks after reach onset) into three 3-week periods' averages corresponding to: prior to reach onset for looking only (we used week 20 for ME), right after reach onset, and the last 3 weeks post-reaching for looking and reaching. For In sum, all three longitudinal infants looked and reached at the drumstick differentially over the 11-week period, however, developmental change over time was only observed in relation to the looking pattern performed after reach onset. Infants increased their visual attention toward the sphere, and in a 6-week span, approximated the distributional looking pattern displayed by 9-month-old infants. Interestingly, for reaching, more frequent first contacts at the sphere were present from the week of reach onset. This reaching trend was maintained over the 6 weeks of reaching and was similar to the distribution of points of contacts displayed by the 9-month-old group.Figure p-values of the statistical analyses performed on these longitudinal data are presented in Tables The corresponding Likewise, Table p = 0.891). Reaching, in that older age group was biased toward the top/left rod area . A similar trend can be seen in the longitudinal infants, although it is present only for week 5 post-reaching. None of the group comparisons between longitudinal and 9-month-old infants revealed significant difference in looking and reaching behavior for any of the object areas and any of the collapsed 3-week periods.In sum, compared to the drumstick that yielded looking and reaching patterns that seemed to gravitate predominantly toward the sphere in all three infants, the plain rod seemed to entice more random trends. Note that for the 9-month-old infants, looking patterns on the rod were also more distributed across all three object pre-defined areas  and the location where the hand made the first contact with the object (area of first touch). The number of trials corresponding to a direct spatial match between the most looked area and the area of first hand contact were normalized as a function of the total number of trials collected for a given object. These data are reported in Figure p < 0.048). It neared significance for MC (p = 0.055) and was not significant for ME (p = 0.209). For the plain rod, there was no significant developmental trend observed. Mann–Whitney tests comparing the 3-week averages of the longitudinal infants with the data of 9 months old group revealed a nearing group difference (p = 0.065) for the first 3 weeks following reach onset in the drumstick. All other comparisons (last 3 weeks for drumstick and plain rod group comparisons) were not significant .We performed a linear curve estimation on these data to assess changes in the rate of spatial look-reach match over time. For the drumstick, the rate of matching between looking and reaching revealed a 2 to 3-fold increase over the observed 6-week period, but reached significance only for AC  and point of first hand-object contact in three infants that we followed weekly over an 11-week period throughout the transition to reaching. With these data, we evaluated three possible scenarios that offered different levels of balance between the respective roles that vision and proprioception could have at the emergence of infant's reaching. The prospective control hypothesis assumed a more predominant role of vision over proprioception, the embodied hypothesis assumed a more predominant role of proprioception or sensory-motor experience over vision, and finally, a more mutually balanced contribution of both vision and proprioception acting in concert was considered as a third possible hypothesis. For each hypothesis, we made specific predictions. Here we discuss our findings against these predictions.Scenario 1 (or the prospective control hypothesis). This scenario assumed that vision would develop its prospective control role prior to reach onset. Reaching would form as a result of the infants increasingly figuring out how to proprioceptively guide their hand toward the area where visual attention is directed. Predictions consistent with this scenario were that (1) vision would reveal specific selective looking trends at the objects in the weeks preceding reach onset, (2) that after reach onset the looking trends would persist, and (3) that change over time would occur in the reaching behavior as a result of the more successful spatial alignment (or mapping) of the action endpoint onto the visually selected object area. For the drumstick, we observed a looking bias prior to reach onset directed toward two object areas—the sphere and the middle rod areas. This looking bias grew more specific in the direction of the sphere after reach onset, while all three infants maintained a relatively steady reaching bias at the sphere from reach onset and thereafter. Thus, for this object, a developmental change was observed in the looking behavior following reach onset but not in the reaching behavior per se, which is inconsistent with this scenario's predictions. Similar trends were not observed for the plain rod. In fact, very few significant results were reported for this plain rod object, suggesting that object shape may have interacted with this perceptual-motor mapping process, a point discussed further below, also in relation to the other objects that we did not present.Scenario 2 (or the embodied account hypothesis). This scenario assumed that infants build an extended, proprioceptive based sense of where to move their arm in space through initially undirected, nonetheless active movement experiences prior to reach onset. As infants happen to touch objects in their surroundings, at first accidentally (and without looking), they discover how to connect their visual attention to the consequential proprioceptive and haptic feel of their arm and hand in the environment. This would allow the formation of some kind of proprioceptive knowledge of how to direct arm movements in space by associating spatial vision to movement perception, leading to reach onset. The predictions we made in relation to this scenario were that infants (1) would not necessarily show specific object-related visual trends prior to reach onset, but (2) would demonstrate a more accurate/consistent spatial aiming ability in their reaching movement from reach onset, as a result of their acquired proprioceptive spatial movement experience in the weeks preceding reach onset. We also expected a change in visual attention toward the touched area (rather than a change in movement aiming toward the looked area) after reach onset as a product of successful reaching. The drumstick results were in line with these predictions. All three infants displayed a greater first touch trend at the sphere from reach onset. They maintained this aiming trend over the post-reaching weeks. Developmental change occurred in the looking pattern after reach onset. Over the post-reaching weeks, visual attention increasingly shifted toward the sphere, the preferred first touch area. Again, similar trends were not observed for the plain rod.Scenario 3 (co-mapping of sight and feel). This scenario assumed that both vision and action would map onto each other following reach onset. With this scenario, we did not predict a dominant trend in looking prior to reaching nor did we predict a dominant trend in reaching from reach onset. Rather, we expected initial random looking and reaching patterns that would reciprocally map onto one another over time. Both vision and reaching would adjust and morph into a more precise perceptual-motor mapping over time. Infants would increasingly try to look to where they aim, while trying to aim to where they look at the same time. None of our data revealed concomitant changes in both looking and reaching over the observed developmental period to support this third scenario.The looking and reaching behaviors documented through the transition of reaching in those three infants seem to point toward the embodied hypothesis, but as noted, this was only for the drumstick-shaped object; these findings did not extend to the plain rod object. Here, we discuss these results in relation to the other objects that we have not presented and evaluate the significance and limitations of these preliminary findings for our understanding of visuo-motor mapping in infants learning to reach.We found the trend reported for the drumstick at first provocative. The long held belief in the infant reaching literature (even from prior embodied accounts) has been that infants have poor control of their arm at reach onset, hence the documented indirect trajectories , the looking and reaching responses were more skewed toward the sphere(s) or cup bowl. Skewed responses appeared stronger when the bigger part of the object was one, as in the drumsticks or cups. For the cups, for instance, looking and reaching were heavily directed at the bowl of the cup, not the handle(s). This trend for the cups was also present in the 9-month- old group (there were no significant differences between groups). Shape features also seemed to engender more developmental changes (as we saw for the drumstick). For example, for the dumbbell-shaped object, two out of the three infants (MC and ME) displayed growing visual attention toward the two spheres located at each end of the rod in the post-reaching weeks compared to the pre-reaching weeks. This developmental change was not as strong as the one reported for the drumstick shaped object due to the fact that, for the dumbbell, visual attention was being increasingly split between two sphere locations (instead of one as in the drumstick). For MC, the sphere/middle rod/sphere looking distributions for the dumbbell went from 24/76/0 on week −5 to 48/13/39% on week 5. For ME, the pattern distribution was 66/34/00 at week −1 and was 45/0/55% at week 5. The looking distributions at week 5 for the dumbbell were not significantly different from those of the 9-month-old group (41/18/41%). Reaching, on the other hand, was already directed toward one of the two spheres more frequently from reach onset . Finally, as a result of two visually attended areas, but only one touched area, the rate of look-reach match for the dumbbell was not showing as a consistent progression over time as reported for the drumstick with one sphere. But this low rate of look-reach match was not void of trends. We reported that for the drumstick, when matches between looking and reaching occurred, they occurred in great majority at the sphere location. For the plain rods, even though there was no strong, consistent progression between looking and reaching matches, when matches occurred, they happened at the middle of the rod area. The same area of match consistency was found for the dumbbell and cup objects. For the dumbbell, when look and reach spatially matched, they occurred 80% of the time on one of the two spheres (only 20% of the matches were performed in the middle rod area), and for the cups, 94% of the look-reach matches occurred at the bowl area . Thus, from all of the above, it appearss that object shape drove infants' reaching responses and visual attention differentially, otherwise we would not have obtained such response trends and regularities within and across objects. Furthermore, a steady reaching trend from reach onset was observed for nearly all objects when distinct shape features were present, while it was not always the case for looking.We also did not expect infants showing points of object contact so similar to those of the 9-month-olds right from reach onset. And we did not expect infants' looking patterns to change so quickly to resemble those of the 9-month-olds in just a few weeks. These findings were surprising but also suggest clues to our understanding of the process of visuo-motor mapping at reach onset.First, we think that these data show that from reach onset infants can project their hand toward a future location in space successfully and can display a certain level of endpoint accuracy, similar to those of more experienced reachers. This supports the interpretation that, infants must have developed some kind of proprioceptive spatial knowledge of their arm movement prior to learning to reach. As discussed earlier, such motor knowledge could have formed as a consequence of accidental events involving the arm and the eyes, which possibly created useful visuo-motor contingencies that helped the development of an extended sense of the arm movement in space (see also Borghi et al., Second, the fact that for the drumstick the greatest point of hand-object contact seemed more consistent over the weeks, while the point of greatest visual attention grew to align with the point of hand-object contact over the weeks, suggests that vision may not have been the main driving factor in setting initial motor goal accuracy. However, as spatial mapping between vision and action strengthened over the weeks, vision may have become more predictive in defining the point of where to bring the hand in contact with the object. Again, this was suggested by the progressive alignment of vision onto the preferred contacted area for the drumstick. Such alignment could reflect an increasing ability of vision to become more selective and more predictive of where the hand is being directed as motor and visual spatial outcomes are being paired repeatedly during early reaching responses. The prospective role of vision could originate from these infants' initial embodied reaching experiences. Indeed, it could be possible that infants' movement experience and associated resulting action's outcomes drove the needs of vision to begin detecting ahead of time where the hand should go. In the case of reaching for the drumstick, infants could have learned to direct their visual attention increasingly toward the sphere area, perhaps because it met some valued outcome. For example, infants may have preferred to touch the sphere because it provided greater haptic experience than the thinner rod to which the sphere was attached. As infants gained experience at reaching and touching, vision became increasingly attuned to these features and began performing more searches for these special features, thereby becoming more selective and predictive for reaching. Such interpretation is consistent with a number of studies on infants' self-produced actions and their understanding of actions in the physical and social world that suggest, in similar ways, that infants' active experiences can drive changes in their attention and perception of the world (Cicchino and Rakison, Future studies are necessary to extend our observations to more infants and wider contexts to examine the validity of the embodied scenario we propose. Most useful, we think, will be studies examining vision during the movement of reaching itself, something we did not do in our longitudinal observations. Such observations will be essential to disentangle the respective role of vision and arm control in infants' first reaching attempts. Prior evidence, in 9-month-old infants, where the recording of infants' eye-movements directed to a target were paired with the arm movement kinematics corresponding to reaching for that same object, pointed to the production of object-specific looking patterns closely matching movement corrections toward that object (Corbetta et al., 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.


Despite several decades of research, electrically powered hand and arm prostheses are still controlled with very simple algorithms that process the surface electromyogram (EMG) of remnant muscles to achieve control of one prosthetic function at a time. More advanced machine learning methods have shown promising results under laboratory conditions. However, limited robustness has largely prevented the transfer of these laboratory advances to clinical applications. In this paper, we introduce a novel percutaneous EMG electrode to be implanted chronically with the aim of improving the reliability of EMG detection in myoelectric control. The proposed electrode requires a minimally invasive procedure for its implantation, similar to a cosmetic micro-dermal implant. Moreover, being percutaneous, it does not require power and data telemetry modules. Four of these electrodes were chronically implanted in the forearm of an able-bodied human volunteer for testing their characteristics. The implants showed significantly lower impedance and greater robustness against mechanical interference than traditional surface EMG electrodes used for myoelectric control. Moreover, the EMG signals detected by the proposed systems allowed more stable control performance across sessions in different days than that achieved with classic EMG electrodes. In conclusion, the proposed implants may be a promising interface for clinically available prostheses. Electromyography (EMG) has been used to control electrically powered hand and arm prostheses since decades . The self-experiment was reported to the local ethics committee who raised no objections.The first implant was located in the distal part of the extensor digitorum muscle, approximately 1/3 of the distance from the wrist to the elbow. The other three implants were inserted 2 month later, after no complications were observed with the first one. They were located on the proximal portion of the extensor digitorum, flexor carpi radialis, and extensor carpi ulnaris muscles. The electrode positions were determined by palpation, and were intended to allow a myoelectric control task (discussed below). The thickness of the epidermis and dermis were identified by ultra-sound imaging and the implants were fabricated with customized length of the linking axis  and with dry electrodes commonly used in active electrode modules of myoelectric hand-prostheses . The Ag/AgCl-electrodes and the dry electrodes were placed directly proximal and distal to the implants, because small displacements in the longitudinal direction with respect to the muscle fibers have a minor influence on the EMG characteristics  biosignal amplifier (OT-Bioelettronica USB-II) with a 16-channel pre-amplifier to minimize the influence of the cable length between the electrodes and the first amplification stage . The sampling rate was 2048 Hz and the amplifier included a 3 Hz high-pass filter to remove DC offsets and a 900 Hz anti-aliasing low-pass filter. All signals were measured with reference the first implant. To compensate for the effect of this unbalanced reference (for Ag/AgCl and dry electrodes) and to reduce the influence of EMG-activity at the reference point, a group-wise common-mean referencing was performed individually for each electrode-type. To further reduce movement-artifacts, noise above the EMG-spectrum and power-line interferences including harmonics, a 4th order Butterworth-bandpass filter (30–500 Hz) and a comb-filter (50 Hz) were applied in software. The tests were conducted 3 month after the implantation of the last implants.EMG signals were acquired with the same amplification, filtering, and acquisition chain for all the electrodes (the novel ones and those used for comparison). Signal amplification was obtained by a battery-powered, low-noise . For the dry and the wet Ag/AgCl electrodes, the impedance was measured over time at 3, 10, 45, and 120 min following placement.Three of the implants were used in the myoelectric control test against the first implant as reference point. All other electrodes were tested against one wet Ag/AgCl electrode close to the implant reference. To correct for the impedance of the reference electrode, this was estimated to be half of the average of the impedance measured from all the electrodes within the same type . The measurement resolution was 100 Ω and all measurements were repeated three times .To qualitatively compare the signals acquired by the different electrode types, a series of wrist-extensions between 10 and 50% maximum voluntary contraction (MVC) force, were performed and the EMG signals were recorded concurrently with all three electrode-types. The forearm was rested on a comfortable arm-rest and the force was measured by a strain gauge which was calibrated relative to 100% MVC. Ten contractions of 5 s duration were recorded per force level, interleaved with periods of 5 s of rest in between the contractions. The amplitude and the frequency spectra were analyzed and the SNRs were estimated by the ratio of the root mean square amplitude of the signals during the contraction phase and during rest for intervals of 3 s. This estimate assumes that the noise level is relatively small so that the root mean square during the contraction can be approximately considered as not influenced by noise.In myoelectric prostheses, the mechanical interference transmitted to the socket and to the integrated electrodes is an important source of signal noise. Therefore, a test was performed for systematically comparing the sensitivity of the electrodes to mechanical vibrations. For this purpose, EMG signals were recorded while the subject used a pneumatic impact driver which delivered strong mechanical vibrations to the forearm. Alternating phases of 15 s mechanical vibrations and 15 s of pauses were applied to the forearm while the subject was asked not to activate his muscles.The performance of myoelectric control algorithms degrades significantly over days without a re-training of the system. This is largely due to the repositioning of the electrodes when donning/doffing the prosthesis . Dry electrodes, which are commonly used in prosthetics, had impedance that was almost three orders of magnitudes greater than the proposed implant and resulted in very large noise levels. Appropriate filtering techniques could reduce the noise level, but the dry electrodes were still outperformed by the implants even with filtering. Moreover, the dry electrodes reacted strongly on mechanical interference, which would make a prosthesis not usable with large vibrations. The implants on the other hand were not influenced by these interferences.Other implants for myoelectric control have been proposed earlier. The IMES (Weir et al., The clearest results on the advantage of the proposed implant are from the real-time myoelectric control for the regression based simultaneous and proportional control of two DoFs. After a short training period, the subject achieved a very good controllability with all three electrode types. However, when the regression model of the first day was applied on a second day, the performance dropped substantially for the dry electrodes and the Ag/AgCl electrodes. In a prosthetic application, this would require a re-training of the algorithm, a significant daily effort, which is a major user-compliant issue. The proposed implant showed stable results across days and would therefore avoid the need for re-training in a prosthetic application. The lack of reliability is one of the most important problems in machine learning based techniques for myoelectric control and it is the main reason for the limited transfer of academic achievements in the past decades into clinical practice. Small shifts in the electrode position and changing of skin conditions are two of the most important factors that degrade the performance under real-world conditions (Jiang et al., It has been shown for a classification based approach that the performance, obtained with intramuscular EMG is similar to that of surface EMG, but targeted placement is required for intramuscular EMG due to the very high specificity (Farrell and Weir, Even though the tolerance of the device to mechanical stress was not explicitly evaluated in this study, we expect a similar tolerance as for cosmetic micro-dermal implants. It remains to be evaluated how the device would behave within a prosthetic socket. In a future application, cavities in the inner prosthetic socket could prevent a direct mechanical contact between the socket and the implant during use. In a future design, the connectors could attach and detach automatically with flexible magnetic connectors. With this connection technology the proposed implant would be also robust against electrode lift-offs that remain a problematic issue in conventional dry electrodes, especially when the volume of the residual limb is changing.In general, the potential risk of infection is another key user-compliant issue for any implant. Our approach has the disadvantage of a permanent link through the skin with potential risks of infection. However, the link through the skin has a diameter of only 0.5 mm and due to the individual adjustment to the thickness of the skin, an antibacterial sealing by the internal and external disks is achieved. Thus, the infection risk of the proposed implant is expected to be not higher than that of frequently used cosmetic micro-dermal implants. To fully prove issues such as safety and increase the statistical reliability of the results, a full clinical evaluation with a larger number of subjects would be required. Also an evaluation with prosthetic users controlling their prostheses with the proposed implant and advanced control strategies should be conducted in the future.We have proposed a novel implanted electrode for myoelectric control that does not require wireless transmission of information or powering as needed in previous approaches (Weir et al., In conclusion, the proposed implants may be a promising interface for current commercial and clinical prostheses.Development and Implantation of the Device: DL Design of the Study and the Experiments: JH, DF, DL, NJ. Executing the Experiments: JH, DL, NJ. Writing Manuscript: JH. Revising Manuscript: DF, NJ, DL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LH and the handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Individual joint deviations are often identified in the analysis of cerebral palsy (CP) gait. However, knowledge is limited as to how these deviations affect the control of the locomotor system as a whole when striving to meet the demands of walking. The current study aimed to bridge the gap by describing the control of the locomotor system in children with diplegic CP in terms of their leg stiffness, both skeletal and muscular components, and associated joint stiffness during gait. Twelve children with spastic diplegia CP and 12 healthy controls walked at a self-selected pace in a gait laboratory while their kinematic and forceplate data were measured and analyzed during loading response, mid-stance, terminal stance and pre-swing. For calculating the leg stiffness, each of the lower limbs was modeled as a non-linear spring, connecting the hip joint center and the corresponding center of pressure, with varying stiffness that was calculated as the slope (gradient) of the axial force vs. the deformation curve. The leg stiffness was further decomposed into skeletal and muscular components considering the alignment of the lower limb. The ankle, knee and hip of the limb were modeled as revolute joints with torsional springs whose stiffness was calculated as the slope of the moment vs. the angle curve of the joint. Independent t-tests were performed for between-group comparisons of all the variables. The CP group significantly decreased the leg stiffness but increased the joint stiffness during stance phase, except during terminal stance where the leg stiffness was increased. They appeared to rely more on muscular contributions to achieve the required leg stiffness, increasing the muscular demands in maintaining the body posture against collapse. Leg stiffness plays a critical role in modulating the kinematics and kinetics of the locomotor system during gait in the diplegic CP. Cerebral palsy (CP) is an irreversible and non-progressive disorder caused by brain injury before, during or shortly after birth . During Human walking is achieved by a highly complex interaction of the force-bearing structures of the musculoskeletal system. While sophisticated models of the system have been used to provide insights into the gait mechanics in children with CP , a simplet al. (2000) [Leg stiffness incorporates information of both the kinematics and kinetics of the lower limb joints because both the leg length and the GRF are affected by the joint angles and moments at any instance of gait , 9, 12. . (2000) , the leg. (2000) . Study o. (2000)  first de. (2000) . Applica. (2000) . HoweverThe purpose of this study was to investigate the leg and joint stiffness, the contributions of skeletal and muscular components, and the associated joint kinematics and kinetics in children with spastic diplegia CP during level walking. It was hoped that the results will provide baseline data on the stiffness control of the lower limb as a whole in healthy children and in children with diplegic CP, which would be helpful in future assessment of the effects of the pathology and the efficacy of relevant treatment methods.This study was approved by the Institutional Research Board of National Taiwan University Hospital (Permit Number: 201305084RINC). All the subjects and their parents/guardians were informed of the procedure and provided written informed consent prior to the study, including enrolment and data collection.a priori power analysis based on pilot results on leg and joint stiffness using GPOWER [Twelve children with spastic diplegia cerebral palsy , and twelve age-matched healthy controls  participated in this study. The CP group was graded 1–3 in the Gross Motor Function Classification System (GMFCS) with slightly limited passive range of motion (ROM) of the hip and ankle, moderate to normal muscle strength and mild to normal muscle tone . Howeverg GPOWER  determinEach subject walked at a self-selected pace on an 8-meter walkway. Each subject wore 39 retroreflective markers for tracking the motions of the body segments  using a With the measured GRF and kinematic data, angular motions and internal moments at the lower limb joints were calculated using inverse dynamics analysis . Each boFe(t)), is defined as the component of the GRF of the limb along the line joining the COP and the hip joint center. The length of the leg spring is defined as the distance between the hip joint center and the COP, and is called the effective leg length (Le(t)). Therefore, the leg stiffness, Kl(t), at time t could be calculated as the gradient of the Fe(t) vs. Le(t) curve as follows  of the force vs. deformation curve. The force applied to the leg spring, called effective GRF ( follows :Kl(t)=dKa(t)), knee (Kk(t)), and hip (Kh(t)) at time t were calculated as the slope of the moment (M(t)) vs. angle (θ(t)) curve of the joint as follows:Similarly, the joint stiffness of the ankle (Fe(t), Le(t), (θ(t)) and M(t) were low-pass filtered using a fourth-order bi-directional Butterworth filter with a cut-off frequency of 10 Hz [Ks,l (t) is the skeletal component in Kl (t); Km,l (t) is the muscular component in Kl (t); Ks(t) is the skeletal stiffness; and Km(t) is the muscular stiffness (t) = 0) lower limb would have the leg stiffness provided solely by the skeletal component. As the angle (φ) increases, the contribution of the muscular component to the leg stiffness increases with the increasing lever arm length available to the effective GRF at the knee =Ktiffness . Based ocos2φ(t);Km,l(t)=)cosφ(t);where φ and double limb support (DLS), using the COP and GRF data measured for each limb. The results were time-averaged over the sub-phases of the stance phase, namely loading response , mid-stance, terminal stance and pre-swing  for each limb. The data from both limbs were then averaged for each trial for each subject. These gait phases were determined using the forceplate and marker data. Temporal-spatial parameters, namely walking speed, stride length normalized to leg length, stride time, cadence and step width, were also obtained. For data presentation, the curves of each angle and moment component at each joint from all the subjects were ensemble-averaged to obtain the means and standard deviations during the gait cycle for each subject group.For each of the calculated variables independent t-tests were performed to compare each of the time-averaged values over the sub-phases between the CP and control groups. The effect of gait speed on the between-group comparisons in all stiffness-related variables was tested using analysis of covariance (ANCOVA) with the gait speed as a confounding factor. All significance levels were set at α = 0.05. All the statistical analyses were performed using SPSS version 20.0 .Compared to the healthy controls, the CP group showed significantly increased stride time and step width, but significantly reduced gait speed and cadence . HoweverCompared to the control group, the CP group showed significantly increased flexion at the hip and knee during most of the stance phase, and increased ankle plantarflexion during terminal stance and the pre-swing phase . The CP Compared with the healthy controls, the CP group showed significantly reduced leg stiffness during loading response and mid-stance, with increased joint stiffness at the hip, knee and ankle . During Compared with the healthy controls, the children with CP walked with a significantly increased muscular component but reduced skeletal component of the leg stiffness during loading response and mid-stance, giving a significantly increased ratio of the muscular to skeletal components . The musThe current study was the first attempt to quantify the leg stiffness and the associated skeletal and muscular components, as well as joint stiffness, during stance phase of gait in children with spastic diplegia CP. In the literature, some studies calculated the leg stiffness as the ratio between the peak GRF and maximum displacement , 27, givDuring loading response or initial DLS, children with CP showed a reduced leg stiffness even though the stiffness of individual joints was increased compared to the control group. This appeared to be a result of the more flexed posture of the lower limbs  while acDuring the pre-swing phase, the reduced leg stiffness but increased joint stiffness in the CP group suggests that the subjects may have a compromised ability for providing a pushing-off force for the subsequent swing. Since the body weight is transferring from the stance limb to the contralateral limb, the leg stiffness and the walking stability during this period did not have to be as high as that during weight acceptance. However, instability in the control of the stance limb during the unloading phase may affect the smooth transfer of the body weight, leading to an increased joint stiffness and muscular component of leg stiffness. The reduced leg stiffness but increased joint stiffness in both limbs during DLS suggest that the children with diplegic CP had altered coordination of the transfer of the body weight and modulation of the leg and joint stiffness between the limbs, leading to a higher risk of imbalance or collapse during weight transfer.During mid-stance, the objective was to ensure smooth progression of the body over the stationary foot, which had to be based on necessary stability of the lower limb. Following loading response, the more flexed posture of the lower limbs during this period also contributed to the reduced leg stiffness in the children with CP, requiring increased muscular components and joint stiffness. This suggests that when subjected to a sudden increase in the GRF, e.g., extra force needed to recover balance, the risk of collapse in these children may increase.During terminal stance, the CP group increased the leg stiffness primarily via increased knee joint stiffness, presumably to prepare for transferring the body weight to the contralateral limb. This would also be helpful for preventing an undesired reduction in toe clearance of the contralateral limb owing to increased shortening of the ipsilateral leg length. This is particularly important in preventing stumbling as their ankle dorsiflexion angles were reduced . It appeThe CP group appeared to rely more on muscular contributions to achieve the required leg stiffness. The more flexed hip and knee increased the lever-arm length of the GRF vector at the joint centers, and thus the joint moments. This also increased the contribution of the musculature system to the leg stiffness but decreased that of the skeletal system . The incWith a more flexed posture during the stance phase, the CP group had to sustain increased loads at the individual joints, as well as increased joint stiffness. However, these changes did not maintain the leg stiffness within the normal range. This appeared to be related to the inability to modulate the leg stiffness to compensate for the symptoms of the neurological changes and the muscle weakness during gait . DecreasThe leg stiffness and related variables proposed in the current study provide a measure for assessing the control of the lower extremities as a whole in supporting the body against collapse during gait, which would be very helpful in the assessment of the effects of the pathology and the efficacy of relevant treatment methods in a clinical setting. This information is not readily available from the individual joint angular and moment data, nor from the EMG data of the relevant muscles obtained using traditional gait analysis. It is thus suggested that the stiffness-related variables should be included in future clinical gait analysis for a more complete assessment of gait in children with CP. As a first step, the current study demonstrated that these variables successfully distinguish the control of the lower limb as a whole between diplegic CP and healthy subjects, providing baseline data for these subject groups. On the other hand, while leg stiffness-related variables integrate both force and displacement information , how theLeg stiffness plays a critical role in modulating the kinematics and kinetics of the locomotor system during gait in diplegic CP. The CP group significantly decreased the leg stiffness but increased the joint stiffness during stance phase, except during terminal stance where the leg stiffness was increased. They appeared to rely more on muscular contributions to achieve the required leg stiffness, increasing the muscular demands in maintaining the body posture against collapse. Therefore, therapeutic decision-making and planning should consider not only the mechanics of individual joints, but also the leg stiffness control. The stiffness-related variables should be included in future clinical gait analysis for a more complete assessment of gait in children with CP.S1 Dataset(CSV)Click here for additional data file.


In Literature fractures of the mandible that involve the condyle ranges from 20% to 35% and various possible surgical options are described according to the varying pathological situations. Up to the present, numerous techniques have been used for the surgical treatment of condylar fractures. In this article we are proposing the combination of two surgical techniques as therapy for extra-capsular condylar fractures with dislocation.From June 2003 to July 2007 30 patients were treated for condylar fractures with the application of a Rigid External Fixator under endoscopic assistance. This method includes a surgical reduction of the fracture with the aid of an endoscope, performing a transcutaneous insertion of a Rigid External Fixator to stabilize the fracture.Out of the total number of patients, 28 reached an optimal result without the need for temporary immobilization of the temporal mandibular joint and pre-auricular cutaneous access, thanks to the decisive aid of the video-endoscope.The endoscope allows perfect control over both the positioning of the external fixator and the surgical reduction, restoring the normal movement of the mandible with a return to full anatomical functioning of the temporo-mandibular joint. This approach avoids possible damages to the facial nerve branches. The rigid external fixation system is better than an internal one, because it is less restrictive in precise anatomical reduction, since with an REF the condylar fragment is kept in the correct anatomical position but is not obliged to maintain that exact position, and therefore it is possible to carry out all the repair mechanisms listed above. Endoscopic assistance allows a good positioning control of the REF although the endoscopy permits an optimal control of the condylemeniscal complex mobility after REF application. In the International Literature, fractures of the mandible that involve the condyle ranges from 20% to 35% . The conUp to the present, numerous techniques have been used for the surgical treatment of condylar fractures: from osteosynthesis using metal wire, to mini-systems with rigid internal fixing, or various types of pin inserted through cutaneous approaches – whether pre-auricular, sub-mandible, trans-parotid or the use of a system of rigid external fixing after an open reduction through preauricular access, introduced in Italy in 1990 by Cascone . Later oFrom June 2003 to July 2007, 32 patients with mandibular condyle fractures  underwent surgical treatment Table . Ages raThe technique proposed envisages tracing the path of the condyle and then repositioning it under video-endoscopic control, by an endoral approach through an incision at the level of the homolateral retromolar trigone, as well as opening the jaw below the periosteum and the posterior border of the mandible to find the fracture focus. Endoscopes with 0°, 30°, 45° and 70° angulations were used according to the type of surgery, with the aid of a Xenon light source [Only in 4 cases was it necessary to position an inter-maxillary blockage for seven days because of the contemporary presence of contralateral intracapsular condylar fracture treated with REF. Five patients requested an additional procedure with a pre-auricular cutaneous approach as described in the original technique proposed by. Out of the total number of patients, 28 reached an optimal result without the need for temporary immobilization of the temporal mandibular joint and pre-auricular cutaneous access, thanks to the decisive aid of the video-endoscope. Out of a total of 32 patients, 4 out of 5 who presented complications did not satisfactorily resolve their problem and of these, 2 underwent new operations to resolve their fractures. The main problems experienced by these 5 patients arose from an inadequate repositioning of the fractured piece with lateral/controlateral movements which were much reduced compared to normal. Nonetheless, the opening of the mouth was satisfactory and the patients’ subjective symptoms did not include pain but only the sensation of a functional impediment in lateral movements; on opening the mouth was a persistent degree of both lateral and protrusive deviation.Fractures of the condyle are still today the subject of much discussion, especially as regards standardizing the therapy, due to the wide variety of forms this may take, and because of the numerous therapeutic methods available.The necessity for a simple classificatory criterion is of fundamental importance to correctly apply any therapy, which must necessarily take into account parameters such as the age of the patient, the intra- or extra-capsular location of the fracture, whether it is mono- or bi-lateral, the kind of dislocation of the stumps and the presence or absence of luxation of the condylar head from the glenoid cavity.ad integrum. In 1983, the various indications for surgical treatment were schematized by Zide and Kent in absolute and relative terms. In the latter case, the possibility of surgical treatment is particularly important in as much as such fractures provoke a reduction in the posterior facial height which must necessarily be recovered through surgery, to give an adequate guide parameter for successive threedimensional reconstruction of the face [Also the age of the patient governs the type of therapeutic treatment. During the years of growth some authors have found a greater capacity for morphofunctional recovery of the fractured condyle in comparison with adult patients. Thus the therapeutic approach can vary not only according to the type of fracture, but also the type of patient. The two main therapeutic directions envisage on the one hand orthopaedic-functional treatment and on the other surgical treatment. Orthopaedic-functional therapy remains the most commonly used by various authors, permitting as it does an optimal functional recovery. Here it must be underlined that an inter-maxillary blockage determines two main problems; as well as immediate morphofunctional limitations due to the complex nature of the temporal-mandibular joint, there are often further psychological problems for the patient. However, despite these, the bibliography is lavish in its support of the efficacy of this particular therapeutic approach. The orthopaedic-functional approach has always been practiced in condylar fractures in the pediatric age, and for intra- and extra-capsular fractures without serious condylar dislocation in adult subjects -8. Delaithe face ,13.Various authors maintain that surgical therapy is indicated in cases of mono-condylar fractures in adults or adolescents, not only where it is impossible to achieve normal occlusion, but also where there is noteworthy dislocation, with an angle of the small fragment greater than 45° . In factUntil only a few years ago, the concept reigned that surgical access had to be such as to allow the most direct approach possible to the dislocated condyle stump.Analysing the evolution of thinking on therapeutic approaches proposed over the last few years, we began to consider condylar fractures a more and more delicate problem, and the therapeutic approach the preferable choice, since it seems to us to be the one which aims at obtaining a morphofunctional recovery leading to a situation which is the most similar to that before the trauma. In this vision, the targeted surgical approach is gathering consensus, but the originality of the method we have introduced lies above all in trying to avoid immobilising the complex temporalmandibular joint system, in making external cutaneous incisions to reduce to a minimum both scarring and lesions of certain branches of the facial nerve, plus the use of a rigid external fixation system (REF), already used extensively in the recovery of fractures in other areas of the body by our orthopaedic colleagues. Endoscopic assistance allows a good positioning control of the REF although the endoscopy permits an optimal control of the condyle-meniscal complex mobility after REF application. Endoscopy nowadays is commonly used in maxillofacial surgery, such as oncology, trauma -21. The All patients granted written specific consent for all video, photographs and personal data to be used in every medical publications, journal, textbook and electronic publications. The study was conducted in accordance with the ethical principles provided by the Declaration of Helsinki and the principles of good clinical practice. Study design, inclusion and exclusion criteria and treatment protocol were reviewed and approved by a council of senior specialists at the Maxillofacial Surgery Department, Sant’Andrea Hospital of Rome, Italy.


Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids  and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases. Cardiovascular disease is a leading cause of morbidity and mortality in Western societies. One of the most encouraging treatment methods to prevent the generation and progression of cardiovascular disease is the elevation of high density lipoprotein (HDL) levels in circulation, as high HDL levels have been found to correlate negatively with the risk of cardiovascular disease. HDL elevation is attainable through inhibition of cholesteryl ester transfer protein (CETP). A novel molecular agent, anacetrapib, fulfills the requirements with an acceptable side-effect profile. In this study, our objective is to gain more detailed information regarding the interactions between CETP and anacetrapib in order to unlock the inhibitory mechanism of the drug that has, to date, remained unclear. Our results point out the primary binding site of anacetrapib and highlight the ability of the drug to regulate the structure-function relationship of those structural regions of CETP that are considered important in CETP inhibition. Our findings could be exploited in the development of new and more efficient molecular agents against cardiovascular disease. Cholesteryl ester transfer protein (CETP) is a 476-residue-long hydrophobic glycoprotein that transports cholesteryl esters (CEs), triglycerides, and phospholipids between high density lipoprotein (HDL) and other lipoprotein fractions in human blood plasma CETP  carries The interest towards CETP and its lipid transfer functions came to the forefront after notable associations between decreased CETP activity, decreased LDL-cholesterol level, increased HDL-cholesterol level, and resistance to atherosclerosis Despite the promising start achieved in the anacetrapib-based CETP inhibition, the precise inhibitory mechanism behind the agent is yet to be proven, regardless of the extensive research efforts its clarification has demanded. Classically, drugs inhibit or promote the functions of an enzyme or a receptor by binding e.g. to the active site thus blocking the binding of a ligand. However, in the case of CETP inhibitors, it has been demonstrated that they promote the formation of CETP-HDL complex, indicating that the current inhibitors do not only compete with CEs and triglycerides in CETP-binding but, additionally, they also hamper the detachment of CETP from HDL surface The above findings and suggestions are insightful and encouraging, but call for better understanding of the inhibitory mechanism of anacetrapib, as well as of the lipid transfer functions related with CETP inhibition. In this regard, our objective is to perform atomistic molecular dynamics simulations to obtain detailed information linking the two processes. Here we have studied the interactions between anacetrapib and CETP with different lipid compositions to gather novel information regarding the inhibition of CETP. Previous studies of HDL-like lipid droplets We find that anacetrapib has a strong affinity for the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of CE to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The present study serves as a solid foundation for future studies concerning interactions between anacetrapib and CETP-lipoprotein complexes.−1, −46.4 kJ mol−1, −48.5 kJ mol−1, and −46.9 kJ mol−1. In order to further validate the binding site of anacetrapib, the recently published X-ray structure of CETP with bound torcetrapib To find the most probable sites from the crystal structure of CETP where anacetrapib would favor to attach, we used the energy-minimized and flexible structure of anacetrapib together with the crystal structure of CETP. Here, the aim was not to identify the possible binding poses of anacetrapib, but rather to show the most probable binding sites of the drug molecule. The results are illustrated in We carried out ten 20 ns atomistic simulations for fully hydrated systems containing CETP with anacetrapib placed outside but in a close proximity with the protein  profiles of 20 ns simulations indicate that the structures do not deviate considerably from the corresponding X-ray structure . The radIt is worth to notice that in S1-helix, S2-1nm, S3-2nm, S7-1N, S8-2N, and S9-1C, the movement of anacetrapib is highly similar, since the drug moves at the N-terminal tunnel opening mainly around residues Arg197, Ser431, Lysh432, Gly433, Ser435, and Hisb462, which is in good agreement with our results based on molecular docking, concerning especially the binding site of the red ligand .The visualization of the simulation trajectories also revealed that while the drug moved at the N-terminal opening, the trifluoromethyl- and methyl groups of the drug oriented close to each other . This inThe present findings based on a self-assembly process highlight the importance of thermal diffusion together with electrostatic and van der Waals interactions during the formation of CETP-inhibitor complexes. Thermal motion predominates for distances above 3 nm between anacetrapib and the tunnel opening of CETP, and only for shorter scales the direct interactions between the molecules become strong enough to drive the complex formation process. Furthermore, the affinity of anacetrapib towards the concave surface of CETP is evident when the distance between the particles is taken into consideration. On the basis of the above findings, we propose that the primary binding site of anacetrapib resides inside the hydrophobic tunnel of the protein, near the residues surrounding the N-terminal tunnel opening, including helix X.Computation of free energy profiles is a major computational challenge in general. The case considered here is not an exception. To get the free energy profiles shown in −1 for anacetrapib, and between 184 and 197 kJ mol−1 for CE (when anacetrapib blocks the path).The obtained free energy profiles  indicateThe free energy barriers found here, as CE is pulled out from CETP, are profoundly high. This results from the transition path that takes CE completely into the water phase. Importantly, −1. The results point towards the important role of helix X in assisting the lipid exchange, and highlight the possible inhibitory mechanism of anacetrapib as the movement of CE outside from CETP could be hindered in the presence of the drug molecule.It is worth to notice that the presence of helix X seems to have an influence on both the binding strength of anacetrapib and the diffusion ability of CE, since both molecules are pulled out from the hydrophobic tunnel more easily when helix X is removed from the structure of CETP. Additionally, when also the drug molecule is removed, the free energy barrier of CE movement towards the water phase appears to be the lowest, 153 kJ mol−1 for the first and −30±20 kJ mol−1 for the latter cycle, where one is tempted to expect for a value of zero. However, the binding site of anacetrapib is not identical in systems where the hydrophobic tunnel is empty  or contains two CEs . To be more specific, the binding site of anacetrapib resides deeper in the hydrophobic tunnel of CETP as the tunnel does not include lipids, while in the presence of these lipids the binding site of the drug molecule resides closer to the N-terminal tunnel opening. Hence, with the present systems, it is not possible to obtain a thermodynamic cycle that would result in a value of zero, and the values of the thermodynamic cycles given above therefore partly stem from this fact, and partly from inadequate sampling. Given the considerable computing resource to generate the data in There are two thermodynamic cycles for binding and unbinding both anacetrapib and CE present in the free energy calculations, namely, the one with the absence of helix X from the structure of CETP (U1/U6/U2/U5) and the other with the presence of helix X in the structure of the protein (U3/U8/U4/U7). With the current data, these cycles result in values of −28±20 kJ molsn-2 chains, experience considerable wobbling in the presence of anacetrapib (L2) compared with the absence of the drug (L1). However, there is a contradiction when comparing the RMSD and RMSF curves of DOPC in simulations L1 and L3. The RMSD appears more stable in L3 , Ω4 (residues 40–50), Ω5 (residues 90–110) and Ω6 (residues 150–170) which were also earlier shown to have high mobility First, for comparison, CETP has previously been reported to have mobile structures with elevated B-factors near tunnel openings including the hinge region of helix X (Gly458-Pro460), and in the N- and C-terminal ends including the loop regions represented with omegas one and two In addition, the observed flexibility of the hinge region of helix X (Glu 461-Ser472) suggests that helix X could play a crucial role in assisting the lipid exchange process. During the lipid exchange process helix X may partly move into the N-terminal CE-binding pocket of CETP to facilitate the export of CE out from CETP 10-helix (extension of the helix) when the drug molecule interacted with the concave surface of CETP , and when either CEs  or anacetrapibs  were present in the hydrophobic tunnel  and 3c tunnel , Table 4On the basis of earlier clinical trials, both anacetrapib and the flawed torcetrapib were shown to increase the binding affinity of CETP towards lipoproteins, especially towards HDL 2+ and Mn2+, into the solution The results showed an evident affinity of anacetrapib towards the concave surface of CETP, especially towards the N-terminal tunnel opening where also helix X resides, and highlighted the importance of electrostatic and van der Waals interactions during the process once the drug was able to migrate to a close enough distance from the tunnel opening. However, the distance between the particles should be taken into consideration in the complex formation, since with too large distances (above about 3 nm) the movement of the drug was noticed to be dominated by thermal motion, eventually resulting in disordered motion. Hence, the question is how the affinity between the particles could be ensured in order to secure the interactions and speed up the complex formation process, as otherwise anacetrapib may experience random motion and may not be suitable for CETP inhibition purposes. For comparison, the formation of CETP-lipoprotein complex has been reported to be modulated by pH, surface pressure, and the introduction of positive divalent ions, such as CaIn addition to the observed affinity, anacetrapib was noticed to align itself to a tighter conformation while moving near the N-terminal opening. This finding together with the evidence of stronger interactions prevailing between the particles when the drug was transferred inside the hydrophobic cavity, indicate the primary binding site of anacetrapib to reside in the tunnel, particularly near the residues surrounding the N-terminal opening, including helix X. Hence, we propose CETP inhibition to be realized when the drug is transferred into the lipid binding pocket.10-helix while anacetrapib moved near the residues that surround the N-terminal tunnel opening. The results provide compelling evidence about the ability of anacetrapib to induce conformational changes to helix X in order to achieve the needed flexibility. How helix X behaves in the presence of the entire HDL-CETP-anacetrapib complex is a question left for additional simulations to be resolved.The regulatory role of helix X has been identified to play an important role in the lipid exchange process, since it has been suggested to act as a lid that conducts the exchange by alternating its open and closed states Another crucial component in the process of neutral lipid exchange are the phospholipids due to their central role both in the binding and detachment of CETP from lipoprotein surfaces. During binding, phospholipids have been proposed to merge into the monolayer followed by a migration away from the tunnel openings to their edges The primary binding site of anacetrapib to reside inside the hydrophobic tunnel is further supported by the free energy profiles that reveal strong attachment between the drug molecule and CETP, especially when two CEs fill the length of the tunnel as shown also previously In conclusion, our results show an evident affinity of anacetrapib towards the concave surface of CETP, especially towards the region of N-terminal tunnel opening. The primary binding site for the drug turns out to reside inside the hydrophobic tunnel, near the residues surrounding the N-terminal opening. When residing in this area, anacetrapib was shown to hinder the ability of CE to diffuse out from the structure of CETP. Additionally, the results point towards the encouraging capability of anacetrapib to influence the molecular interactions between phospholipids and helix X, both of which represent the structural regions of CETP important for lipid exchange between lipoproteins, thus giving support for the competency of pharmacological CETP inhibition.The view presented in this article paves the way for extending the scope of computational studies to gain a deeper understanding concerning the pharmacological ways to inhibit CETP and to modulate HDL levels. In this regard, simulations concerning the interactions between HDL-CETP-inhibitor –complex are ongoing (work in progress). The novel understanding could be used in the development of new molecular agents in the fight against the progression of cardiovascular diseases.Here we consider systems where anacetrapib interacts with CETP, which is either empty or carries a number of lipids in its transfer pocket. First, anacetrapib  is an orFor all molecules considered in this study , we used the official distribution force field OPLS-AA −1. Four conformations with the highest binding free energy for anacetrapib are shown in −1, −46.4 kJ mol−1, −48.5 kJ mol−1, and −46.9 kJ mol−1. More detailed technical information for the applied program, AutoDock Vina, can be found in Prior to molecular dynamics simulations, we used molecular docking calculations to determine the initial configurations for the simulated systems. The purpose of the calculations was not to identify the possible binding poses of anacetrapib, but rather to explore the most probable sites from the crystal structure of CETP where the drug would desire to attach in terms of the lowest binding energy. The constructed box covered the two tunnel openings and the hydrophobic tunnel of CETP. CETP-bound lipids were not present. A flexible anacetrapib molecule with 9 rotatable bonds was used in the calculations. In total, 1000 runs were carried out with default settings, namely, the maximum number of binding modes to generate/export was set to 9, and the maximum energy difference between the best ligand pose and other ligand poses was set to 12.6 kJ molBased on the docking data see , we consThe second group consisted of five systems with anacetrapib placed inside the lipid binding pocket to study the conformational changes of CETP induced by the drug. In the first simulation system (L1), the two CEs and anacetrapib were removed from CETP, thus only the two DOPC molecules remained inside the protein . In the The third group consisted of eight systems directed to umbrella sampling simulations. In the first four systems, one anacetrapib molecule was pulled out from CETP through the N-terminal tunnel opening. The interior lipid composition and the presence of helix X in the structure of CETP were varied between the systems. In the first two systems, helix X was removed and the tunnel was either empty U1,  or contaThe systems belonging to the first group were solvated with ∼100,000–160,000 water molecules with counter ions, while in the second and third groups about 100,000 and ∼30,000–60,000 water molecules, respectively, were used for solvation. Altogether, the systems included ∼125,000–500,000 atoms. The number of water molecules used in the simulations depends on the size of the simulation box: the greater the distance between CETP and anacetrapib, the greater the size of the simulation box and, hence, the number of water molecules. The size of the simulation box originates from the requirement of periodic boundary conditions. Before any simulation was started, energy minimization was performed for each system using the steepest descent method with 500 steps −1 nm−2 with a pull rate of 0.003 nm ns−1. After pulling simulations, umbrella sampling was conducted with a total of 25 and 47 umbrella windows when the pulled molecule was anacetrapib and CE, respectively. The umbrella windows were selected at 0.1 nm intervals from the original location of the molecules inside the hydrophobic tunnel towards the water phase. Each window was simulated for 160 ns where the first 100 ns were used for equilibration. These parameters were chosen based on a systematic study for increasing equilibration and simulation times. Altogether, the umbrella sampling simulations required about 600 core-years of computing time. In these simulations, both molecules were restrained to the middle of every umbrella window by a harmonic potential with the same force constant as used in the pulling simulations. The restraints were applied only along the reaction coordinate defined by the vector connecting the reference pull group and the pulled molecule, and the molecules were free to move in the other directions. In order to keep the reaction coordinate along the vector and to prevent CETP from rotating and tilting, position restraints were applied to a carbon atom of Ser115 and to a carbon atom Gly413. These atoms reside in the N- and C-terminal ends of the protein, respectively.Prior to conducting umbrella sampling simulations, eight pulling simulations were performed in order to generate a series of configurations that served as the starting configurations for umbrella sampling. In the pulling simulations, either anacetrapib or the N-terminal CE, depending on the system, was pulled out from the structure of CETP through the N-terminal tunnel opening. Residues Cysh9, Arg10, Ile11, and Thr12 were used as the reference pull group due to their parallel location with the N-terminal tunnel opening. The force constant applied either to the center of mass of anacetrapib or to the last carbon atom in the acyl chain of CE was 2000 kJ molThe simulations were performed under NpT conditions  with the GROMACS software package using the version 4.5.4 for the first and second simulation groups and version 4.6.1 for the third simulation group DSSP (define secondary structure of proteins) The root mean square deviation (RMSD) was used to evaluate the deviation of the structure of the simulated system from the initial starting structure over the course of simulation. RMSD was calculated by applying the g_rms tool of GROMACS.The radius of gyration was used to measure the size and compactness of a molecule. It is defined as the mean square distance of each particle in the structure with respect to its center of mass. The radius of gyration was calculated by applying the g_gyrate tool of GROMACS.The root mean square fluctuation (RMSF) of atomic positions was used to discover and evaluate the most flexible regions of CETP. RMSFs were calculated by fitting the simulated structure to a reference structure followed by the calculation of the average distance deviation from the reference structure. Typically, the residues of a protein that fluctuate the most can be found in loop regions. For purposes of comparison with experimental data, the RMSFs can be converted into B-factor values. RMSF was calculated by applying the g_rmsf tool of GROMACS.Interaction energies between different molecules were calculated by applying the g_energy tool of GROMACS. The tool calculates the contributions of the energies automatically from the simulation trajectory.Weighted histogram analysis method (WHAM) is a standard technique to compute the potential of mean force (PMF) along the reaction coordinate for a molecule Statistical uncertainty of the PMF can be estimated by applying the bootstrap analysis. The idea is to generate new hypothetical observations, that is, a bootstrapped trajectory for each umbrella histogram, thus yielding a new set of histograms for the corresponding umbrella window that are subsequently applied in WHAM to calculate a bootstrapped PMF. The process is repeated for each bootstrapped trajectory when a large number of bootstrapped PMFs can be obtained. The applied bootstrapped sample size was 200.rDA) and a criterion for the angle α (between acceptor-donor-hydrogen triplet αADH),Hydrogen bond formation has a remarkable role, e.g., in the stabilization of the secondary structure of a molecule. In this study the formation of hydrogen bonds is analyzed between CETP and anacetrapib in order to see where and how tightly anacetrapib binds. The formation of hydrogen bonds is analyzed between all possible donors D and acceptors A. OH and NH groups are regarded as donors while O is always an acceptor. The determination for the existence of a hydrogen bond is done by using a geometrical criterion Click here for additional data file.Dataset S1Derived charges for anacetrapib, based on its topology file.(DOC)Click here for additional data file.