The effects on children’s behavior of visual information presented by augmented reality (AR) were investigated. A human-like AR character was presented standing in one of two physical pathways to children aged 5–7 years old and 8–10 years old before they completed a filler task. After the task, the children were required to walk through one of the two pathways to obtain a reward. Both the 5–7- and 8–10-year-olds chose the pathway that was not associated with the AR character more frequently than the pathway that was. Subsequently, adult participants tested in a similar manner showed no significant bias in pathway selection. Taken together, these results suggest that the presentation of an AR character within the present experimental setting affected the behavior of children aged from 5–10 years but not that of adults. The results are discussed in the context of developmental changes in sensitivity to insubstantial agents , the reality of information displayed by AR technology, and differences in the methods of AR presentation . Technologies that expand the human sensory experience, such as virtual reality (VR) and augmented reality (AR), have been rapidly popularized during the last decade. In general, VR completely replaces sensory information from the natural environment with artificial information; e.g., fully masking one’s vision with computer-generated visual stimuli using a head-mounted display. In contrast, AR superposes artificial sensory information on information from the natural environment; e.g., overlaying a computer-generated avatar onto a scene of the real world in real time using a camera and smartphone display.1. Furthermore, against a background of the worldwide spread of personal portable electronic devices (PED), including smartphones and tablets, a variety of applications and games have adopted AR for PED users, resulting in a variety of uses for these technologies and the involvement of many people. For example, Pokémon GO, which is one of the most popular AR games, has been downloaded more than 800 million times2.In today’s society, opportunities to be involved with these types of technology are increasing for adults as well as children. A variety of consumer VR headsets have been released by many companies at reasonable prices, and some of these VR headsets are designed for young children. For example, children 7 years of age and older are allowed to use the Nintendo Labo VR Kit3. Additionally, adult observers watching a human-like AR avatar via a head-mounted device tend to control their walking path to avoid the space in which the AR avatar appears, just as they do for a real human model4. Although many studies have demonstrated the advantages of using AR for pragmatic purposes, such as the potential merits of AR in educational situations8, few studies have investigated the effects of AR on children’s behaviors using a scientific approach. Therefore, the present study investigated whether visual information displayed by AR, such as a human-like agent, would have any significant effect on children’s behavior under experimental situations.The present study investigated whether AR experiences would influence children’s behavior. Previous research has shown that information presented by AR technology has considerable effects on the mental and behavioral responses of human adults. For example, adult observers subjectively report more entertaining and realistic experiences under a gaming situation with an AR-generated avatar than with a similar avatar displayed as a 2-D- or 3-D-modeled cartoon characteret al.9 found that the verbal suggestion of the presence of an insubstantial agent alters behavior in children aged 5–6 years and 8–9 years9. In that study, children were instructed to engage in a very challenging game task in which successfully putting a sticky ball in the central area of a target earned them a reward. However, it was essentially impossible to succeed in this task without cheating because the children were required to follow three stringent rules during the game: throw the ball from a position 6 feet away from the target, use the non-dominant hand to throw the ball, and stand with their back to the target when throwing the ball. Prior to engaging in the task, the children were divided into three groups according to the task conditions: with an invisible agent, with adult supervision, and with no supervision. Under the invisible agent condition, each child was told, “An invisible girl will stay with you in this room and observe your behaviors.” They then engaged in the task while remaining alone in the room. Under the adult supervision condition, each child engaged in the task under the supervision of an adult, who stayed in the room and observed the child’s behavior during the game. Under the no supervision condition, the children were given no specific instructions about an observer, and they engaged in the game task in the room alone. The latency to full cheating behavior during the game task  was measured as the dependent variable. Children’s cheating behavior was reduced in both the 5–6- and 8–9-year-old groups by the presence of an actual human observer as well as by an invisible agent such that the mean latency to full cheating behavior was significantly longer under the invisible agent and adult supervision conditions than under no supervision.Although the full range of effects of AR agents on children’s behavior remains unknown, Piazza et al.9, our research group conducted a preliminary study to investigate the effects of an AR character on children’s behavior10 . In our study, 5–7-year-old and 8–10-year-old children engaged in the same task as that used by Piazza et al.9 under the following experimental conditions: (1) the AR agent condition, in which a three dimensional (3D) AR cartoon agent were shown to the children prior to the game; (2) the TV agent condition, in which the same cartoon agent as used for the AR condition was shown to children on a TV screen prior to the task; and (3) the no agent condition, in which no agent was shown to children. The results indicated that, in the younger group, the mean latency to full cheating behavior was shorter under the AR than under the other two conditions; thus, that the younger children’s cheating behavior appeared to be promoted by the pre-presentation of the AR character. Although the reasons for this finding were unclear, these results imply that visual information presented by AR has some impact on young children’s behavior.Based on the findings of Piazza et al.9 (9–14 children per group), this number is insufficient to generalize the results (cf.11). The second issue is associated with the validity of the game task used in the preliminary study. Only 10 of the 72 participants exhibited full cheating behaviors in the game task in the preliminary study, whereas 18 of 67 participants showed full cheating behaviors in the study by Piazza et al.9 . Thus, the number of children who exhibited full cheating behavior in our preliminary study was relatively small. This discrepancy implies that the preliminary findings might have been biased by the behavior of only a few participants. More specifically, only two of 24 children engaged in full cheating behavior under the no agent condition in the preliminary study, whereas 10 of 24 children did so in the study by Piazza et al.9 Furthermore, differences in responses to this particular task may exist among various cultures and societies; e.g., Japanese children may be less responsive to the game. Taking the limitations of the preliminary study into consideration, the present study evaluated the effects of visual information provided by AR on behaviors in children using a simple experimental task with a larger sample size.Although the preliminary study conducted by our research group demonstrated that visual information presented by AR potentially affects children’s behaviors, the study had several limitations that should be noted when interpreting the results. First, the preliminary study included six groups (two age groups × three agent conditions) with 12 children in each group; thus, a total of 72 children took part in the study. Although the sample size per group was comparable to that of Piazza The present study was approved by the Ethics Committee for Human Research at Niigata University, and all experiments were conducted in accordance with the principles of the Declaration of Helsinki.10. The parent(s) of each child provided written informed consent prior to the experiment and confirmed that the participants had no visual deficits or known history of any developmental disorder.Experiment 1 included 24 younger children  and 24 older children . Three additional children participated in the experiment but were excluded from the final sample due to the following experimenter errors: (1) the experimenter forgot to ask the parents about the AR experiences of two children and (2) the experimenter wrongly recruited and tested a non-naïve child who had participated in a preliminary study by our research groupAll experiments were conducted in a room  displayed the AR character to the participants using a custom application developed by the Unity 3D game engine  with an AR library for Unity . Using the application on the tablet, the participants could see the AR character, which was a 3D-modeled cartoon avatar of a boy, superimposed on the real scene. When a participant turned the back camera of the tablet toward an AR marker on the floor of an aisle, the AR character appeared on the tablet screen, just as if the character were standing on the AR marker. The height of the AR character in the simulated space that was superimposed on the real environment was approximately 80 cm.https://assetstore.unity.com/packages/3d/characters/cute-male-3-18090), which is distributed on the Unity asset store (https://www.assetstore.unity3d.com). Model data for the actions of the avatar were adopted from the “Taichi Character Pack” package  and implemented in the application by a built-in animation control system of the game engine for the 3D avatar . Variations in the actions of the avatar included putting his right hand on the back of his head and turning his head to the right side (‘idle_00’ of the data package), standing straight (‘idle_01’), looking around to the right and left and then looking down and kicking the ground gently with his right foot (‘idle_10’), looking at the participant and waving his right hand (‘greet_02’), and scratching his head with his left hand (‘scratchhead_00’). These actions were serially performed in accordance with a preprogrammed sequence . One stroke of the action sequence was approximately 16 sec, and the sequence ran repeatedly during use of the application. Once the application was started, the action sequence automatically started and continued running regardless of the avatar’s visibility. Thus, even when the avatar was invisible , the action sequence continued in the background in real time.The avatar of the AR character was “Male3_shortHairNGAvatar” and was adopted from the “Cute Male 3” package  and began to talk about “an invisible friend” who was somewhere in the experimental room as follows: “Today, I would like to ask you to cooperate in a game for our experiment. But, before we start to do an experiment, I want to introduce a friend to you. His name is George. Because he is so shy about showing himself to others, he usually makes himself invisible. So, we can’t see him with our eyes. But he is surely in this room and watching us right now.” Then, the experimenter handed a tablet to the participant and gave additional instructions about how to visually find George as follows: “He is actually friendly and loves to show himself on the tablet’s camera. So, you can see George by using this tablet device. Let’s look for George using this tablet.” Next, the experimenter gently urged the participant to look at both of the AR markers on the floor. The AR character appeared on either the AR marker on the window side or the one on the door side of the room  at least once, then that child was categorized as an ‘AR experiencer’.The position of the experimenter (either at the window or door sides), the AR character (either at the window or door sides), and the type of marker for the AR character  than in older children (8–10-year-olds). However, a two-tailed Fisher’s exact test revealed that the number of children who chose the aisle without the AR character compared to the number of children who chose the aisle with the AR character did not significantly differ between the younger and older groups , which indicates that the tendency of pathway selection did not differ between the younger and older children. It is possible that prior experience with AR technology might have affected the pathway selection of the children and, thus, all participants (N = 48) were divided into two groups based on previous AR experience  to compare the tendency for pathway selection between these two groups. Eighteen of 26 AR experiencers and 16 of 22 non-AR experiencers selected the aisle without the AR character and a two-tailed Fisher’s exact test revealed that the tendency for pathway selection did not significantly differ between the AR experiencers and non-AR experiencers . These results indicate that prior experience with AR technology had no effects on the pathway selections of children in the present experiment.In Experiment 1, 19 of 24 younger children (5–7-year-olds) and 15 of 24 older children (8–10-year-olds) chose to pass through the barrier using the aisle that was not associated with the pre-presentation of the AR character  more participants selected the pathway without the AR character (N = 34) than that with the AR character (N = 14). Thus, the results of Experiment 1 suggest that the 5–10-year-old children tended to avoid the pathway where the AR character pre-appeared and that the visual information presented by AR technologies may have had a significant impact on behavior in children in this age group.The results of the present analyses assessing the participants’ ages and previous experiences with AR technology suggest that there was no valid reason to divide the participants into subgroups. Thus, the data of the younger and older children were combined into a single group (N = 48) for further statistical analyses. Two-tailed binomial tests were conducted to examine whether the number of participants who chose the pathway with the AR character differed significantly from the number of participants who chose the pathway without the AR character and revealed that significantly ; one participant was excluded from the final analyses, because that individual recognized the actual aim of Experiment 2. However, based on verbal reports made during the debriefing session after Experiment 2, none of the other participants had recognized the actual aim of Experiment 2. Although written informed consent was obtained from all participants prior to Experiment 2, the consent forms were updated and reconfirmed soon after the experiment because the procedure for Experiment 2 contained a mild deception, described below.The experimental setting for Experiment 2 was almost identical to that for Experiment 1 except that a different cover story was used for showing the AR character to the participants. It would be inappropriate to apply the children’s story from Experiment 1  to the adult participants. Hence, the cover story was modified to be more realistic and pragmatic for adult participants.Briefly, the main experimenter (experimenter) escorted a participant to the experimental room and led that individual to a chair in front of the table in A1. Another experimenter (assistant) was already sitting in a chair in A2, preparing a visual pseudo-experiment on a laptop computer. Then, the experimenter introduced the assistant to the participant  and the assistant greeted the participant . Next, the experimenter told the participant to, “Have this chair, please”, to gently urge the participant to sit in the chair. After the participant was seated, the experimenter stood by either the right or left side of the participant  and provided instructions for the pseudo-experiment. Next, the experimenter asked the participant to go to A2 to engage in the pseudo-experiment. Then, the assistant suddenly said “Oh, I am having trouble with a computer program for the experiment. I will have it fixed soon, so please wait there for a moment”. The experimenter confirmed this story and told the participant “OK, now we need to wait for a while. I would like to offer you a chance to try our new experimental material, which is under development: an AR character for a child study. Please try it; we welcome your comments about the character”.Next, the experimenter handed a tablet to the participant, and asked him/her to look at both AR markers on the floor. The AR character appeared either at the AR marker on the window side or at the one on the door side; the side was counterbalanced across participants. After the participant successfully observed the AR character on the tablet screen, the assistant said to the participant “The problems have been fixed. Please come over here and let’s start the experiment.” The pathway selected by the participant for the transition from A1 to A2 was recorded and used as the dependent variable. The typical length of time between the end of observing the AR character and the moment at which the participants passed through the barrier was less than 1 min. Soon after the participant reached the assistant, a debriefing that described the true objective of Experiment 2 was provided to each participant, completing the experiment.The position of the experimenter (either at the window or door sides), the AR character (either at the window or door sides), and the type of marker for the AR character . These results imply that the presentation of an AR character had no effect on the behavior of adults in the present experimental setting.In Experiment 2, 14 of 24 adult participants chose to pass through the barriers using the aisle that was not associated with the pre-presentation of the AR character Fig. . A two-tThe primary aim of the present study was to investigate whether visual information displayed using AR technology would have any effects on children’s behavior. In Experiment 1, 5–7-year-old and 8–10-year-old children were shown an AR cartoon character, pre-presented on the floor in one of two aisles. When the children were required to pass through one of these aisles, they more frequently chose the aisle not associated with the pre-presentation of the AR character than that associated with the AR character. Although this tendency in pathway selection seemed to be more remarkable in the younger children than the older children see Fig. , this di13; however, the experience of imaginary companions is not completely extinct among 9-year-olds. In other words, children may have a stronger tendency than older individuals, such as adults, to treat insubstantial and unreal agents  as real agents. In the present study, the children (but not the adults) might have perceived some sense of “reality” from the AR agent and, thus, avoided passing through the aisle occupied by this “real” agent.It is possible that the presence of an AR character had a significant effect on pathway selection only in the children because young children tend to have a higher sensitivity to insubstantial agents than older individuals. For example, young children often engage in steady communication with imaginary companions  or characters that are embodied in inanimate objects . Additionally, it has been reported that the experience of having imaginary companions by Japanese children peaks at approximately 4 years of age and then significantly declines beginning at 7–9 years of ageet al.4 reported that information displayed by AR technology has a significant effect on adults’ behavior, the present results did not reveal any significant effects of AR-generated information on pathway selection in adult participants. This discrepancy may be due to differences in the timing of watching the AR information. The previous study measured participants’ responses in real-time during the AR experiences whereas the present study examined participants’ behaviors after they had observed the AR information. Presumably, the real-time presentation of AR information had a stronger effect on behavior in adults than the non-real-time presentation of paradigm AR information in the present study.Although Lee 14 found that behaviors of adult participants are also significantly modulated by the presentation of AR agent even when the behaviors occur just after the presentation of the AR agent. In that experiment, an AR agent was presented to each participant in an experimental room that had two empty chairs. The AR agent walked toward one of the chairs, sat on it, and introduced her/himself (the gender of the AR agent was matched with each participant) to the participant while the participant watched the agent’s behaviors via a head-mounted AR device. Then, the participant was instructed to sit in one of the two chairs: the empty chair or the chair occupied by the AR agent. Most participants chose to sit in the empty chair, even though they had removed the headset before choosing where to sit . These findings demonstrate that even the non-real-time presentation of AR information can have a significant effect on adult behaviors. However, they appear to be inconsistent with the present results from Experiment 2, which showed no significant effects of the AR character on the behaviors of adult participants.On the other hand, a very recent study by Miller, Jun, Herrera, Yu Villa, Welch, and Bailensonet al.14 and those from Experiment 2 in the present study. For instance, the difference in the length of the time period between observing the AR character and the selection of the walking behavior might have affected the results. In Miller et al.14, the participants were asked to choose a chair immediately after taking off the head-mounted display used to present the AR agent whereas the adult participants in the present study typically made their pathway selection and came across the barrier within a few tens of seconds to a minute after the end of the AR character presentation. The longer time period between the observation of the AR character and the choice of behavior in the present study may have weakened the response to the AR character in the adult participants.There are several possible explanations for the inconsistencies between the results of Miller et al.14 had a very realistic appearance and spoke to the participants whereas the AR agent in the present study was a human-like but relatively unrealistic cartoon character and did not say anything. Additionally, Miller et al.14 employed head-mounted equipment that provided an immersive visual experience to present the AR agent whereas the present study used a tablet device held in the hands of the participants to present the AR avatar . Because the use of head-mounted devices for VR/AR in young children is very controversial15, head-mounted devices were not used to present AR characters in the present study. It is plausible that the visual stimulus used by Miller et al.14  provided a more real perception and/or feeling of the AR agent than the stimulus used in the present study. Thus, the qualitative differences  among these studies may have affected the present results of the adult participants. Alternatively, apart from the reality of the AR agent, technical differences in the AR presentation (head-mounted display vs. tablet device) per se might have been related to the discrepancies between the previous studies14 and the present results. It is likely that an avatar presented by a head-mounted device and one presented by a hand-held device have different effects on human behavior. It will be interesting for future studies to compare the effects of information presented by AR via different technologies  on human behavior.Another factor that could have contributed to these discrepancies involves qualitative distinctions between the visual stimuli used in the respective studies. The AR agent used in Miller 14). Alternatively, the lack of change in the adults’ behavior in the present study may be specific to AR information presented by hand-held devices, such as a tablet. Thus, future studies should assess the effects of more realistic AR agents  displayed by a variety of AR devices  on observers’ behavior as well as the developmental trajectories of these behavioral effects.Taken together, the present findings indicate that visual information presented via AR technology can have a significant effect on behavior in childhood (approximately 5–10 years of age). Although the adult participants in the present study did not significantly change their behavior in response to the AR character, it is possible that these non-significant results were due to the insufficiently lifelike nature of the AR agent. For example, previous studies have shown that a realistic AR avatar presented by a head-mounted device has a significant effect on the behavior of adult observers (cf.


This pilot study assessed a novel intervention to enhance both walking and executive function in older adults. The primary hypothesis was that eighteen sessions of frontal lobe tDCS combined with walking rehabilitation would be feasible, safe, and show preliminary efficacy. Eighteen participants were randomized to one of three intervention groups: active tDCS and rehabilitation with complex walking tasks (Active/Complex); sham tDCS and rehabilitation with complex walking tasks (Sham/Complex); or sham tDCS and rehabilitation with typical walking . Outcome measures included multiple tests of walking function, executive function, and prefrontal activity during walking as measured by functional near infrared spectroscopy (fNIRS). Of the three groups, the Active/Complex group demonstrated the broadest improvements across outcome measures including for prefrontal activity. The functional range of prefrontal activity in this group was increased considerably, as conceptualized by the Compensation Related Utilization of Neural Circuits Hypothesis. Frontal tDCS is a promising adjuvant to walking rehabilitation.


Prostate cancer (PCa), a type of malignancy that arises in the prostate gland, is the most commonly diagnosed neoplasm and the second leading cause of cancer-related deaths in men. Acquisition of resistance to conventional therapy is a major problem for PCa patient treatment. Androgen receptor (AR) signaling pathway is necessary in the pathogenesis of prostate cancer, and there is a heightened interest in finding novel AR antagonists that target AR and its regulatory pathways. In our search for novel androgen receptor antagonists, we focus on the Traditional Chinese Medicine (TCM), which has been used for thousands of years to prove effective in the treatment of cancer. In this study, we collected 653 traditional Chinese medicine prescriptions that have certain therapeutic effect to prostate cancer, including the prescriptions and even the folk prescriptions. After summarizing the frequency of herbs and gathering the natural products contained in these prescriptions, we built a natural products database to do computer-aided virtual screening and drug-like evaluation to find potential AR antagonists. Totally 25 compounds were submitted to experimental biological activity tests. Through the MTT cell proliferation experiment, 5 chemicals were found to inhibit the proliferation of LNCaP cells in a concentration-dependent manner. Especially, MoL_11 was found to have good antagonistic activity and significantly inhibit fluorescence enzyme activity by the AR reporter gene experiment. Finally, the molecular dynamics simulation method was used to study the interaction between the most active compound MoL_11 and the wild-type and F876L mutant androgen receptor (WT/F876L AR), and it was found that F876L AR could not cause resistance to MoL_11. Prostate cancer (PCa), a common non-cutaneous cancer, accounts for almost one-fifth of new cancer diagnoses, and is the second leading cause of cancer-related deaths in men in the United States  is a ligand-dependent trans-regulatory protein that belongs to the steroid receptor in the nuclear receptor superfamily  has been extensively used as an alternate treatment for prostate cancer. Many herbal extracts have been shown to inhibit the development of prostate cancer. Generally, TCM achieves therapeutic effects by targeting multiple physiological pathways, including AR, exerting pharmacological activities by numerous active agents and components. The complex active substances in herbs are still elusive and not accurately detected solely by routine methods  has widely used in lead compound identification, which exhibits undefeatable advantage and has become very popular in drug discovery and design based on its time, cost, resources, and labor saving .We counted the herbs contained in the prescriptions collected above, and reserved the herbs with high frequency (≥10) for the succeeding research. Among different prescriptions, the herbs with high frequency should play important roles in anti-prostate cancer, including anti-AR effects. Then the existing chemical ingredients of each Chinese medicine were inquired by using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)  was downloaded from RCSB Protein Data Bank  >500 and calculated Log P (CLogP) >5, compounds are more likely to have poor absorption or permeability  docking was performed to execute the additional molecular docking. The structures of these compounds obtained above were given to the corresponding OPLS_2005 force field, and then were converted to their most probable charged states at pH 7.0 ± 2 and different tautomer were generated using the LigPrep module in Schrödinger . The compounds with docking scores higher than enzalutamide were chosen for further analysis.The aim of this study is to obtain potential AR antagonists with rich diversity, so the hierarchical clustering method is used by the canvas module. The tanimoto coefficient based on binary fingerprints is used as a molecular similarity measure. The clustering similarity threshold was set to 0.98. Then we consider the following factors to choose suitable compounds, receptor-ligand binding mode, ligand steric hindrance, molecular skeleton, hydrocarbon group, N-heterocyclic compound, amino and amide, halogen, carboxyl, ester groups and so on.http://www.pymol.org) to mutate position 876 from phenylalanine to leucine based on the WT AR to obtain mutant F876L AR. In the docking parameters, the radius of the active binding site is set to 12, and the docking pose is set to 10. Finally, the best conformation with the most optimal docking score is selected for molecular dynamics (MD) studies.The wild-type androgen receptor (WT AR) 2AXA and mutant receptor F876L AR were docked with the small molecules Enz and the most promising compound, respectively, by the CDOCKER module in Discovery Studio 2.5 software. We used Pymol . pcDNA3.1-AR and pMMTV-Luc were reorganized by Changsha Youbao Biological Co., Ltd. Lipofectamine3000 (Invitrogen) and Dual-Glo Luciferase Assay System (Promega) was purchased. Thiazolyl tetrazolium (MTT) was purchased from Solarbio Science& Technology. Enzalutamide (MDV-3100) was purchased from J&K. All other chemicals were obtained from Wuhan Tianzhi Biological Co., Ltd.LNCaP (Human prostate cancer cell line) was purchased from Hangzhou Qiannuo Biotechnology Co., LTD . Cos-7 (African green monkey kidney cell transformed with SV40) was kindly provided by Stem Cell Bank of the Chinese Academy of Sciences . LNCaP cells were propagated in RPMI 1640 medium (Hyclone) supplemented with 10% FBS and 1% penicillin—streptomycin. Cos-7 cells were grown in DMEM medium (HyClone) supplemented with 10% FBS and 1% penicillin-streptomycin. Cell cultures were maintained in a humidified atmosphere  was supplemented to each well, and followed by another 4 h of incubation. Then 150 μl of three linked dissolved solution per well was added to dissolve the crystal of formazan for overnight . Afterwards, the absorbance at 570 nm was measured by a microplate reader, and the ICAR-antagonist nature of test compounds was exhibited by luciferase reporter gene assay using Cos-7 cells. The Cos-7 cells were seeded in 24-well plates at a density of 40,000 cells per well and incubated for 24 h, pcDNA3.1-AR, pMMTV-Luc and pRL-SV40 vectors were co-transfected into Cos-7 cells using lipofectamine 3000 according to the manufacturer's instructions. After transfection for 24 h, cells in each well were treated with 10 nm DHT or 10 nm DHT with 10 μm compounds, and placed in the incubator for another 24 h. Then cells were harvested with 150 μL of cell passive lysis buffer, Luciferase activity was determined with luciferase assay systems (Promega) following the manufacturer's protocol to detect the AR mediated transcriptional activity.The result of the experiment shows the average value of the results from three independent repeated experiments. Statistical analysis was performed using the GraphPad Prism 5.0 statistical software . Data are expressed as means ± standard deviation (SD). Statistical analysis was carried out using one-way ANOVA followed by Tukey's multiple comparison, with p < 0.05 considered statistically significant.After statistical analysis of the TCM prescriptions, we found that 82 herbs have high frequency (≥10) in the prescriptions and we summarized them in Virtual screening techniques have become a reliable and relatively inexpensive technique for the discovery of lead compounds and have been widely used in hit identification. Docking power is a critical indicator of docking reliability for reproducing the experimental combination mode of antagonists  as the positive control, LNCaP cell was treated with different concentrations of enzalutamide and compounds for 120 h.50 value of 17.21 μM, while the compound MoL_11 obtained by virtual screening has an IC50 value of 35.00 μM, which is about twice of that for Enz. Among all the tested compounds, the IC50 value for Mol_11 is the smallest, and the activity is relatively good. MoL_11 is derived from Gardenia jasminoides Ellis, also known as Isoimperatorin. The IC50 values of MoL_12, MoL_16, and MoL_19 increase in sequence. MoL_12 is suzilactone, which is found in Cistanche deserticola Ma and Panax ginseng C. A. Mey. MoL_16 is perillyl alcohol, present in Salvia miltiorrhiza Bunge. MoL_19 is the original opioid, and Panax ginseng C. A. Mey., Cortex Phellodendri Chinensis all have this ingredient. The IC50 value of MoL_21 is close to 5 times that of enzalutamide, which is orange peel olein and is present in the Cortex Phellodendri Chinensis.The MTT results indicated that Enz and the five compounds can inhibit the growth of LNCaP cells in a dose-dependent manner, shown in 50 values, the most active enzalutamide has the largest cdocker interaction energy, followed by the second best activity MoL_11, then MoL_16 with the third docking value, but its activity value is in the fifth place, At the same time, MoL_21 with the worst activity value, ranked second to last in cdocker interaction energy. The best cdocker interaction energy of MoL_10 has no activity value in the MTT experiment among all the compounds in From the cdocker interaction energy in The dual luciferase reporter gene can detect the expression and regulation of AR protein. PMMTV-Luc was constructed as a reporter gene plasmid, which contains 5,005 bp base pairs and 216 bp MMTV-LTR promoter sequences. MMTV-LTR is a long terminal repeat sequence of mouse breast tumors. It has four ARE sequences and can be activated as a steroid response element. pcDNA3.1-AR, pMMTV-Luc and pRL-SV40 vectors were co-transfected into Cos-7 cells, and then the dual luciferase reporter gene kit was used to detect whether the compound acted on AR.Through the MTT cell proliferation experiment, five small molecules with significant inhibitory effects on cells were selected to study AR reporter gene experiments. DHT was used as the model group, and enzalutamide was used as the positive control group. With reference to the model group, the positive control group had a significant inhibition of AR expression, as shown in From the above experimental results, we know that compound Mol_11 can be a promising lead compound deserved further study. Then we conducted MD simulations to investigate the interaction of Mol_11 with AR, taking Enz as a compare. We did four different MD analyses as stated in section 2.1.7, i.e., Enz/WT AR, Enz/F876L AR, Mol_11/WT AR, and Mol_11/F876L AR.After 100 ns molecular dynamics simulation of the systems, the root mean square deviation (RMSD) was used to analyze and evaluate the stability of the entire simulation system. The RMSD values of the protein skeleton structure, amino acid residues within 5 Å of the active pocket and ligand atoms were calculated, as shown in From the RMSF value analysis chart of the last 10 ns MD trajectory in Taking the RMSD of the ligand atom as the abscissa and the RMSD of the amino acid residues within 5 Å of the active pocket as the ordinate, the protein conformation was extracted through the lowest energy point to find the dominant conformation.The α Helix-12 (H12) is a stretch of folding protein (residue 892–908 region) on the ligand binding domain of androgen receptor, and is a molecular switch for AR activation and inactivation. When AR is combined with androgen, the conformation of the H12 position changes, thereby covering the hormone-binding pocket and forming the activation functional area 2 (AF-2). This surface can recruit co-activators to promote AR transcription. If the small molecule destroys H12 and keeps it away from the binding pocket area, it is difficult to form an activation functional area, and then it will inhibit the transcription of AR and achieve the purpose of treating disease. This process is difficult to find directly in biological experiments, so it is further explained with the help of molecular dynamics simulations. Next, the action mode of the small molecule was observed through the H12 conformational change.The combined free energy of each system is listed in The total binding free energy was decomposed to each amino acid, looking for key amino acid in the interaction between small molecules and proteins. From −1 to −3.1034 kcal • mol−1, which proves that the interaction between MoL_11 and H12 is enhanced, which makes H12 deviate from the binding pocket, expose the binding site, and destroy the formation of the co-activation site, which is consistent with the analysis of the binding mode diagram of In Due to the complex composition of traditional Chinese medicine, the mechanism of action is not clear. In this study, virtual screening of anti-prostate cancer traditional Chinese medicine ingredients is performed to obtain potential antiandrogens. The biological activity of the selected compounds was verified by biological experiments, and the small molecule MoL_11 was found to own the best activity. The molecular dynamics simulation method was used to study the mechanism of action between MoL_11 and wild-type AR/ F876L AR. It was found that MoL_11 will not cause resistance to F876L theoretically. The newly discovered compound, Isoimperatorin, can be a lead compound used for potential anti-PCa drug discovery.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.in vitro evaluation of hit compounds. YS and JS helped to do the in vitro evaluation of hit compounds. ZZ carried out molecular-dynamics simulations. XW guided the in vitro experiments. JL conceived and coordinated the study. HL helped to discuss the results. All authors analyzed the results and approved the final version of the manuscript.WH undertook structure-based virtual screening and 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.


Anti-Androgen Receptor (AR) therapy holds promise for a subset of AR expressing triple-negative breast cancer (TNBC) patients. However, current AR assays are suboptimal in detecting the dynamic range of AR expression, contributing to its controversial role in TNBC disease prognosis. This study is aimed at evaluating the feasibility of qRT-PCR to sensitively and robustly detect AR mRNA levels for prognostication.mRNA expression profiling was performed on FFPE blocks from a retrospective cohort of 101 TNBC patients using qRT-PCR and compared with AR protein expression by immunohistochemistry . Statistical analyses included Spearman’s rank correlation, Chi-square and Kaplan-Meier analyses. Distant Metastasis Free Survival was used as the end point in survival analysis.p = 0.02), high FOXA1/GATA3 (p < 0.05) and distant recurrence. AR mRNA positive patients had poorer DMFS . DMFS dropped further to 26% (p = 0.006) in AR (+)/high FOXA1/GATA3 patients. AR mRNA expression together with node positivity had the worst DMFS  compared to patients who were either positive for any one of these, or negative for both AR and node status. Low Ki67 mRNA with AR mRNA positivity also had poorer DMFS  compared to patients expressing low Ki67 with no AR mRNA expression.AR mRNA expression was observed in 34/101 patients (34%) whereas 12/80 cases (15%) were positive by IHC. qRT-PCR could thus detect more AR positive patients as compared to IHC, with 75% (9/12) concordance between the two methods. Co-expression of GATA3 and FOXA1 mRNA was observed in 85 and 88% of AR mRNA positive tumors, respectively. AR mRNA positivity was significantly correlated with age at disease onset (qRT-PCR was more sensitive and reliable in detecting the dynamic expression levels of AR compared to IHC and this variation could be explained by the higher sensitivity of the former method. High AR mRNA expression was strongly associated with expression of AR protein, high FOXA1/GATA3 mRNA, and with poor prognosis. qRT-PCR was more efficient in detecting the AR positive cases compared to IHC. A distinct signature involving high GATA3/FOXA1, low Ki67, and node positivity in AR mRNA positive tumors correlated with poor prognosis. Thus, AR mRNA screening can serve as an effective prognostic marker along with offering potential targeted therapy options for TNBC. The incidence of triple-negative breast cancer (TNBC) varies from 6.7 to 27.9% in different countries, with the highest percentage reported in India . TNBC exA phase II trial MDV3100–11) has demonstrated the clinical utility of the AR antagonist enzaliutamide in a large cohort of locally advanced AR-positive TNBC  was combined with node status (N+ or N0), DMFS of AR(+)/N(+) patients were significantly lower  as comp = 0.001) as compared to low proliferative tumors with no AR expression (DMFS of 75%)  tumors was associated with poorer prognosis  Fig. d.We have previously used a combination of biomarkers, clinical parameters and a machine learning algorithm to develop CanAssist Breast, a cost-effective prognostic test for hormone positive breast cancer –24. The There are very few studies that have investigated AR mRNA expression in BC patients and none of these studies probed into correlation of mRNA expression with disease prognosis , 31. In Our study also found correlation between AR expression and prognosis to be dependent on the method of detection used. AR positivity by qRT-PCR correlated with poor prognosis while positivity by IHC correlated with a better prognosis, as reported in previous studies , 37. ThiResults of the correlation analysis of AR mRNA with clinicopathological features revealed that only age and grade of the tumors correlated with AR positivity. Correlation of age with AR positivity has been reported in previous studies by IHC methods . The corEvidences show that AR expression in TNBC is associated with enrichment of hormone-regulated pathways, including steroid synthesis and androgen/estrogen metabolism  similar The results of this study indicate that TNBC comprises a subset of AR (+) tumors with luminal marker expression. qRT-PCR is a sensitive method to detect low transcript copies of all these markers in tissues which lacks protein expression. AR mRNA expression accompanied by its co-regulators, FOXA1, and GATA3 indicates a luminal phenotype. These data also indicate AR expression itself is an independent marker for poor prognosis in TNBC. Concurrent evaluation of AR and its co-regulators suggests that the three-marker combination of AR/FOXA1/GATA3 could be superior to AR alone as a prognostic marker. However, this requires further examination in a larger cohort since the AR/FOXA1/GATA3 cases are a small subset of AR (+) cases in the current cohort. Therefore, AR positivity in TNBC patients offers alternative targeted therapy options for better management of the disease.Additional file 1 : Table S1. Primer and probe sequence of all the genes in the study. Table S2: FOXA1 and GATA-3 co-expression at different threshold of AR mRNA. Table S3: Comparison of AR qRT-PCR positive cases with AR Protein Expression by IHC. Table S4: Concordance between AR protein expression and AR mRNA at different thresholds of expression. Table S5: Univariate and Multivariate Analysis using Cox proportional hazard methodAdditional file 2 : Figure S1: Analysis of assay efficiency: PCR efficiency of each gene was assessed using 10- point serial dilutions (log2) (1:2 with nuclease free water) of cDNA generated from a pooled RNA test sample. Starting concentration for GAPDH, RPLPO and FOXA1 assays was 2^3 and that of target assays was 2^6. Efficiencies of GAPDH and RPLPo ; Efficiencies of FOXA1, AR. Ki67 and GATA-3 respectively . The assay was considered linear if the deviation from linearity, i.e. the difference between the best and the linear regression model, did not exceed 1 Cq value. The efficiency was calculated for each gene using the formula mentioned in supplementary methods and was ranging from 87.14 to 106%. Figure S2: Concordance between Ki67 protein and mRNA Expression. IHC was performed on 21 cases which were also positive for AR by qRT-PCR. The bar graph of the correlation analysis indicated a 62% concordance between the two methods. Figure S3: Effect of AR protein and different AR mRNA threshold levels in TNBC prognosis. (a) The distant metastasis free analysis of patients with (+) and (−) AR protein expression by IHC. (b) DMFS of TNBC patients stratified based on AR low/no (< 1) vs high (> 10.0) mRNA levels.Additional file 3: Supplementary method. Primer and probe effciency study.


This article is inspired by a pseudo Oxford-style debate, which was held in Tel Aviv University, Israel at the International Conference on Virtual Rehabilitation (ICVR) 2019, which is the official conference of the International Society for Virtual Rehabilitation. The debate, between two 2-person teams with a moderator, was organized by the ICVR Program committee to address the question “Will virtual rehabilitation replace clinicians?” It brought together five academics with technical, research, and/or clinical backgrounds—Gerry Fluet, Tal Krasovsky, Anat Lubetzky, Philippe Archambault, W. Geoffrey Wright—to debate the pros and cons of using virtual reality (VR) and related technologies to help assess, diagnose, treat, and track recovery, and more specifically investigate the likelihood that advanced technology will ultimately replace human clinicians. Both teams were assigned a side to defend, whether it represented their own viewpoint or not, and to take whatever positions necessary to make a persuasive argument and win the debate. In this paper we present a recapitulation of the arguments presented by both sides, and further include an in-depth consideration of the question. We attempt to judiciously lay out a number of arguments that fall along a spectrum from moderate to extreme; the most extreme and/or indefensible positions are presented for rhetorical and demonstrative purposes. Although there may not be a clear answer today, this paper raises questions which are related to the basic nature of the rehabilitation profession, and to the current and potential role of technology within it. But first, we must define virtual reality (VR). Jaron Lanier, who is credited with first coining the term virtual reality, has defined it in many ways, some more poetic than others ),A final but alarming point is safety: what happens when things go wrong? HMDs, for example, have the advantage of providing 3D, stereoscopic vision, increasing realism and sense of presence. However, a still unsolved issue is the possibility of appearance of symptoms of cybersickness, such as nausea, vertigo and disorientation . AlthougTo summarize the “no” side, despite the incredible advancement of technologies in recent years, we can safely state that “technologies are only as good as their makers” . The issTechnology has been advancing at an exponential rate for many decades (Moore’s Law), hence where we will be in 10–20 years in not yet known. For the rehabilitation field, it is unknown whether we will have a contemporary Luddite rebellion in our future or instead an age of technophilia, which will allow for a rapid adoption of virtual rehabilitation. The points raised in this debate highlight the complexity of the issues surrounding this question. Prior to the debate, 100% of the clinicians, scientists, and technologists in the audience all voted ‘no’. However, a post-debate vote revealed the audience was split in their support for either side. We believe that this is due to the fact that while the current state of affairs clearly supports a “no” (as evidenced by the fact that VR has not replaced clinicians), considering the possibilities, the future state of affairs may suggest a “maybe” (Fig. are the driving force [In the process of technological change, humans ng force . Clinica


Actual motor competence (MC), perceived motor competence (PMC), and health-related fitness (HRF) exhibit a dynamic and reciprocal relationship in child populations, but little is known about the nature of these relationships in young adulthood. The purpose of the study was to assess these relationships in a sample of college-aged males. A total of 55 participants enrolled in an undergraduate Kinesiology course completed the study. Perceived motor competence (PMC) was assessed with the Physical Self-Perception Profile questionnaire; MC was assessed using maximum throw and kick speed and maximum jump distance; HRF was assessed with a two-minute push-up test, two-minute sit-up test, and the Multistage 20-m Shuttle Run Test. Pearson’s bivariate correlations were calculated to assess relationships among PMC total score, MC scores, and HRF scores. Two separate indices were calculated to create composite total MC and total HRF scores used for subsequent analyses. Significant correlations were found between PMC total score, MC index, and HRF index. Multiple linear regressions were used for analyzing predictive measures for HRF and PMC scores. From the two regression models, significance varied among total MC scores, PMC scores, and HRF individual measures. These findings may suggest that relationships among MC, HRF, and PMC strengthen over developmental time in young adult males. Many factors contribute to the capability of an individual to perform skilled movement. Motor competence (MC) is a global term that describes an individual’s level of ability to perform gross fundamental motor skills such as throwing, kicking, jumping, and running . MC has In 2008, Stodden and colleagues  proposedAlthough Stodden and colleagues  contend A total of 57 male participants from a university in the northeast region of the United States were recruited to participate via an undergraduate Kinesiology course with an associated laboratory session. Participants were recruited from their scheduled lab session, each of which held approximately 20 students. Students were given the option to participate in the study or complete the regularly scheduled lab assignment for the week in which testing was scheduled. Students that chose to participate in the study earned the same number of points that could have been earned by completing the lab assignment. Exclusion criteria included having sustained an orthopedic injury in the last six months or the presence of any known cardiorespiratory conditions. A total of 55 men completed all measures for the study . Two students who were absent the day of testing opted to complete the lab assignment rather than participate in the study. Prior to the onset of the study, participants completed an informed consent and approval was obtained by the university’s Institutional Review Board .Participants completed the informed consent, the Physical Self-Perception Profile, and had their height and weight taken by a member of the research team during a previously scheduled class time one week prior to the testing day. The MC tests and the HRF tests, respectively, were completed in a large indoor recreation area that was easily accessible for all participants. Four separate data collection sessions were conducted over the course of a single day, with 12–15 participants in each collection session. Upon arrival to the recreation area, all participants performed a 10-min, unstructured (but required) warm-up that consisted of jogging around the area, stretching, and/or throwing/kicking with the available equipment. MC testing took place immediately following the warm-up period. Throwing speed was measured first, followed by kicking speed. Participants completed the jumping task with a member of the research team while waiting to complete the kicking and throwing measures. After all participants completed the three MC tests, the HRF portion took place. The push-up test was conducted first, followed by the sit-up test. Both tests had alternating turns for their partners, which allowed for adequate rest time between the MC assessments and push-up assessments. If participants requested a longer rest time, it was granted. Last, the Multistage 20-m Shuttle Run Test was conducted.Age and sex were self-reported on a brief survey attached to the informed consent. Participants’ standing height and weight were directly measured using a portable stadiometer  and a portable scale , respectively. Height was measured to the nearest 0.1 cm and weight was measured to the nearest 0.1 kg.The revised version of the Physical Self-Perception Profile  was usedGross MC was assessed in a single testing session using product (outcome) scores of three tasks: throwing, kicking, and jumping; this approach has been used in other studies to examine MC in a young adult population ,26. PartParticipants completed three tasks to assess HRF: push-ups, sit-ups, and the Multistage 20-m Shuttle Run Test . The push-up test and sit-up test followed the same protocol used in the U.S. Army Physical Fitness Test . A timedNext, abdominal strength and endurance were assessed using a two-minute sit-up test . A membe−1 and increased by 0.5 km/hr−1 each minute. When participants were further than 2 m from the line when the beep sounded (or when participants self-selected to stop running), their test was completed and the number of total laps ran was recorded. Verbal encouragement was given by the research team and the non-active partner throughout the test. After the active participant completed the test, the partners switched roles and the test was repeated.The final measure of HRF was the Multistage 20-m Shuttle Run Test  , which wAll data were analyzed using IBM SPSS version 25.0 . Prior to data analysis, all variables were assessed for normality of distribution using a Shapiro–Wilk test. Means and standard deviations were calculated for age, height, weight, and BMI. Likewise, means and standard deviations were also calculated for MC, HRF, and PMC measures. An MC index was created as an “overall” MC score for each participant by averaging maximum scores on throwing, kicking, and jumping. Similarly, an HRF index  was calculated for each participant by averaging the number of push-ups, sit-ups, and laps completed. A series of Pearson’s bivariate correlations were calculated on individual variable scores . Pearson’s bivariate correlations were also calculated for the MC and HRF index scores and PMC scores. Lastly, two multivariate regressions were calculated based off significant findings from the correlation analyses. The first regression analysis used PMC scores and the MC index to predict HRF index scores; the second analysis used individual HRF measures to predict PMC scores.2. Most were right-hand (89.1%) and right-foot (85.5%) dominant. Results from the Shapiro–Wilk test indicated that all outcome variables were normally distributed (p > 0.05). Means and standard deviations of physical characteristics and outcome measures are presented in r = 0.352, p < 0.01); jumping and throwing ; and jumping and kicking . Throwing was not significantly correlated with any other variable outside of MC measures. Within HRF variables, sit-ups and laps ran  were significantly, moderately correlated, whereas sit-ups and push-ups were not (p > 0.01). Jumping and push-ups were both significantly correlated to PMC score .Pearson’s bivariate correlations among individual MC and HRF variables and the PMC total score are presented in r = 0.355, p < 0.05).A second series of Pearson’s bivariate correlations was conducted for the three main constructs of the study: MC index, HRF index, and PMC total score . The onl2 = 0.167, F = 4.56, p = 0.015).A multiple linear regression was conducted  and reve2 = 0.183, F = 3.37, p = 0.027).A second multiple linear regression was conducted  with indn = 55). Compared to a similar cohort of college-aged males, our sample demonstrated poorer throwing, kicking, and jumping [The purpose of this study was to investigate relationships among MC, HRF, and PMC in male Kinesiology students ( jumping . Lower s jumping .n = 1045) motor skill proficiency in comparison with their adolescent  object control skills, locomotor skills, and cardiorespiratory fitness [Significant correlations among the individual MC scores, individual HRF scores, and PMC scores existed . We cont fitness . Their f fitness , it is rr = 0.48, r = 0.49, r = 0.59, respectively). Our lack of findings here could be attributed to the order of testing. By the time participants completed the sit-ups, they had warmed up, completed five trials of throwing, five trials of kicking, and 2 min of push-ups, all of which likely created fatigue in their abdominal region. Even though testing measures only accumulated about 90 min in total, mental fatigue could have also been a factor. Activities or exercises performed the previous day were not recorded and could have left participants fatigued.The sit-up variable was only significantly related to cardiorespiratory endurance; there were no other significant relationships within HRF measures. This contradicts Stodden et al. , who four = 0.350, p < 0.01), and push-ups , which indicates individuals who are more skilled have higher perceptions of competence compared to their less-skilled counterparts. Jumping far or having the ability to perform more push-ups can require practice and training over time. If participants know they have not practiced skills like jumping or push-ups (or recently incorporated them into a training regimen), then they may perceive themselves as less likely to be able to do them and vice versa. These findings oppose a previous study by Wang et al. [Results indicated that PMC was significantly correlated to both jumping . It is not surprising that males who have higher physical perceptions of themselves also are highly physically fit. Males tend to value the effects of hypertrophy and muscle strength when exercising, which is evident in their outward appearance [When MC and HRF were considered as total constructs , correlapearance . Therefopearance  who founThe study was not without limitations. Females were not assessed in the sample and therefore, findings cannot be generalized to this population. The sample was also comprised exclusively of Physical Education and Coaching majors, so participants’ performance on the variables that were measured may not be representative of the general population. Additionally, the protocol used to collect HRF data relied upon participant counts, and while they were instructed on proper protocol and monitored by the research team, we acknowledge that some error in recorded scores may have occurred. We also could not control for the motivation of the participants. While verbal encouragement was given to all participants during the HRF and MC testing, and participants were instructed to put forth their best effort, it is likely that some participants were inherently more motivated to perform than others. It is plausible that individuals who were less motivated were inherently less physically fit or less inclined to give their best effort. Though our data suggest that the relationship between MC, HRF, and PMC exists in young adulthood, these findings cannot be confirmed without longitudinal data. Furthermore, physical activity was not assessed in the current study, but should be included in future studies to further understand how these health variables influence the trajectory towards overall health. Future research should consider including a young adult sample of both males and females in longitudinal studies of this nature, as the few longitudinal studies that exist only track MC, HRF, and PMC in childhood. It would also be important to examine the MC/HRF/PMC relationship in young adults from fields other than Physical Education and Coaching.In summary, our study indicates that there are relationships among MC, HRF, and PMC in a college-aged male sample, but these relationships are expressed differently depending on individual skills and the type of measurement used. As expected, males reported high scores on the Physical Self-Perception Profile questionnaire. Males’ HRF was significantly correlated to their PMC total scores, indicating that men who perceived themselves as being physically competent were actually competent. MC and PMC total scores were predictive indictors of their HRF, and individual measures of HRF were predictive of their PMC total score. Overall, our findings suggest that MC, HRF, and PMC are related in a male college population which can help define the importance of practicing motor competency and maintaining a healthy lifestyle for younger populations.


Mechanistic studies indicated that lnc-TSI specifically inhibited the phosphorylation of Smad3 and subsequent EMT by binding with the MH2 domain of Smad3 to block the interaction between Smad3 and TGF-β receptor I in ccRCC cells. In a cohort of 150 patients with ccRCC, expression of lnc-TSI in tumors was negatively correlated with phosphorylated (p)Smad3 and activated EMT markers. Patients with expression of tumor lnc-TSI greater than or equal to the median at radical nephrectomy had a higher survival rate compared to those with lnc-TSI below the median during follow-up. These findings reveal a new regulatory mechanism of ccRCC metastasis and suggest a potential molecular target for the development of anti-cancer drugs.The transforming growth factor-β (TGF-β)/Smads signal plays an important role in cancer metastasis by mediating the epithelial-mesenchymal transition (EMT) in cancer cells. lnc-TSI is a recently identified long noncoding RNA that negatively regulates the TGF-β/Smads signal. The present study was conducted to test the hypothesis that lnc-TSI inhibits metastasis in clear cell renal cell carcinoma (ccRCC) by regulating the TGF-β/Smad3 pathway. Herein, we show that lnc-TSI was upregulated in ccRCC cells and tissue and was associated with activation of the TGF-β/Smads signal. Depleting lnc-TSI enhanced tumor cell invasion and metastasis   TGF-β/Smad3 signaling has been found to play an important role in cancer metastasis. Wang et al. show that lncRNA lnc-TSI inhibits cancer cell invasion and tumor metastasis via specifically inhibiting the phosphorylation of Smad3 and subsequent EMT, which could be potentially applied in treatments for cancer metastasis. We demonstrated for the first time that lnc-TSI inhibited the metastasis of ccRCC via inhibition of the phosphorylation of Smad3 and the subsequent EMT. This discovery adds to our understanding of the complex regulation of EMT in ccRCC and provides a potential new therapeutic target for this malignant tumor.GSE14994 (n = 37) and GSE6344 (n = 20) were downloaded and analyzed for the expression of TGF-β1 mRNA. TGF-β1 transcription was upregulated in ccRCC tissue compared to that in renal tissue from normal subjects  with TGF-β1. Cells exposed to TGF-β1 showed increased invasiveness D and prein situ hybridization (FISH) assay (The expression levels of lnc-TSI in ccRCC cell lines (Caki-1 and 786-O) were significantly upregulated as compared to a normal human tubular epithelial cell line (HK2) G and 1H.H) assay J.Furthermore, knockdown of Smad3 significantly reduced lnc-TSI promoter activity K. ChromaOur previous study showed that lnc-TSI inhibits TGF-β1 signaling by specifically hindering the phosphorylation of Smad3 in tubule epithelial cells.Immunofluorescence staining showed that knocking out lnc-TSI increased Smad3 nuclear translocation, while forcing expression of lnc-TSI attenuated TGF-β1-induced Smad3 nuclear translocation in Caki-1 cells C. To furin vitro followed by immunoblotting assays. The results showed that lnc-TSI specifically bound with Smad3, but not with other TGF-β1 signaling-related proteins, such as SARA, Smad2, Smad4, Smad7, and TβRI  and colony formation assays showed that there was no significant effect of knocking out or overexpressing lnc-TSI on cell proliferation  compared to those with lower expression (expression index < median) at nephrectomy (p = 0.007) G. Cox reIn this study, we identified a lncRNA, lnc-TSI, which was expressed in ccRCC cells and functioned as a negative regulator for tumor metastasis. In ccRCC cells, lnc-TSI specifically inhibited the phosphorylation of Smad3 and subsequent EMT by binding with the MH2 domain of Smad3 to block the interaction between Smad3 and TβRI. lnc-TSI knockout promoted ccRCC metastasis whereas its overexpression drastically inhibited ccRCC metastasis. More importantly, in a cohort of 150 patients with ccRCC, expression levels of tumor lnc-TSI at radical nephrectomy was negatively correlated with pSmad3 and EMT markers in ccRCC tissue. Lower expression of lnc-TSI was significantly associated with a decreased risk of survival rate during follow-up, suggesting that lnc-TSI may serve as a potential therapeutic target for ccRCC.,TGF-β-induced EMT has been implicated in tumor progression and metastasis in many types of carcinomas, including ccRCC.,in vitro and attenuated ccRCC lung metastasis in vivo. Alternatively, overexpression of lnc-TSI inhibited ccRCC cell invasion and tumor metastasis. To further test the effect of lnc-TSI expression in human ccRCC, we analyzed lnc-TSI expression in ccRCC tissue at the time of radical nephrectomy in 150 patients with ccRCC. We found that the expression of lnc-TSI seemed higher in patients with a tumor diameter ≤7 cm or with AJCC grades I and II. However, these results need to be further confirmed due to the small sample size of patients with larger tumors or with AJCC grades III and IV in our study population. The expression of tumor lnc-TSI was correlated with the expression of pSmad3 and the alterations of EMT markers, which suggested that lnc-TSI may regulate the phosphorylation of Smad3 and subsequent EMT in human ccRCC. More importantly, patients with lower expression of tumor lnc-TSI at radical nephrectomy had lower survival rates, compared to those with higher lnc-TSI expression, during a 30.7-month mean follow-up, suggesting that the expression level of tumor lnc-TSI may predict the prognosis of patients with ccRCC.ccRCC is the most common subtype of kidney cancer, and it can metastasize to various organs.Until now, the therapeutic design that targets the TGF-β pathway in clinical trials for cancer treatment has been focused on antibodies, antisense oligonucleotides, and kinase inhibitors for TGF-β or its receptors.There are limitations in this study. First, we did not evaluate the effects of lnc-TSI on other subtypes of RCC such as papillary and chromophobe, and the results may not generalize to these renal carcinomas. Second, the relationship between lnc-TSI expression and tumor metastasis or recurrence after radical nephrectomy and clinical survival analysis needs to be validated in a larger cohort study in the future.In summary, our study showed that lnc-TSI was a critical modulator of human ccRCC metastasis. A potential molecular mechanism underlying the effect of lnc-TSI is the negative regulation of the TGF-β/Smad3-induced EMT by the inhibition of phosphorylation of Smad3 through blocking the interaction between TβRI and Smad3. lnc-TSI could serve as an independent predictor for long-term mortality in patients with ccRCC and may represent a potential therapeutic target to inhibit the metastasis of ccRCC.2. For TGF-β1 stimulation, cells were serum-starved overnight when they reached 65%–75% confluence, and then they were incubated with various concentrations of recombinant human TGF-β1  for the indicated time periods  and human RCC cell lines (Caki-1 and 786-O) used in this study were commercially available . Caki-1 cells were cultured in McCoy’s 5a modified medium  supplemented with 10% fetal bovine serum (FBS) . 786-O cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific). Cells were maintained at 37°C in a humidified incubator containing 5% CO periods E and S1FTo generate lnc-TSI knockout cells with CRISPR-Cas 9 gene editing, two sgRNAs targeting the gene of lnc-TSI located on chromosome 21 were cloned into U6-multiple (sgRNA)-SV40-EGFP. A Cas9-expressing vector was constructed as a Lenti-Cas9-Puro vector . For lnc-TSI knockout CRISPR-Cas9 clone 1, the two sgRNA sequences were as follows: sgRNA1, 5′-TTCCAGCCAGGTATCAGAGT-3′, sgRNA2, 5′-CACTGTGGGCATCTCAACCC-3′. For lnc-TSI knockout CRISPR-Cas9 clone 2, the two sgRNA sequences were as follows: sgRNA1, 5′- GAGCTAATGCTGGTGCCGTG-3′, sgRNA2, 5′-GGCTGTCGAGGCTGCAGCGA-3′. Both lentivirus-sgRNA and lentivirus-Cas9 were customized by GeneChem and applied to co-transfected target cells according to the manufacturer’s instructions.For ectopic expression of lnc-TSI, the full length of lnc-TSI cDNA was amplified by PCR and cloned into pCDH-EF1-MCS-Puro vector to generate the overexpressing plasmid. The empty pCDH-EF1-MCS-Puro vector or pCDH-EF1-MCS-Puro-lnc-TSI overexpressing vector was used to produce lentivirus in HEK293T cells with the packaging plasmids pMD2.BSBG and PSPAX2. Infectious lentiviruses were collected at 72 h after transfection of plasmids and filtered through 0.45-μm polyvinylidene fluoride (PVDF) filters. To generate lnc-TSI overexpressing ccRCCs, cells were incubated with lentiviruses and polybrene  followed by puromycin selection for 72 h.For transient transfection to knock down Smad3, 50%–80% confluent Caki-1 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Sequences of siRNA  targeting Smad3 were as follows: siSmad3-1, 5′-GUGAGCAGAACAGGUAGUAUUAC-3′; siSmad3-2, 5′-GAGCCUGGUCAAGAAACUCAA-3′.−ΔΔCT).Total RNA (1 μg) was extracted using TRIzol (Thermo Fisher Scientific) and reverse transcribed into cDNA using a PrimeScript RT reagent kit  according to the manufacturer’s protocol. Real-time PCR was performed with a SYBR Premix Ex Taq II kit (Takara) on the LightCycler 480 system  with the following procedure for 40 cycles: 95°C for 2 min, 90°C for 20 s, and 60°C for 1 min. The sequences of the lnc-TSI primers were 5′-AATCTGGTGTGGACAAACGC-3′ (sense strand) and 5′-AACAGATGCTTCCGAATGCC-3′ (antisense strand). The sequences of the E-cadherin primers were 5′-GCTGGACCGAGAGAGTTTCC-3′ (sense strand) and 5′-CAAAATCCAAGCCCGTGGTG-3′ (antisense strand). The sequences of the N-cadherin primers were 5′-ATGGGAAATGGAAACTTGATGGC-3′ (sense strand) and 5′-CAGTTGCTAAACTTCACTGAAAGG-3′ (antisense strand). The sequences of the Vimentin primers were 5′-GACGCCATCAACACCGAGTT-3′ (sense strand) and 5′-CTTTGTCGTTGGTTAGCTGGT-3′ (antisense strand). The sequences of the Snail primers were 5′-TCGGAAGCCTAACTACAGCGA-3′ (sense strand) and 5′-AGATGAGCATTGGCAGCGAG-3′ (antisense strand). The sequences of the β-actin primers were 5′-TGAAGTGTGACGTGGACATC-3′ (sense strand) and 5′-GGAGGAGCAATGATCTTGAT-3′ (antisense strand). The relative expression of lnc-TSI was normalized by β-actin and calculated using the CT method  according to the manufacturer’s instructions. RT-PCR was performed with the separated nuclear and cytoplasmic fractions.Cells were fixed in 4% formaldehyde at room temperature for 15 min. Then, the cells were permeabilized in PBS containing 0.5% Triton X-100 for 10 min on ice. Applying the digoxigenin (DIG)-conjugated oligodeoxynucleotide probes  for lnc-TSI and U6, hybridization was performed in hybridization solution   overnight at 52°C in a humidified chamber. After hybridization, cells were washed in 2× standard saline citrate (SSC) for 10 min at 52°C, and then in 50% deionized formamide/2× SSC at 52°C for 30 min. The target RNA was detected with fluorescein-conjugated antibody against DIG (Roche) overnight at 4°C. Finally, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride and images were obtained by a confocal laser-scanning microscope .Paraffin-embedded tissue sections were dewaxed and rehydrated followed by digestion with trypsin  and fixation in 4% paraformaldehyde. A DIG-labeled locked nucleic acid (LNA)-modified lnc-TSI probe (Exiqon) was diluted in hybridization solution (Boster) to create a final concentration of 25 nM. The sections were hybridized with the diluted probe at 52°C for 16 h. After extensive washing, sections were incubated with anti-DIG monoclonal antibody  overnight at 4°C and then dyed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate . The staining index of lnc-TSI was calculated based on the intensity and proportion of lnc-TSI-positive cells in three random fields under a ×40 objective. The proportion of positively stained cells in each field was graded as follows: 0, no positive cells; 1, ≤25%; 2, 26%–50%; 3, 51%–75%; and 4, >75%. The staining intensity of positive stained cells in each field was recorded as follows: 0, no staining; 1, light blue; 2, blue; and 3, dark blue. The staining index for each field was determined as follows: staining index = staining intensity × proportion grade of positively stained cells. The final staining index for each case was the average of the three random fields.Northern blots were performed using a DIG luminescent detection kit (Roche) according to the manufacturer’s instructions. Briefly, total RNA (2 μg) was mixed with RNA loading buffer  and heated at 65°C for 10 min. After cooling on ice for 10 min, the samples were loaded on an agarose (1.5%)-formaldehyde (2.2 M) gel at 60 V for 4 h. The separated RNAs were then transferred to a positively-charged nylon membrane (Thermo Fisher Scientific) and crosslinked with UV light. DIG-labeled LNA-modified probes (Exiqon) complementary to the target genes were hybridized to the membrane at 55°C overnight. The sequences of the probes were as follows: lnc-TSI, 5′-ACATCTCTTAATCAGCGAATCA-3′; ACTB, 5′-CTCATTGTAGAAGGTGTGGTGCCA-3′.The human ccRCCs tissue microarray (HKidE180Su02), which included 150 carcinoma tissues and 30 matched adjacent non-tumor tissues, their clinical data at time of radical nephrectomy, and the mortality during a mean of 30.7 months of follow-up, were provided by Shanghai Outdo Biotech . All patients consented to an Institutional Review Board-approved protocol that allowed comprehensive analysis of discarded tumor samples (Ethics Committee of Shanghai Outdo Biotech).To determine whether lnc-TSI was transcribed by Smad3, we cloned the promoter of lnc-TSI between −1 and −2000 into the pGL3-enhancer vector . Caki-1 cells were transiently transfected with pRL-TK-Renilla luciferase plasmid and pGL3-enhancer carrying lnc-TSI promoter. After 24 h, cells were collected and luciferase activity was measured with the Dual-Luciferase reporter assay system  according to the manufacturer’s instructions. Firefly luciferase activity was divided by Renilla luciferase activity in every sample to determine the transfection efficiency that was used to normalize the data.A ChIP assay was performed using a Magna ChIP HiSens chromatin IP kit (Merck Millipore) according to the manufacturer’s instructions. Caki-1 cells pretreated with TGF-β1 (10 ng/mL) for 2 h were fixed with 1% formaldehyde for 10 min at 37°C. The fixed cells were quenched with glycine and collected for sonication in an ultrasonic cell disruptor  to generate DNA fragments ranging from 300 to 600 bp. IPs were performed with an antibody against Smad3 . The binding of the promoter region of lnc-TSI to immunoglobulin G (IgG) or Smad3 was quantified using real-time PCR. Precipitated DNA fragments were detected with specific primers as follows: sense, 5′-ACAGACGGGAGTCGAGTGTA-3′, antisense, 5′-CAGACATGGGCAATCCCACT-3′.Cells were lysed in IP lysis buffer (Thermo Fisher Scientific) containing protease inhibitors. Cell lysates were immunoprecipitated by anti-Smad3 . The immunoprecipitated complexes were captured by protein A magnetic beads (Thermo Fisher Scientific) on a magnet. After washing five times with IP lysis buffer, the immunoprecipitated proteins were separated by SDS-PAGE and detected by western blot.in vitro using a MEGAscript T7 high-yield transcription kit (Thermo Fisher Scientific), labeled with biotin using a magnetic RNA-protein pull-down kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 5 pmol of biotin-labeled RNA in RNA structure buffer  was heated at 95°C for 2 min, put on ice for 3 min, and then left at room temperature for 30 min for proper RNA secondary structure formation. Folded RNA (5 μg) was mixed with ccRCC cell lysates (5 mg) in 500 μL of Pierce IP lysis buffer (Thermo Fisher Scientific) and incubated at room temperature for 2 h. After gentle washing, streptavidin magnetic beads (50 μL) were added to each sample and incubated for 1 h. Finally, beads were washed carefully with IP lysis buffer five times and boiled in 1× loading buffer. The retrieved proteins were detected by western blot.The full length of lnc-TSI cDNA was amplified by PCR, the complementary sequence or the delete mutations of lnc-TSI and LacZ cDNA were synthesized , and each sequence was cloned into the pcDNA3.1 vector (Thermo Fisher Scientific). Stem-loop A delete mutation was conducted by deleting the nt 310–333 of lnc-TSI. Stem-loop B delete mutation was conducted by deleting the nt 338–412. Stem-loop C delete mutation was conducted by deleting the nt 451–534. Biotin-labeled RNAs were transcribed To determine which domain of Smad3 binds lnc-TSI, the full-length or truncated fragments sequence was separately cloned into a pCDH-CMV-EF1  construct with a FLAG tag at the N terminus. Caki-1 cells were transfected with plasmids carrying either the truncated fragments or the full length of Smad3. RNA pull-down was conducted on the transfected cells with biotin-labeled lnc-TSI as described above.in vitro as mentioned above. Then, the complementary sequence and biotin-labeled lnc-TSI at different molar ratios in RNA structure buffer were heated at 95°C for 2 min, put on ice for 3 min, and then left at room temperature for 30 min for proper RNA secondary structure formation and interaction. RNA pull-down was performed as mentioned above after lnc-TSI was preincubated with its complementary sequence.To determine whether the complementary sequence to lnc-TSI inhibits the interaction of lnc-TSI with Smad3, the complementary sequence of lnc-TSI was transcribed The extracted proteins were separated by 10%–12% SDS-PAGE, transferred to PVDF membranes (Merck Millipore), and probed with primary antibodies against Smad2 , Smad3 , Smad4 (Merck Millipore), pSmad2 , pSmad3 , Lamin A/C , SARA , TβRI (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), E-cadherin , N-cadherin , Vimentin , and Snail . After incubating with secondary antibodies, the antigen-antibody reactions were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific).To detect the location of the Smads complex, the nuclear and cytoplasmic fractions of ccRCC cells were isolated with a Paris kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Western blot was performed with the separated nuclear and cytoplasmic fractions.Cells were fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100, and probed with the specific primary antibodies against Smad3 , N-cadherin , and E-cadherin  overnight at 4°C, followed by incubation with secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). The nuclei were counterstained with DAPI. Actin filaments were stained with DyLight 594 phalloidin . Images were obtained using confocal microscopy . To quantitatively evaluate the nuclear translocation of Smad3, cells with positive nuclear Smad3 staining were calculated in 10 random fields under a ×40 objective, and then the quantity for each sample was summed up. To analyze the co-localization of lnc-TSI and Smad3, 786-O cells were subjected to a FISH assay, then fixed for 15 min in 4% formaldehyde and subjected to immunofluorescence staining.Paraffin-embedded tissue sections were dewaxed and rehydrated followed by inhibiting endogenous peroxidase with hydrogen peroxide (3%). The slides were incubated overnight at 4°C with primary antibodies against pSmad3 , E-cadherin , N-cadherin , Vimentin , and TGF-β1 (Santa Cruz Biotechnology). After extensive washing with PBS buffer, the slides were stained by an EnVision/horseradish peroxidase (HRP) kit . The staining index for pSmad3, E-cadherin, and N-cadherin in human ccRCC samples was calculated based on the intensity and proportion of positive cells in three random fields under a ×40 objective. The proportion of positively stained cells in each field was graded as follows: 0, no positive cells; 1, ≤25%; 2, 26%–50%; 3, 51%–75%; and 4, >75%. The staining intensity of positive stained cells in each field was recorded as follows: 0, no staining; 1, light brown; 2, brown; and 3, dark brown. The staining index for each field was determined as follows: staining index = staining intensity × proportion grade of positively stained cells. The final staining index for each case was the average of the three random fields.Cells were seeded into six-well plates at a concentration of 1,000 cells per well and incubated for 7 days until colonies were clearly formed. Then, the cells were fixed and stained with 0.5% crystal violet solution. Colonies containing >50 cells were counted.Cells were seeded into 96-well plates at a concentration of 200 cells per well and incubated for the indicated time points. Then 20 μL of MTS solution (Promega) was added to each well, followed by incubation at 37°C for 1 h. The absorbance at 490 nm was quantitatively measured in a microplate reader .ccRCCs cells were seeded in six-well plates and grown to confluence. The adherent cells were scraped using a pipette tip to create a scratch, washed with PBS twice, and the medium was replaced with serum-free medium. Images were captured at 0, 24, and 48 h after the initial scratch and the widths of scratches were measured.5 cells in serum-free medium was added to the upper inserts of the Transwell migration chambers . For TGF-β1 stimulation, TGF-β1 was added to the medium of the upper inserts to make a final concentration of 10 ng/mL. The lower chambers were filled with 500 μL of culture medium supplemented with 10% FBS. Cell mobility was detected after the indicated incubation period, and cells were fixed and stained with 0.1% crystal violet. The cells that penetrated the membrane were counted in five random fields.To assay the migration ability of ccRCC cells, a cell suspension containing 1 × 105 cells in serum-free medium was added to the upper inserts of the Transwell migration chambers . For TGF-β1 stimulation, TGF-β1 was added to the medium of the upper inserts to make a final concentration of 10 ng/mL. The lower chambers were filled with 500 μL of culture medium supplemented with 10% FBS. After the indicated incubation period, cells were fixed and stained with 0.1% crystal violet. The cells penetrated the Matrigel, and membranes were counted in five random fields.The upper inserts of the Transwell migration chambers (BD) were pre-coated with 50 μL of Matrigel (Thermo Fisher Scientific) overnight at 4°C. Then, 200 μL of cell suspension containing 1 × 106 cells in 200 μL of PBS were injected into the mice via the tail vein. The mice were imaged 21 days or 2 months later with luciferase-based bioluminescent imaging and analysis system  after injection of 100 μL of d-luciferin (15 mg/mL)  10 min before imaging. After imaging, lungs were harvested from euthanized mice and fixed in paraformaldehyde for the subsequent experiments.All animal experiments were approved by the Sun Yat-sen University Laboratory Animal Care and Use Committee. Male BALB/C nude mice (3-4 weeks old) were purchased from Vital River Laboratories  and applied for experimental lung metastasis experiments. Caki-1 cells with lnc-TSI overexpressed or knocked out were transfected with luciferase reporter plasmid  and elected by G418 (200 μg/mL). 2 × 10post hoc test. To compare variables that were not normally distributed, we conducted the Mann-Whitney U test. Spearman’s rank correlations were applied for analyzing the associations between lnc-TSI expression and pSmad3 or the EMT marker expression. Kaplan-Meier survival analysis was used to demonstrate the prognostic relevance of lnc-TSI in a univariate analysis. Cox proportional hazards regression analysis was applied to analyze the independent factors on the survival prognosis of patients with ccRCC. p <0.05 was considered statistically significant.Continuous variables are expressed as means ± SD. Categorical variables are shown as numbers (percentages). Data were tested for normality using the Kolmogorov-Smirnov test. To compare normally distributed continuous variables, we conducted a two-tailed Student’s t test or one-way ANOVA with a Tukey’s P.W., W.C., and T.M. performed the experiments, analyzed the data, and drafted the manuscript. Z.L. and C.L. assisted with the experiments. Y.L. provided suggestions to the study design and writing of the paper. F.F.H. designed and financed the study and wrote and edited the manuscript. All authors approved the final manuscript.The authors declare no competing interests.


Precise insertion of pedicle screws is important to avoid injury to closely adjacent neurovascular structures. The standard method for the insertion of pedicle screws is based on anatomical landmarks (free-hand technique). Head-mounted augmented reality (AR) devices can be used to guide instrumentation and implant placement in spinal surgery. This study evaluates the feasibility and precision of AR technology to improve precision of pedicle screw insertion compared to the current standard technique.Two board-certified orthopedic surgeons specialized in spine surgery and two novice surgeons were each instructed to drill pilot holes for 40 pedicle screws in eighty lumbar vertebra sawbones models in an agar-based gel. One hundred and sixty pedicles were randomized into two groups: the standard free-hand technique (FH) and augmented reality technique (AR). A 3D model of the vertebral body was superimposed over the AR headset. Half of the pedicles were drilled using the FH method, and the other half using the AR method.p = 0.389) but for novice surgeons .The average minimal distance of the drill axis to the pedicle wall (MAPW) was similar in both groups for expert surgeons .Expert surgeons showed 0 primary drill pedicle perforations (PDPP) in both the FH and AR groups. Novices showed 3 (7.5%) PDPP in the FH group and one perforation (2.5%) in the AR group, respectively (p > 0.05). Novices showed significant differences of SSPP between most groups: 6-mm screws, 18 (45%) vs. 7 (17.5%), p = 0.006; 7-mm screws, 20 (50%) vs. 10 (25%), p = 0.013; and 8-mm screws, 22 (55%) vs. 15 (37.5%), p = 0.053, in the FH and AR group, respectively. In novices, the average optimal medio-lateral convergent angle (oMLCA) was 3.23° (STD 4.90) and 0.62° (STD 4.56) for the FH and AR set screws (p = 0.017), respectively. Novices drilled with a higher precision with respect to the cranio-caudal inclination angle (CCIA) category (p = 0.04) with AR.Experts showed no statistically significant difference in average secondary screw pedicle perforations (SSPP) between the AR and the FH set 6-, 7-, and 8-mm screws (In this study, the additional anatomical information provided by the AR headset superimposed to real-world anatomy improved the precision of drilling pilot holes for pedicle screws in a laboratory setting and decreases the effect of surgeon’s experience. Further technical development and validations studies are currently being performed to investigate potential clinical benefits of the herein described AR-based navigation approach. Precise insertion of pedicle screws for spinal instrumentation is paramount to achieve primary stability in fusion surgery and to avoid possibly catastrophic complications including permanent nerve or vascular injury. Anatomic landmarks supported by fluoroscopic image guidance are utilized to determine safe pedicle screw trajectories without endangering closely adjacent neurovascular structures. The reported accuracy of pedicle screw placement varies considerably depending on the applied technique of screw placement and patient-specific factors including spinal deformity. Image-based intraoperative techniques such as 2D and 3D fluoroscopy or CT-based navigation increase the precision of pedicle screw placement but significantly increase radiation exposure to the patient and operating room personnel –7. OtherAugmented reality (AR) is a rapidly emerging technology providing the user with computer-generated information superimposed to real-world environment. Although its application in orthopedic and spine surgery today remains limited, AR was gradually introduced in different experimental medical and surgical settings –15. AdvaEighty identical sawbone models of a third lumbar spinal vertebra  were embedded in an agar-based gel  mimicking exposure of the surgical field in lumbar fusion surgery through a posterior approach. The vertebrae were embedded in different orientations by changing their angulation and rotation by approximately ± 15° in a random direction  scans  of the vertebral sawbone models were acquired and a 3D triangular surface model was generated using a commercially available software  Fig. .Fig. 33After drilling, CT scans of the vertebrae were acquired. The pilot holes of each vertebra were marked with pencil leads  to facilitate identification of the trajectories in the CT scan.The following measures were used to assess the quality of pedicle screw placement in the multi-planar reconstructions of the CT scans using standard PACS software . The minimal distance of the drill axis to the pedicle wall (MAPW) was defined as the shortest distance between the drill axis and the pedicle wall that could be measured along the entire trajectory. Larger MAPW values correspond to a more centric drill, which allows to use a bigger screw diameter without perforation of the pedicle cortex.Two measures, namely the number of primary drill pedicle perforations (PDPP) and secondary screw pedicle perforations (SSPP), were introduced to quantify drill perforation of the pedicle wall. The PDPP measure was defined as the perforation of the drill axis itself, represented by a two-dimensional (2D) line. Three SSPP measures were defined by cylinders having outer diameters equal to 6-, 7-, and 8-mm pedicle screws, respectively.The optimal medio-lateral convergent angle (oMLCA) was defined as the angle between the sagittal plane of the vertebral body and the pilot hole that would result in the best centricity of a screw within the pedicle. In case of our vertebra model, the angle was measured and calculated to be 15°. Based on the oMLCA and the measured true medio-lateral convergence angle (tMLCA) of the pilot hole, the deviation of the optimal medio-lateral convergence angle (doMLCA) was calculated as the difference between tMLCA and oMLCA. Positive values correlate with greater convergence of the pilot hole.The cranio-caudal inclination angle (CCIA) of a pilot hole was defined as the angle between the endplate  and the pilot hole. A CCIA of 0° parallel to the endplate was considered to be optimal. Positive CCIA correlated with a more caudal direction of the screw. More caudally directed screws were rated acceptable and not considered as a surgical failure, because only deviation towards the endplate are associated with increased risk of endplate perforation and early screw fatigue or failure . With ret tests were used for each skill level separately to compare FH vs. AR navigation. The interaction term of navigation method and surgeon’s level of experience was added to the model to detect potentially opposite effects of the navigation technique depending on the level of experience of the surgeon. The number of screw perforations for different screw diameters depending on the navigation method was analyzed for both levels of experience separately using Fisher’s exact test. The deviation of the orientation between placed screw and planned screw depending on the navigation technique was compared for both levels of experience separately with independent samples t tests. Level of significance was set at p < 0.05.Statistical analysis was conducted with SPSS . To measure the effect of the navigation method and the skill level of a surgeon on MAPW, independent samples p = 0.389) but higher in the screw trajectories set by the novice surgeons   Fig. . This reExpert surgeons showed a PDPP with 0 perforations in both the FH and AR groups. Novices showed a PDPP of 3 perforations (7.5%) in the FH group and one perforation (2.5%) in the AR group, respectively. There was no significant difference between FH and AR navigation for both experts and novices Table .Table 1The average SSPP for 6-mm screws were 2 perforations (5%) in the FH group and 3 perforations (15%) in the AR group for experts. For 7-mm screws, 3 perforations (15%) occurred in both groups, and for 8-mm screws, the SSPP were 8 perforations (20%) and 6 perforations (15%) in the FH and AR groups, respectively.p = 0.006). For 7-mm screws, the difference between the FH and AR set screws was 20 perforations (50%) versus 10 (25%) perforation, respectively (p = 0.013). For 8-mm screws, the SSPP were 22 perforations (55 %) in the FH and 15 perforations (37.5 %) in the AR set screws, respectively (p = 0.053).For novices Table , the avep = 0.489) , respectively.The average doMLCA was − 0.51° ± 4.20° and 0.14° ± 3.98° in the FH and AR set screws, respectively, for experts (89) Fig. . In noviThere were no significant differences of the CCIA between the AR and the FH set screws nor in the hands of experts nor novices, respectively Fig. . Interesp = 0.04) with AR. The number of low- and medium-precision screws decreased  towards an increase high-precision category (from 9 to 20).Novices drilled with a higher precision with respect to the CCIA category  fluoroscopic and three-dimensional (3D) computed tomography (CT) renderings to guide operative approach in the perioperative setting. Augmented reality has already been used as an effective tool for training and skill assessment of surgical residents . CompareHowever, in this study, the AR and FH techniques showed no significant difference in terms of primary or secondary screw perforation, average MAPW, average doMLCA, or cranio-caudal inclination angle in the hands of the expert surgeons. Several studies demonstrated significantly higher accuracy of pedicle screw placement using other navigation systems providing three-dimensional information to the surgeon compared to the free-hand technique , 23, 24.However, projecting the required information directly into the line of sight of the surgeon is considered the natural progression of these well-established methods mitigating the errors associated with attention shift by directly projecting the navigation guidance onto the surgical field , 28.In this study, the surgical procedure was simulated in a laboratory setting using third lumbar sawbone models placed apart from each other without soft tissue covering the posterior structures of the vertebra. We hypothesize that the simplicity of the model used in this study might be one contributing factor explaining why the additional three-dimensional information provided by the AR headset did not influence the precision of screw placement in the expert group. The simplicity of the experimental setup however allowed elimination of potential biases such as variation in anatomy or bone quality.This study has limitations. The AR navigation technique per se demonstrated technical challenges that still need to be overcome. The exact manual overlay of the 3D model with the real environment, also known as registration, and its correction in case of drift, is still time-consuming. The pronounced bony landmarks were medio-lateral and less so cranio-caudal, making latter alignment of the 3D model with the real anatomy more difficult. In a real operating room setting, the described registration approach would be impractical. Various methods of registration have been described including ultrasound-based techniques, reflective markers mounted on spinous processes, and non-invasive skin placed markers –29. HoweHowever, in this study, surgeons were able to perform the manual registration as previously described and the drilling of 40 pedicles in 40 different vertebras in approximately 60–90 min.Also, this study focused on superimposition of anatomy, while the surgeon had to imagine the optimal trajectory. For the future, adding a virtual representation of the optimal drilling axis could further improve usability and precision.In this study, the additional anatomical information provided by the AR headset superimposed to real-world anatomy improved the precision of drilling pilot holes for pedicle screws in a laboratory setting for surgeons not experienced in spine surgery. Further technical development and validations studies are currently being performed to investigate potential clinical benefits of the herein described AR-based navigation approach.