Augmented reality (AR) Head-Mounted Displays (HMDs) are emerging as the most efficient output medium to support manual tasks performed under direct vision. Despite that, technological and human-factor limitations still hinder their routine use for aiding high-precision manual tasks in the peripersonal space. To overcome such limitations, in this work, we show the results of a user study aimed to validate qualitatively and quantitatively a recently developed AR platform specifically conceived for guiding complex 3D trajectory tracing tasks. The AR platform comprises a new-concept AR video see-through (VST) HMD and a dedicated software framework for the effective deployment of the AR application. In the experiments, the subjects were asked to perform 3D trajectory tracing tasks on 3D-printed replica of planar structures or more elaborated bony anatomies. The accuracy of the trajectories traced by the subjects was evaluated by using templates designed ad hoc to match the surface of the phantoms. The quantitative results suggest that the AR platform could be used to guide high-precision tasks: on average more than 94% of the traced trajectories stayed within an error margin lower than 1 mm. The results confirm that the proposed AR platform will boost the profitable adoption of AR HMDs to guide high precision manual tasks in the peripersonal space. Visual Augmented Reality (AR) technology supplements the user’s perception of the surrounding environment by overlaying contextually relevant computer-generated elements on it so that the real world and the digital elements appear to coexist ,2. PartiAR has proven to be a key asset and an enabling technology within the fourth industrial revolution  ,6,7. AR Similarly, one of AR’s most common applications is during stages related to product manufacturing ,14,15,16AR technology allows the user to move and interact with the augmented scene removing the need to shift attention between the digital instructions and the actual environment . Publishtransparent interface between the user and the environment—a personal and mobile window that fully integrates real and virtual information” [Head-Mounted Displays (HMDs) are emerging as the most efficient output medium to support complex manual tasks performed under direct vision . This is owing to their ability to preserve the user’s egocentric perception of the augmented workspace and so allow the hands-free interaction with it ,19. The rmation” .One of the largest obstacles to the successful adoption of the existing technology for guiding high precision manual tasks is the inability to render proper focus cues: indeed, the majority of commercial HMD systems offer the AR content at a fixed focal distance outside the peripersonal space (>1 m), thus failing to stimulate natural eye accommodation and retinal blur effects. Recent research studies show that this not only leads to visual fatigue, but also to a proven reduction in user performance in completing a task, which requires keeping both the real and the virtual information in focus simultaneously, for example, to integrate virtual and real information for a reading task , or to cThe lack of perceptual conflicts, the accurate calibration mechanisms, system ergonomics, and low latency are the basic requirements that an AR headset should comply in order to be used as a reliable aid to high-precision manual tasks such as in surgical or industrial applications .These requirements have been recently translated into a working prototype of a new-concept AR headset developed within the European project VOSTARS . The ovThe AR platform comprising the software framework and an early version of the custom-made HMD were thoroughly described in a recently published paper . In the These results were very encouraging and they supported the claim that the wearable AR framework could represent an effective tool in guiding high-precision manual tasks.Along the same line of reasoning, with this work, we show the results of a user study whose goal is to provide a conclusive answer as to whether the AR platform under video see-through (VST) modality can be an effective tool in guiding complex 3D trajectory tracing tasks on 3D-printed replica of planar structures or more elaborated bony anatomies.This section provides a detailed description of the hardware and software components. All components are depicted in The custom-made hybrid video-optical see-through HMD fulfills strict technological and human-factor requirements towards the realization of a functional and reliable aiding tool for high-precision manual tasks. The HMD was assembled by re-working and re-engineering a commercial OST visor (ARS.30 by Trivisio ).As described in , the keyNotably, the collimation optics of the display were re-engineered to offer a focal length of about 45 cm, which constitutes, when used for close-up works, a defining and original feature to mitigate the vergence-accommodation conflict and the focus rivalry. The HMD was incorporated in a 3D printed plastic frame together with a pair of Liquid Crystal shutters and a pair of front-facing USB 3.0 RGB cameras . The steThe computing unit is a Laptop PC with the following specifications: Intel Core i7-8750H CPU @ 2.20 GHz with 12 cores and 16 GB RAM . Graphic card processing unit (GPU) is a Nvidia GeForce RTX 2060 (6GB) with 1920 CUDA Cores .The software framework is conceived for the deployment of VST and OST AR applications able to support in situ visualization of medical imaging data and specifically suited for stereoscopic AR headsets; the key function of the software, under VST modality, is to process and augment the images grabbed by the stereo pair of RGB cameras before they are rendered to the two microdisplays of the visor. The grabbed frames of the real scene are processed to perform a marker-based optical tracking, which requires the identification of the 3D position of the markers both in the target reference frame and camera reference frame.The augmented scene is generated by merging the real camera frames with the virtual content  ensuring the proper locational realism. To accomplish this task, the projection parameters of the virtual viewpoints are set equal to those of the real cameras and the pose of the tracked object defines the pose of the virtual content in the scene .The software is capable of supporting the deployment of AR applications on different commercial and custom-made headsets.The CUDA-based architecture of the software framework makes it computationally efficient.The software provides in situ visualization of task-oriented digital content.The software framework is highly configurable in terms of rendering and tracking capabilities.The software can deliver both optical and video see-through-based augmentations.The software features a robust optical self-tracking mechanism  that relies on the stereo localization of a set of spherical markers.The AR application achieves an average frame rate of The main features of the software framework can be summarized as follows :The softA 2D curve (79 mm in length) (T1).A 3D curve (130 mm in length) describing a closed trajectory on a convex surface (T2).A 3D curve (223 mm in length) describing a closed trajectory consisting of a series of four curves on concave and convex surfaces (T3).Three trajectories with different degrees of complexity were implemented to test the system accuracy:T1 was designed to test the system on a simple planar phantom simulating, for instance, an industrial manufacturing process that requires cutting flat parts to specific shapes; T2 and T3 were drawn on two anatomical surfaces , and they simulate complex surgical incision tasks.Creo Parametric software was used to design the three trajectories . T1 was The three trajectories were represented with dashed curves (0.5 mm thickness) and saved as .wrl models to be imported by the software framework and displayed as the virtual content of the AR scene.As previously mentioned, the accurate AR overlay of the virtual trajectory to the physical 3D-printed models is achieved by means of a tracking modality that relies on the real-time localization of three reference markers; for this reason, three spherical markers (11 mm in diameter) were embedded in the CAD model of phantoms as shown in Ten subjects, 3 males and 7 females, with normal visual acuity or corrected-to-normal visual acuity (with the aid of contact lenses) were recruited from technical employees and University students. The experimental setting is shown in The subject was asked to perform the “trajectory tracing” task three times for each trajectory, and to report any perceptible spatial jitter or drift from the AR content. The trajectories were administered in random order.The accuracy of the trajectories traced by the subjects was evaluated by using templates designed ad hoc to match the surface of the phantoms . The temThree templates were designed for each phantom, with different wide inspection windows, to evaluate three different levels of accuracy: given that the virtual trajectory, as well as the pencil line, have a 0.5 mm thickness, inspection windows measuring 1.5 mm, 2.5 mm, and 4.5 mm in width were designed to test a 0.5 mm, 1 mm, and 2 mm of accuracy level, respectively. We considered as successful only those trials in which the accuracy was ≤2 mm. Indeed, 1–2 mm accuracy is regarded as an acceptable range in many complex manual tasks such as in the context of image-guided surgery . When thIn the experiments, stripes of graph paper were used to estimate the cumulative length of the traced trajectory within the inspection windows . In thisA 0.5 mm pencil was used to draw the perceived trajectory on a masking tape applied over the phantom surface; the tape was removed and replaced at the end of each trial after the evaluation of the user performance.Subjects were instructed that the primary goal of the test was to accurately trace the trajectories as indicated by the AR guidance; time to completion in tracing the trajectory was recorded using a stopwatch. At the end of the experimental session, subjects were administered a 5-point Likert questionnaire to qualitatively evaluate the AR experience .The SPSS Statistics Base 19 software was used to perform statistical analysis of data. Results of the Likert questionnaire were summarized in terms of median with dispersion measured by interquartile range  in answering tendencies among subjects with different levels of experience with VR, HMDs, and VST-HMDs . For item 2 and item 7, the agreement level varied according to the expertise of the subject: for item 2, the less experienced participants, namely with no or limited experience with AR and HMDs, and, for item 7, the participants with limited experience with HMDs and VST-HMDs expressed their agreement, whereas the remaining subjects (those with more experience) strongly agreed with both items.Results of the Likert Questionnaire are reported in p > 0.05) in accuracy performances and normalized completion time between participants with different levels of experience with VR, HMDs, and VST-HMDs.Recent literature shows that HMDs are emerging as the most efficient output medium to support complex manual tasks performed under direct vision. Despite that, technological and human-factor limitations still hinder their routine use for aiding high-precision tasks in the peripersonal space. In this work, we show the results of a user study aimed to validate a new wearable VST AR platform for guiding complex 3D trajectory tracing tasks.The quantitative results suggest that the AR platform could be used to guide high-precision tasks: on average, more than In this study, the main criterion adopted to select the participants was to include subjects with different levels of experience with VR, HMDs and VST-HMDs, as the 3D trajectory tracing task was general purpose. To apply the proposed AR platform to a more specific industrial or medical application, usability tests with the final users should be performed after having defined, for each specific trajectory tracing task, the most appropriate strategy to track the target 3D surface AR registration strategy. In the field of image-guided surgery, we are currently designing the most appropriate tracking/registration strategy to perform AR-guided orthognathic surgery. In order to properly register the planned 3D osteotomy to the actual patient in the surgical room, we have adopted an innovative patient-specific occlusal splint that embeds the three spherical markers for the inside-out tracking mechanism. For this specific application, we are planning to perform an in vitro study recruiting several maxillofacial surgeons with different level of expertise in orthognathic surgery to test, on patient-specific replicas of the skull, an AR-guided osteotomy of the maxillary bone.As regards the display  latency caused by the VST mechanism, we have a direct measure of the frame rate of the tracking-rendering mechanism (i.e.,  studies ,31,32, wEven if these results should be confirmed considering a larger number of subjects sample from end users for each specific application, we believe that the proposed wearable AR platform will pave the way for the profitable use of AR HMDs to guide high precision manual tasks in the peripersonal space.


AR). It is organized in eight exons located on the X chromosome. Hundreds of genetic variants in the AR gene have been reported in CAIS. They are distributed throughout the gene with a preponderance located in the ligand-binding domain. CAIS mainly presents as primary amenorrhea in an adolescent female or as a bilateral inguinal/labial hernia containing testes in prepubertal children. Some issues regarding the management of females with CAIS remain poorly standardized . Basic research will lead to the consideration of new issues to improve long-term well-being . An expert multidisciplinary approach is mandatory to increase the long-term quality of life of women with CAIS.Complete androgen insensitivity syndrome (CAIS) is due to complete resistance to the action of androgens, determining a female phenotype in persons with a 46,XY karyotype and functioning testes. CAIS is caused by inactivating mutations in the androgen receptor gene ( Androgen insensitivity syndrome  is a main disorder (or difference) of sex development (DSD) with a 46,XY karyotype . AIS is In this paper, some aspects related to the molecular genetics, diagnosis and management of complete AIS (CAIS) are summarized, and some controversial aspects are discussed, taking into account some findings from basic research.The androgen receptor (AR) protein belongs to the superfamily of nuclear receptors also designated as NR3C4 . The AR protein consists of a 920 amino acid sequence, has a molecular mass of 110 kDa and is organized in eight exons (indicated as A–H or 1–8) and seven introns. The AR receptor is a single-stranded polypeptide consisting of four main structural domains . Co. CoAR muAR mutations (about two-thirds) are of germline origin inherited from asymptomatic mothers; in other cases, CAIS is due to somatic and de novo mutations [AR gene, but in the presence of the phenotype as well as biochemical data suggesting AIS, an altered signaling pathway due to the impairment of some coactivators or some postligand binding factor has been suggested. However, mutations in cofactor genes have not been found in the large majority of such cases; if so, this hypothesis will require more investigations [The majority of utations . It has igations .Females with CAIS present with a normal external female phenotype in girls and women with a 46,XY karyotype and normally functioning testes ,2,3,8,9.Today, the diagnosis of CAIS can also arise because of a mismatch between the prenatal sex  and the phenotype at fetal ultrasound scans or at birth. Some series reported this clinical presentation in about 3% , while iIn consideration of the X-linked transmission, it is recommended to perform a karyotype in prepubertal female relatives of a proband, given the possible recurrence of CAIS within the same family .A delay between the initial clinical suspicion and the definitive diagnosis is still present despite advances in molecular techniques in our and other’s experience ,6. DelayMalignant transformation of the gonads is the most feared complication in women with CAIS ,8,9; timGonadectomy has been performed prior to definitive molecular diagnosis in some females, and early gonadal removal may still occur in prepubertal girls ,17,18. HPostponing gonadectomy until after the age of majority requires an accurate follow-up. Döhnert et al.  proposedHypergonadotropic hypogonadism is obviously present in adolescent and adult females with CAIS and removed gonads ,2,3. AdeThere is no unique HRT protocol or any evidence-based data on the optimal hormone formulations, route of administration, doses and monitoring parameters in women with CAIS. Various formulations of estrogens are used to induce puberty or adult HRT. The more adequate formulations are likely oral micronized or transdermal (gel or patch) bioidentical 17-β-estradiol ,26. The Because testosterone is the main steroid hormone secreted by testes in women with CAIS, it may be an alternative therapy to estrogens for these females ,26. ReceProgestins are not indicated due to the absence of a uterus and possible negative effects on bone mineral density (BMD) , but sevInternational surveys should be performed to give evidence-based indications for optimal HRT in women with CAIS and removed gonads. Specific hormonal profiles of these women should be considered when monitoring HRT . Since tAndrogens are involved in the growth, development, peak achievement and maintenance of bone mass . Thus, aAR expression in cultured osteoblasts, which was later confirmed both in vivo and in vitro in osteoblasts and osteocytes. Other authors underlined the role of androgen-enhanced tissue-nonspecific alkaline phosphatase expression mediated by AR for the initial formation of hydroxyapatite crystals during osteoblastic mineralization. AR was found to preserve the number of trabeculae independently from GH/IGF1 effects, acting directly on osteocytes or indirectly by inhibiting osteoclastogenesis through interaction with osteoblast precursors, but it seems to have no direct role on osteoclasts [Experimental data in AR knockout (ARKO) mice demonstrated a phenotype with reduced bone mass and decreased stiffness. Microstructural analysis revealed a significant loss of bone volume and trabecular number with a shift towards bone resorption . In 1989eoclasts ,31. GonaFew data are available on bone health in CAIS , due to AR gene (Some studies have shown reduced BMD in women with CAIS according to both female and male reference values . Study sAR gene  with theThe effectiveness of HRT in normalizing BMD values in gonadectomized women with CAIS remains an unsolved issue . RecentlDespite the administration and good adherence to classic HRT, many women with CAIS reported a decrease in psychological well-being and sexual functioning after gonadectomy ( and persFurther longitudinal randomized trials are needed to identify the advantages of testosterone replacement therapy with respect to classical estrogen therapy in terms of psychological and sexual well-being in women with CAIS.Recent studies of ARKO mouse models uncovered new cell-type- or tissue-specific actions of AR .A decrease in myelocytes/metamyelocytes and mature neutrophils were found in bone marrow cells of ARKO mice. The defect in granulopoiesis occurs during the transition between the proliferation of precursors and the maturation of neutrophils; such defects together with a high rate of apoptosis lead to increased susceptibility to acute bacterial infection . ImpaireImpaired AR signaling can be responsible for an increased risk of metabolic syndrome, diabetes and cardiovascular diseases. ARKO mice became obese with increased body white adipose tissue mass without an increase in food uptake or dyslipidemia at ages beyond 10 weeks old; hyperinsulinemia, hyperleptinemia, hyperglycemia, hypoadiponectinemia, increased serum levels of triglycerides and free fatty acids and impaired glucose tolerance occurred with aging . These dThe ARKO mice model presents with a decreased heart size, smaller volume and wall thickness of the left ventricle and significantly reduced aortic NO synthase expression. Taken together, these observations seem to indicate that AR disruption may play some role in promoting atherosclerosis. It also seems that the AR might play some role in the initiation or progression of abdominal aorta aneurism .Data on these items are largely absent for humans with CAIS. Some of our previous data in a small group of women with CAIS and intact or removed gonads suggested that altered AR signaling may increase body fat and affect some metabolic parameters , but theAR plays key roles during male sex differentiation in utero and produces pleiotropic effects during extrauterine life; the understanding of these roles and effects has been expanded by recent basic researches ,2,3,50.


An inexperienced therapist lacks the analysis of a patient’s movement. In addition, the patient does not receive objective feedback from the therapist due to the visual subjective judgment. The aim is to provide a guide for in-depth rehabilitation therapy in virtual space by continuously tracking the user’s wrist joint during Leap Motion Controller (LMC) activities and present the basic data to confirm steady therapy results in real-time. The conventional Box and Block Test (BBT) is commonly used in upper extremity rehabilitation therapy. It was modeled in proportion to the actual size and Auto Desk Inventor was used to perform the 3D modeling work. The created 3D object was then implemented in C # through Unity5.6.2p4 based on LMC. After obtaining a wrist joint motion value, the motion was analyzed by 3D graph. Healthy subjects  were enrolled in this study. There was no statistically significant counting difference between conventional BBT and system BBT. This indicates the possibility of effective diagnosis and evaluation of hemiplegic patients post-stroke. We can keep track of wrist joints, check real-time continuous feedback in the implemented virtual space, and provide the basic data for an LMC-based quantitative rehabilitation therapy guide. In recent years, stroke, traumatic brain injury, dementia, and so on have been increasing due to increased life expectancy ,2,3. AppHowever, conventional rehabilitation therapy is tedious and environmentally limited. In addition, it requires experience and skills from the therapist ,15,16. TThe Leap Motion Controller , a new device for rehabilitation therapy, can avoid limitations of existing methods . Using LHowever, LMC rehabilitation training is still focused as a therapeutic approach. In addition, an inexperienced therapist cannot provide objective feedback to the patient by analyzing only the visual judgment of the patient’s movement. Thus, neither the therapist nor the patient receives proper feedback. The therapist needs various techniques and skills to identify patients, motivate them to participate in therapy programs, use appropriate rehabilitation techniques, and make system developments available to perform effective rehabilitation therapy. In the objective 3D analysis of the wrist joint using LMC, it is important to secure medical image data in the subjective rehabilitation field. Therefore, the objective of this study was to provide a guide for in-depth rehabilitation therapy in virtual space by continuously tracking a user’s wrist joint during LMC activities and resent basic data to confirm steady therapy results in real-time.Healthy adults  in their twenties (22.71 ± 1.65 years) who had no upper extremity musculoskeletal disease during the last three months were included in this study. Experiments were conducted on all participants after receiving experimental methods and prior informed consent.The Box and Block Test (BBT) was developed by A. Jean Ayres, and Patricia Holser Buehler uses blocks to assess the hand motor ability of adult cerebral palsy . It is sAugmented reality (AR) is a technique that has advantages of sensory engagement, interaction, and autonomy . AR can Z-axis representing the depth of field and depth of the super-wide angle of 150 degrees expressed in a 3D space. This makes it possible to accurately measure the motion of the same user as the actual one. LMC is a special device for hand movement recognition [Rehabilitation therapy using LMC can apply patient-tailored therapy in virtual space and suggest an effective rehabilitation system that can confirm the therapeutic performance. Also, LMC can recognize ten fingers up to 0.01 mm and track motion at 200 frames per second, enabling precise operation. In addition, actual hand motion is recognized as the coordinate of the ognition . It consWe used Unity5.6.2p4, a game engine, and integrated development environment, to visualize LMC in virtual space. Unity is a development tool primarily used in VR and AR content. It supports various platforms and specific functions basically. As a VR development platform, it is possible to create a 3D virtual space and display coordinates of the 3D virtual space tracked by LMC on the screen, including an additional engine that can interface with the LMC . The actWhen the system starts, a timer is displayed on the screen so that the user can check the time b. From tt-test was performed (p < 0.05) to compare the means of the two methods.All statistical analyses were performed using SPSS version 21.0 . It was assumed that there was no significant difference between conventional BBT and system BBT. The number of boxes moved in 1 min from the conventional BBT and system BBT were counted. In addition, Z-score standardization was performed to compare the data size of each group and make the data distribution similar to each other. Then, an independent Unlike existing rehabilitation therapy methods, we implemented BBT, an upper extremity rehabilitation therapy performed in virtual space. LMC can substitute expensive equipment because it is easy to implement individualized rehabilitation therapy. In addition, it can be repeatedly performed by the user alone, and the result of therapy can be seen. The system developed in this study can analyze the entire treatment process from start to finish of the movement by continually tracking the movement of the user’s hand while graphically displaying motion patterns when the user is performing system BBT. In addition, it can provide data to diagnose the patient with a small movement pattern during the test. This can be used as an objective indicator to identify the level of functional recovery by numerically representing the behavior of the patient undergoing rehabilitation therapy. In addition, unlike conventional upper limb rehabilitation system studies, it is a system that enables objective evaluation for diagnosis that is appropriate for the purpose of therapy. In addition, the user can perform rehabilitation therapy without space limitation in a comfortable environment because there is no need for expensive hardware equipment. Therefore, it is possible to implement home-rehabilitation programs that can be practiced at home by implementing the upper extremity rehabilitation therapy used in the virtual space. This can lead to a patient’s interest in a safe environment, an objective graph, and a clear goal awareness of the patient’s function recovery process.The result of the motion pattern can be obtained by 2D graphing with MATLAB using 3D coordinates of each finger obtained from the motion result of system BBT as shown in Stretch measurement is an efficient diagnostic method for ROM and muscle strength evaluation of patients with joint and muscle contraction. In addition, it can be an important indicator of objective hand tremor analysis of central nervous system diseases such as cerebral palsy, ataxia, and Parkinson’s disease. X is a numerical value, m is an average, and σ is a standard deviation. The result of the standardization process of conventional BBT and system BBT implemented in AR is shown in We performed a Z-score standardization process to make the distribution of data similar in order to show how the data of each group were different in terms of differences in the mean standard. Data of the two groups can then be viewed on the same basis . The Z-sWe enrolled 48 healthy participants  in their twenties (22.71 ± 1.65 years) in order to objectify movement tracking and diagnosis of patients after tracking the upper extremity rehabilitation therapy model in virtual space using LMC. Our results demonstrate that the system BBT is a quantitative diagnostic evaluation method by providing an objective judgment index using a coordinate change of each finger and palm of the hand through user motion tracking in 3D virtual space. In the rehabilitation field until now, the patient’s movement has been monitored using physical devices. Subjective analysis is then performed by the therapist for diagnosis and evaluation. Therefore, it is difficult to analyze the effect of individual therapy due to the different environments of each patient . In addiHowever, it is difficult to objectively evaluate the complexity of muscle tension, and synergy movement and associated reaction. Therefore, rehabilitation therapy should be made objective and accurate with a state change of the current therapy process. During activities of daily living (ADL), the hand function of the upper extremity region is the most important part ,7. BehavLMC-based rehabilitation therapy has a limit in that it only performs analysis based on the visual judgment of the therapist using only existing contents. However, the system BBT developed in this study showed similar results to the actual conventional BBT. The objective graph of the user’s movement pattern showed that the evaluator could quantitatively analyze it. Therefore, system BBT can be applied to nervous system patients in the clinic so that the therapist can recognize any abnormal pattern based on objective data. The results of this study can also be used as basic data for evidence-based diagnostic evaluation. The future work can be applied to medical research that can be evaluated by classifying the degree of recovery according to the type of upper extremity disease by recording the tracked wrist joint of the developed system BBT.


How empathically people respond to a stranger’s pain or pleasure does not only depend on the situational context, individual traits and intentions, but also on interindividual factors. Here we ask whether empathic responses towards unknown others are modulated by behavioural similarity as a potential marker of genetic relatedness. Participants watched two supposed human players who were modelled as having a strong (player LP) or weak (player NLP) tendency to lead in social situations executing penalty shots in a virtual reality robot soccer game. As predicted, empathic response were modulated by shared behavioural traits: participants whose tendency to lead was more similar to player LP’s tendency to lead experienced more reward, and showed stronger neural activity in reward-related brain regions, when they saw player LP score a goal, and participants whose tendency to lead was more similar to player NLP’s tendency to lead showed stronger empathic responses when they saw player NLP score a goal. These findings highlight the potentially evolutionary grounded role of phenotypic similarity for neural processes underlying human social perception. Importantly, significant target-perceiver effects can arise not only between personally known others, but also between strangers.Complex modern societies require individuals to interact benevolently not only with personally acquainted others but also with complete strangers. How readily individuals respond empathically to the needs and desires of their fellow human beings does not only depend on the situational context and individual traits and intentions, but also on interindividual factors. According to David Kenny4. According to kin selection theories individuals should not only act selflessly towards their close kin, but also unfamiliar others if they share a sufficiently large proportion of their genes5. Genetic relatedness to unfamiliar others is thought to be signalled through phenotypic similarity: If I help someone who is similar to me the chance is high that I help someone who shares some of my genes (“phenotype matching”6). However, currently there is little evidence that human empathic responses towards unknown others are modulated by phenotypical similarity between target and perceiver.Sociobiological accounts of human social behaviour surmise that selfless  social behaviour has emerged through kin selection10.Neuroimaging studies have examined whether humans show stronger empathic pain responses towards another person’s pain when they perceive the other person as belonging to their own ethnicity than when they perceive the other person as belonging to a different ethnicity . Although participants in these studies typically showed stronger pain-related neural responses towards targets of their own ethnicity than towards targets of a different ethnicity it remains unclear whether this response bias was due to innate behavioural tendencies or whether it arose from learned  preferences towards members of one’s own ethnicity11 investigated whether people experience more empathic reward when they see another person succeed who they perceive as sharing their own attitudes than when they see another person succeed who they perceive as having dissimilar attitudes. Perceived similarity was manipulated by the use of pre-recorded videos in which two supposed contestants verbally expressed either socially desirable or socially undesirable attitudes on social and ethical issues. After seeing the videos, participants watched the two supposed contestants betting on cards. Participants reported to experience more empathic reward, and showed stronger neural activity in reward-related brain regions, when they saw the contestant win who they rated as more similar to themselves than when they saw the other contestant win.Using a different approach, Mobbs and colleagues13. Thus, phenotypic similarity inferred from a target’s verbal statements might not constitute a valid cue to genetic relatedness for the perceiver, questioning whether the observed modulation of empathic responses indeed reflected kin selection mechanisms. Second, due to the way similarity was manipulated , similarity ratings were significantly higher for one (the desirable) than for the other (the undesirable) target across all participants, making it difficult to disentangle the roles of social desirability and similarity. Finally, target-perceiver similarity was assessed by self-report, which might be skewed by a perceiver’s tendency to see them as being more similar to the target modelled as having socially desirable traits13 .This study provided first evidence that empathic responses might be modulated by perceived attitudinal similarity. However, it also leaves some key questions unresolved. First, verbally expressed attitudes often reflect desirable rather than actual individual traits11, except our the targets differed in a non-verbal behavioural pattern that (in Western societies) is not strongly valued by social norms . Participants were told that they would see video clips of two human players interacting in a virtual reality robot soccer penalty shoot out (by moving two humanoid robots), and that the common goal of the players was to defeat the soft-ware driven goalie and to score as many goals as possible. Unbeknownst to the participants, the supposed players were modelled to differ in how often they took the lead and executed a penalty shot: One player (LP) was modelled as having a strong tendency to lead and executed 75 percent of the shots, and the other player (NLP) was modelled as having a weak tendency to lead and executed 25 percent of the shots  and that are not strongly valued by social norms . For this, we used an experimental approach similar to that used by Mobbs and colleaguesots Fig. . All othWe predicted that (a) the degree of self-reported empathic reward and (b) the strength of neural responses in reward-related brain regions elicited when a participant saw a player successfully converting a shot into a goal  would be predicted by shared behavioural traits: the more similar a participant’s self-reported tendency to lead was to one player’s tendency to lead (relative to the other player’s tendency to lead), the stronger the participant’s empathic response towards this player (relative to the other player) should be.tendency-to-leadLP = 0.61 +/− 0.04 [mean +/−  s.e.m.], tendency-to-leadNLP = 0.25 +/− 0.03, Δ tendency-to-leadLP-NLP = 0.36 +/− 0.06, Cohen’s d = 1.2; T[23] = 5.7, p < 0.001, Fig. 1B). In contrast, participants disagreed on which player they found more likable, and for which player they felt more empathic reward. About 40 percent of participants rated player LP as more likable than player NLP, and about 25 percent rated player NLP as more likable than player LP , indicating that participants did not generally prefer one behavioural pattern over the other. About 33 percent of participants reported to have experienced more reward when they saw player LP score a goal, and about 15 percent reported to have experienced more reward when they saw player NLP score a goal , indicating that participants also differed in whose players’ success they shared more empathically.Button presses recorded during fMRI indicated that participants quickly learned that the two supposed players (LP and NLP) differed in how often they took the lead and executed a penalty shot  ranged from 0.36 to 1 . On average, similarity scores were slightly higher for player LP than for player NLP, but this effect was small and statistically not significant .Overall, participants rated their tendency to lead as slightly weaker than player LP’s tendency to lead and as stronger than player NLP’s tendency to lead (empathic rewardLP-NLP) were explained by individual differences in behavioural similarity to player LP and Player NLP (Δ similarityLP-NLP)  were removed from both variables .Supporting the first part of our hypothesis (a), across participants individual differences in self-reported empathic reward for player LP and player NLP  and in a second large cluster in the medial orbitofrontal cortex (mOFC) increased when a participant saw that they had correctly predicted the outcome of a trial (relative to when they saw that they had not correctly predicted the outcome)  predicted differences in empathic-reward related neural activity  in these ROIs. This was the case in the left VS and mOFC  were removed . Thus, in line with the second part of our hypothesis (b), individual differences in behavioural similarity predicted individual differences in empathic-reward related neural activity in the left VS and mOFC .To examine whether differences in similarity scores predicted differences in neural responses in reward-related brain regions when a participant saw a player successfully score a goal  we extracted contrast estimates from three spherical ROIs (regions of interest) centred at the most significantly activated voxels in the left and right VS and mOFC, respectively, in the analysis of own reward. We then tested whether differences in similarity scores , suggesting a role of this region for detecting and/or representing self-similarity. No correlation between individual differences in behavioural similarity and individual differences in neural responses to a player’s failure  were observed, neither at a conventional voxel-wise height threshold of p = 0.001 (no extent threshold), nor when the voxel-wise height threshold was lowered to p = 0.010 (using an extent threshold of k = 10 voxels).A post-hoc whole brain analysis, searching for brain regions outside the VS/mOFC whose activity would be modulated by differences in behavioural similarity revealed a cluster in the ventral anterior cingulate cortex (vACC) . Thus, we used a behavioural similarity measure that was based on two independent questionnaires that did not directly assess perceived similarity.The current study aimed to test whether empathic responses towards unknown others are modulated by behavioural similarity. For this we sought to implement a measure of similarity that did not require participants to directly rate self-similarity, because self-ratings might be skewed by social demandsTo ensure the external validity of this measure we performed a supplemental study with two groups of participants in which participants (i) interacted with one another, (ii) rated how similar each participant was to themselves, and (iii) completed the two tendency-to-lead questionnaires used to compute similarity scores in the main study. Analysis of these data showed that a significant amount of variance in self-rated similarity was explained by similarity scores computed from the two questionnaires  or a weak (player NLP) tendency to lead in social situations executing penalty shots in a virtual reality robot soccer game. As predicted, we found a significant three-way interaction: participants whose self-reported tendency to lead was more similar to player LP’s tendency to lead experienced more reward, and showed stronger neural activity in reward-related brain regions, when they saw player LP score a goal than when they saw player NLP score a goal, and vice versa. These findings suggest that empathic responses towards unknown others are modulated by behavioural similarity, and partly support with kin-selection theories of human social behaviour5.Observational studies suggest that human selfless behaviour is modulated by genetic relatedness13. Thus, the current study extends previous studies that observed significant phenotype-dependent target-perceiver effects in neural responses towards others’ pain10 or pleasure11 by showing that phenotype-dependent modulation of empathic responses is not restricted to physical10 or attitudinal11 similarity but can be induced by similarity of behavioural traits that are not strongly valued by social norms.Importantly, the modulation of empathic responses observed in the current study cannot easily be explained by learned preferences or social norms that might value some behavioural traits higher than others. First, although the vast majority of participants perceived player LP as having a stronger tendency to lead than player NLP, participants disagreed on which player was more likable. Second, participants had very similar socio-cultural backgrounds but showed different neural responses when they saw one or the other player score a goal. Third, similarity scores were computed based on two independent questionnaires that did not directly assess perceived similarity, making them robust against biases that can be induced by social demands15. In addition to these predicted effects we observed a significant phenotype-dependent modulation of neural activity in a cluster in the ventral anterior cingulate cortex (vACC) which was linked to the modulation of neural activity in the left VS and mOFC. The vACC is part of a larger brain region that has been found to play an important role in representing others in relation to oneself, the ventromedial prefrontal cortex (vmPFC) Specifically, activity in a region of the vmPFC that partly overlaps with the cluster in the vACC detected in the current study has been shown to increase when people make judgments about others who they believe share their own preferences and attitudes (compared to when they make similar judgments about others they perceive as dissimilar to themselves)25. Furthermore, greater attitudinal similarity was associated with stronger functional coupling between the vACC and VS in the study by Mobbs and colleagues11. Together, these findings point towards a potential role of the vACC/vmPFC for detecting and/or representing self-similarity and modulating prosocial responses accordingly.Participants in the current study showed significant phenotype-dependent modulation of self-reported empathy and neural responses in left VS and mOFC, two brain regions that also responded when participants saw that they had correctly predicted the outcome of a trial and that are regarded core regions of the brain’s reward system28. Interestingly, the mPFC also seems to play a role in mediating these effects30. This suggests that similarity-induced target-perceiver effects and in-group/out-group effects might be mediated by similar neural mechanisms, even though sociobiological theories assume that they might have emerged through different evolutionary processes (e.g2.). Clearly, more research is needed to better understand the neural processes underlying interindividual effects in social responses towards unfamiliar others and how they translate to related concepts such as social proximity and distance (e.g.31), interindividual attraction (e.g.32), feelings of affection (e.g.34) and social bonds (e.g.36). Integrative studies examining different factors with larger sample sizes than the current and previous studies11 are needed to gain a deeper insight into the neural processes that mediate target-perceiver effects in human social perception and behaviour.Significant target-perceiver effects have also been observed in studies examining in-group/out-group effects in empathic pain and pleasure. In these studies participants typically show stronger responses to another person’s pain or pleasure when they believe that this person to their own social group than when they believe that the person belongs to a different social groupIn sum, the findings of the current study show that significant phenotype-dependent interindividual effects between unfamiliar others can arise from differences in behaviour that are not strongly valued by social norms. We believe that a deeper understanding of the role of such potentially genetically grounded interindividual effects in empathic responses towards others will not only enhance our understanding of the neural processes that govern human social perception, but might also affect our view on human social behaviour in a rapidly changing and increasingly anonymous social world.Declaration of Helsinki and participants gave written informed consent before participation. The study was approved by the Ethics committee of the Universität zu Lübeck, Germany.Twenty-nine participants with no record of neurological or psychiatric disorders (assessed by self-report) were recruited at the University of Greifswald, Germany. Data of two participants were excluded because these participants did not believe in the cover story or did not use all response options in all conditions, and data of three other participants were excluded because of significant head movement during fMRI (>3 mm within a run or >10 mm between runs). The final data set included data from 24 participants . The study was conducted in accordance with the Participants were told that during fMRI they would see a series of pre-recorded video clips showing two humanoid robots that were driven by two human players (participants in a previous study) interacting in a virtual robot soccer penalty shoot-out. Participants were further told that the human players were team mates who had the common goal to defeat the software-driven goalie and to score as many goals as possible. To keep the participants’ attention focussed on the supposed players’ behaviour, and to obtain data to localize reward-related neural activity in the participants’ brain, participants were told that their task would be to predict the players’ behaviour in each trial more accurately than the software that supposedly controlled the goalie.Each trial commenced with a start scene (5 s) showing the two humanoid robots standing side-by-side at the penalty spot Fig. . Robots http://psychtoolbox.org)39 in Matlab .To familiarize participants with their task and the robots’ appearance they completed a number of practise trials in the scanner (with robots with eye and chest mark colours not used in the main experiment) and passively viewed a series of portrait pictures [1.5 s] of the two robots used in the main experiment (18 pictures per robot) before the main experiment started. The series of portrait pictures was shown once again after the main experiment. FMRI data of the pre- and post portrait picture runs were not used in the current study. Stimulus presentation and response logging were controlled with the Psychophysics toolbox  using simulated NAO robots 3.0  and robot-behaviour assignments were counterbalanced across participants.Four different series of 128 pseudorandomized video clips (randomly assigned to different participants) were compiled with the restriction that each of the 16 possible configurations of start position and block direction in two consecutive trials occurred once within overlapping blocks of 16 trials . A simple rule determined which robot executed the shot in each trial: The robot assigned to player LP executed the shot whenever its start position was on the right side of the penalty spot , used to correct for image distortions, were obtained from each participant before functional imaging.MRI data were obtained on a 3 Tesla scanner equipped with a 32 channel head coil at the University of Greifswald, Greifswald, Germany . Functional imaging was divided into 4 runs (320 scans per run). Functional data were acquired with a single-shot gradient echo T2*-weighted EPI sequence covering the whole brain . To allow for T1 saturation each run was preceded by acquisition of five scans which were not included in the analysis. A T1-weighted anatomical image , used to register functional data into MNI spaceAssertiveness and Dominance scales of the International Personality Item Pool41  and assessed the participants’ own tendency to lead in social situations. All rating were obtained on 5-point scales ranging from [1]  to [5] (very much) and were linearly transformed to range from [0] to [1] for further analysis and visualization. Individual similarity scores were computed for each participant and player by computing the absolute difference between the participant’s rating of the player’s tendency to lead  and their rating of their own tendency to lead (IPIP), and contrasting this difference with 1 ([1 – |tendency-to-leadparticipant - tendency-to-leadplayer|]). For all measures, individual difference scores for player LP and NLP were obtained by subtracting individual scores for player LP with individual scores for player NLP.After imaging participants completed two questionnaires. The first questionnaire comprised five questions relating to the participant’s perception of each of the two supposed players, placed next to a picture of the respective player’s robot. The first two items assessed how likeable the participant found the supposed player and how much vicarious reward they had experienced when they saw that player score a goal. The other three items related to the supposed player’s tendency to lead (assertiveness-influence-aggressiveness). Ratings for the last three items were averaged (separately for each participant) to a tendency-to-lead score for each supposed player. The second questionnaire comprised 23 self-report items taken from the start scene [5 s] – response screen [2.5 s] – video clip [2 s]). All contrasts of interest (see below) were based on parameter estimates for video clips (showing the shot being executed).MRI data were analyzed with SPM8  using Matlab . Preprocessing followed standard procedures and included (i) concurrent spatial realignment and correction of image distortions, (ii) normalization into standard MNI space , and (iii) resampling at a spatial resolution of 3 × 3 × 3 mm³. BOLD (blood oxygen level dependent) responses during each trial were estimated using a GLM for each participant that accounted for first-order autocorrelations and low-frequency drifts (high-pass cut-off 128 s). Each GLM included three regressors per trial , modelling the three parts of each trial , and Δ neural activity goal-no goalLP-NLP . To correct for the fact that player LP executed more shots than player NLP . Because the participants’ individual response selection did not always result in a completely balanced two-by-two-by-two design (Table own reward) and each player’s trial-by-trial success in scoring a goal (empathic reward) were not related , thus no correction was necessary. Individual contrast images were spatially smoothed (8 mm isotropic Gaussian kernel) for further analyses.Two contrasts of interest were computed for each participant: Δ LP Table , only pagn Table  individu15 that showed stronger neural activity when participants saw that they had correctly predicted the outcome of a trial than when they saw that they had not correctly predicted the outcome of a trial . Next, to test whether in these regions neural responses to a player’s success would be modulated by behavioural similarity, we placed three spherical ROIs (6 mm radius) at the most significantly activated voxel in the left/right ventral striatum and mOFC, respectively, and averaged voxel-wise contrast estimates Δ neural activity goal-no goalLP-NLP across all voxels in each ROI. These mean contrast estimates were used for univariate correlation analyses with Δ similarityLP-NLP. This two-step approach avoided overestimation of effect sizes that might occur in mass univariate analyses42. For completeness, we also report coordinates and statistical values for the highest activated voxel within each ROI in corresponding voxel-wise analyses  did not exceed α = 0.050. Exact p values are given for p > 0.001 and p < 0.200.Student’s T statistics were used for all tests. Degrees of freedom (df) for each test are given in square brackets. Pearson’s product-moment correlation coefficient was used to estimate correlation strengths. Behavioural effects and effects based on mean contrast estimates for ROIs were tested two-sided, and voxel-wise effects were tested one-sided. For the whole brain analyses, statistical maps were thresholded at a voxel-wise height threshold of p = 0.001 and effects were assessed at cluster level, correcting for family-wise errors [FWE] according to Random Field TheoryOIs Fig.  effects Supplemental material.


Terrestrial vertebrates have evolved hard skin appendages, such as scales, claws, feathers, and hair that play crucial roles in defense, predation, locomotion, and thermal insulation. The mechanical properties of these skin appendages are largely determined by cornified epithelial components. So-called “hair keratins,” cysteine-rich intermediate filament proteins that undergo covalent cross-linking via disulfide bonds, are the crucial structural proteins of hair and claws in mammals and hair keratin orthologs are also present in lizard claws, indicating an evolutionary origin in a hairless common ancestor of amniotes. Here, we show that reptiles and birds have also other cysteine-rich keratins which lack cysteine-rich orthologs in mammals. In addition to hard acidic (type I) sauropsid-specific (HAS) keratins, we identified hard basic (type II) sauropsid-specific (HBS) keratins which are conserved in lepidosaurs, turtles, crocodilians, and birds. Immunohistochemical analysis with a newly made antibody revealed expression of chicken HBS1 keratin in the cornifying epithelial cells of feathers. Molecular phylogenetics suggested that the high cysteine contents of HAS and HBS keratins evolved independently from the cysteine-rich sequences of hair keratin orthologs, thus representing products of convergent evolution. In conclusion, we propose an evolutionary model in which HAS and HBS keratins evolved as structural proteins in epithelial cornification of reptiles and at least one HBS keratin was co-opted as a component of feathers after the evolutionary divergence of birds from reptiles. Thus, cytoskeletal proteins of hair and feathers are products of convergent evolution and evolutionary co-option to similar biomechanical functions in clade-specific hard skin appendages. Keratin intermediate filaments form the cytoskeleton of vertebrate epithelia . The basTerrestrial life of amniotes  was associated with the evolution of hard skin appendages such as scales, claws, feathers, and hair . These aSequence analysis has shown that keratins expressed in hair and claws/nails of mammals and in claws of reptiles contain high amounts of cysteine residues which form disulfide bonds to other proteins and thereby establish the rigid cytoskeleton that is crucial for the mechanical resistance of the skin appendages . ComparaHere, we screened genome sequences of sauropsids for genes encoding cysteine-rich keratins. We identified homologs of HAS keratins and a previously uncharacterized group of cysteine-rich type II keratins that are conserved in all clades of sauropsids. Comparative genomics and results of gene expression analyses suggest a model in which the evolution of high cysteine contents in different subgroups of keratins was instrumental in the evolution of hard skin appendages, including feathers.Gallus gallus, Taeniopygia guttata, Struthio camelus australis, Apteryx australis), alligators , turtles (Chrysemys picta bellii), lizard (Anolis carolinensis), and snakes , amphibians , and fishes . Additional keratins were predicted from transcriptome data of the axolotl (Ambystoma mexicanum) and a caecilian (Microcaecilia unicolor). In mammals such as humans, type I keratin genes KRT9 through KRT40 are localized in one gene cluster (KRT1–KRT8 and KRT71–KRT86) and KRT18 (type I) are localized in another gene cluster    online tantelli)  online aantelli)  online, antelli) . The comKRT14 gene in representatives of all major clades of sauropsids. These cysteine-rich type I keratins of the green anole lizard were previously termed HAS keratins  keratin gene cluster, two to four genes encoding keratins with more than 20 cysteine residues are localized on the 3′-side of the keratins . Three Hkeratins  online akeratins . In the ke genes A. All HBke genes B.Phylogenetic analysis showed that HAS  and HBS KRT9-like genes (named KRT9L1-6) which encode keratins with smaller numbers of cysteine numbers (KRT78LT) which encodes a K78-like keratin with an unusually long tail domain  and more than 50 cysteine residues  , are fla numbers . Likewis numbers . In the residues  online. rthologs  online. Danio rerio) . Molecular phylogenetics showed that these keratins of medium to high cysteine content are neither closely related to each other nor to the cysteine-rich keratins of amniotes   online aopicalis  online aamniotes  online, Xenopus tropicalis   analysis of HBS1 mRNA in the skin of different body sites of chicken at embryonic day 18 showed high expression levels of HBS1 in feathers A. HBS2 aFinally, we compared the number of hair keratins, HAS and HBS keratins in species from different branches of the phylogenetic tree of tetrapods to infer the evolutionary history of these keratins . The preThe results of this study reveal that, in addition to the previously characterized evolution of type I and type II “hair keratins” in early amniotes , two sepChrysemys picta). Although further characterization of individual HAS and HBS keratin genes is warranted, our data strongly support the general conclusion that the cysteine content has increased in these two clades of sauropsid keratins that are not orthologous to hair keratins.Our analysis of genes that encode cysteine-rich keratins was made possible by the determination and publication of genome sequences of many sauropsids . AlthougOur analysis of the sauropsid keratins extends the findings of previous studies  by exploAt the level of individual cysteine residues within the intermediate filament domain, which is the amino acid sequence region that can be faithfully aligned, convergent evolution has likely occurred at several amino acid positions of type I keratins (HAS and type I hair keratins) and type II keratins (HBS and type II hair keratins) but the majority of cysteine residues are present at positions specific for only one phylogenetic clade of keratins  online. The immunolocalization of the chicken HBS1 keratin in feathers supports the hypothesis that this keratin and probably also other sauropsid-specific cysteine-rich keratins contribute to the cytoskeleton of hard skin appendages in sauropsids. Keratin filaments are known to be present in feathers  but specThe increase of the cysteine content in different clades of keratins is an example of convergent evolution driven by the selection for stable protein–protein interactions, that is, cross-linking via disulfide bonds. This ability to become cross-linked is a crucial feature of structural proteins of hard skin appendages of terrestrial vertebrates which require mechanical resilience in a dry environment. The evolution of high cysteine contents in keratins was paralleled by the evolutionary rise of cysteine contents in other cornification proteins, such as high-sulfur keratin-associated proteins (KRTAPs) in mammals and epidermal differentiation cysteine-rich protein (EDCRP), epidermal differentiation protein containing DPCC motifs (EDDM), and CBPs in sauropsids . The emeThe convergent evolution of cysteine-rich keratins in sauropsids differs from most cases of molecular convergence in the literature because the convergently evolving genes  resided in the same genome. Both hair keratin orthologs, which functioned as claw keratins , and theIn conclusion, the results of the present study suggest that convergent evolution of cysteine-rich keratins accompanied and possibly facilitated the parallel evolution of hard skin appendages in mammals and sauropsids. The expression of a HBS keratin in cornifying feathers and the presence of similarly cysteine-rich orthologs in featherless reptiles indicate that co-option of these cross-linkable keratins contributed to the evolution of skin appendages in sauropsids.Gallus gallus) (Alligator sinensis) (Chrysemys picta bellii) (Anolis carolinensis) (Protobothrops mucrosquamatus) (Homo sapiens) were investigated for the presence or absence and sequence integrity of type I and type II keratin genes. In addition, genes encoding cysteine-rich keratins were identified in the genomes of the Tibetan frog (Nanorana parkeri) , zebrafish (Danio rerio), kiwi , ostrich , zebra finch (Taeniopygia guttata), American alligator (Alligator mississipiensis), green sea turtle (Chelonia mydas), Burmese python (Python bivittatus), and platypus (Ornithorhynchus anatinus). Sequences were obtained from the GenBank database of the National Center for Biotechnology Information (NCBI), United States . Keratin cDNAs were identified in the axolotl (Ambystoma mexicanum) and a caecilian (Microcaecilia unicolor). Basic Local Alignment Search Tool (BLAST)  , Chineseinensis) , painted bellii) , anole linensis) , venomouuamatus) , and humparkeri) , clawed  (BLAST)  was used (BLAST) . The conwww.timetree.org; last accessed October 31, 2019)  .All animal procedures were approved by the Animal Care and Use Committee of the Medical University of Vienna (66.016/0014-II/3b/2011), and all these procedures were conducted according to the guidelines established by this committee. Fertilized chicken eggs were incubated according to published protocols . Hatched® technology (LC480). The primer pairs HBS1-s (5′-TGGCCCTGGACATTGAGATT-3′) and HBS1-a (5′-TGGCTCCAGTCTTCACAGAG-3′), and K5-s (5′-TGGAGAGATGGCCCTGAAAG-3′) and K5-a (5′-CCACCTGTACTGAGACCTCC-3′) were used for the amplification of chicken HBS1 and K5, respectively. Quantities of target relative to reference transcripts were calculated according to a published mathematical model . Quantitative PCRs were performed using the LightCycler 480 DNA SYBR Green I Master Kit (Roche Applied Science) and the LightCycleral model . The staOther HBS keratin mRNAs were amplified by semiquantitative PCRs using the primers HBS2-s (5′-ACGCTGTCAGGAGAAGGAAC-3′), HBS2-a (5′-TTCTCACACAGCAAGCAGTC-3′), HBS3-s (5′-GAAGTCCCACCAGCAAGAGA-3′), and HBS3-a (5′-GTGGCGATCTCAATGTCCAG-3′), an annealing temperature of 60 °C and 32 amplification cycles. The ubiquitously expressed apoptotic protease, caspase-3, was amplified as a control gene using the primers CASP3-s (5′-TGGCGATGAAGGACTCTTCT-3′) and CASP3-a (5′-CTGGTCCACTGTCTGCTTCA-3′).Antisera against HBS1 keratin were raised by immunizing mice six times with 100 µg of the synthetic peptide SAASCVGREVLSGADGLQC, corresponding to amino acid residues 553–571 of HBS1 keratin, coupled to keyhole limpet hemocyanin (KLH) according to a published protocol .About 50 µg protein per lane were electrophoresed through an ExcelGel SDS gradient 8–18% polyacrylamide gel  and blotted onto a nitrocellulose membrane. The membrane was incubated with anti-HBS1 antibody (1:500) or, for comparison, with mouse anti-GAPDH  at 4 °C overnight. Then the membrane was incubated with sheep anti-mouse immunoglobulin  coupled to horseradish peroxidase for 1 h at room temperature, and bands were visualized using the enhanced chemiluminescence system .Immunohistochemistry was performed according to published protocols  with modmsz279_Supplementary_DataClick here for additional data file.


Androgen receptor (AR) expression is emerging as a prognostic biomarker in breast carcinoma (BCa). The study aimed to determine the prevalence of AR expression by immunohistochemical analysis among a cohort of Sri Lankan women with early BCa and to evaluate its association with clinicopathological features including immunohistochemical molecular subtype and early survival.We studied the clinical and pathological features and immunohistochemical profile of 141 women undergoing primary surgery for early BCa, followed by standard adjuvant therapy. AR status was assessed by immunohistochemistry in all cases. Overall survival (OS) and disease-free survival (DFS) was determined. The relationship between AR expression and clinical and pathological parameters and immunohistochemical molecular subtype was analyzed using Student T test and chi-square tests. Cox regression analysis was used to analyze the prognostic impact of AR expression.p > 0.05). Ductal carcinoma in situ(p = 0.003), oestrogen receptor (ER) (p = 0.001) and progesterone receptor (PR) (p = 0.001) positivity and luminal IHC molecular subtype(p = 0.016) were significantly associated with AR-positive status. AR-negative status was significantly associated with tumour necrosis > 50%(p = 0.031), moderate to extensive lymphocytic infiltrate at the tumour margin(p = 0.025) and basal triple negative breast carcinoma(p = 0.016).AR expression was seen in 40.8%(95%CI 33.10–49.07%) of the BCa study cohort. None of the clinical data studied showed a significant association with the AR status(p = 0.047), specifically in postmenopausal women (p = 0.030). In ER-negative tumours, AR positivity was associated with worse DFS (p = 0.036). On multivariate analysis, TNM stage and ER/AR status were predictive of survival. ER-positive/AR-positive (ER+/AR+) tumours demonstrated better OS than ER-positive/AR-negative (ER+/AR-) tumours(p = 0.015). ER-negative/AR-positive (ER−/AR+) tumours (p = 0.014) had a worse DFS than ER-negative/AR-negative (ER−/AR-) tumours.The mean duration of patient follow-up was 46.70(95% CI 46.495–46.905) months (3.89 years). On univariate analysis, AR-positivity was associated with better OS among ER-positive tumours(AR prevalence obtained was low. AR positivity was associated with positivity for ER and PR. On multivariate analysis, apart from TNM stage only ER/AR status were predictive of OS and DFS, with concordant expression of ER/AR demonstrating a better, early survival. Breast carcinoma (BCa) is the commonest malignancy among women in Sri Lanka and worldwide. In 2010, 27% of women diagnosed with cancer in Sri Lanka had BCa with a lifetime risk of 2.5% in the population [BCa can be categorized into several molecular subtypes based on the surrogate immunohistochemical marker expression. Oestrogen receptors (ER), progesterone receptors (PR), HER2, Ki67 and basal markers  are analyzed by immunohistochemistry (IHC). Based on these results the tumours are categorized into Luminal A, Luminal B, HER2 enriched, triple negative and basal like subtypes [AR gene is located in the X chromosome at position Xq11–12. It is known to produce its effects through 3 major pathways which contribute to AR function in BCa: genomic signaling, non-genomic signaling and signaling via cross talk with growth factors and cytokines [Expression of androgen receptors (AR) in BCa has generated considerable interest in the recent past as a prognostic biomarker. The human androgen receptor ytokines . Differeytokines . This exytokines –10. It iytokines , 11.Systematic reviews and meta-analysis have shown AR positivity to be associated with favorable clinical outcomes , 13. HowData regarding the prevalence and the impact of AR expression in BCa and survival are mostly from the west and none from Sri Lanka. Only a few studies have been done in Asia; in Iran, India, Thailand and China –17.We aimed to determine the prevalence of androgen receptor expression by immunohistochemical analysis among a cohort of Sri Lankan women with early breast carcinoma, and to evaluate its association with clinical and pathological features including immunohistochemical molecular subtype and early survival.Ethical approval for the study was obtained from the Ethical Review Committee of the Faculty of Medicine, University of Colombo (EC-17-088). The study population comprised all women undergoing surgery for invasive breast carcinoma at the National Hospital of Sri Lanka from June 2012 to December 2014. Inclusion criteria were women undergoing mastectomy or wide-local excision for an invasive breast carcinoma of early stage. Early stage breast carcinoma was defined as cancer confined to the breast with or without regional lymph node involvement, and absent distant metastasis . PatientThe study population comprised 301 patients. One hundred and forty one cases were selected from 179 cases fulfilling the inclusion and exclusion criteria.The clinical parameters evaluated included ethnicity, age, body mass index, parity, history of breast feeding, menopausal status, family history of BCa and use of oral contraceptives and hormone therapy. The clinical data was collected by the investigators through patient interviews. The following information was retrieved from the pathology reports: type of surgery, tumour size, margin involvement and lymph node status. Slides of cases that had been reported as having a positive margin were reviewed to confirm the presence of margin involvement. Only cases showing the presence of tumour on the ink were classified as having a positive margin. The haematoxylin and eosin stained slides of the tumour were evaluated by the investigators for the following parameters: histological type, histological grade, necrosis, ductal carcinoma in situ (DCIS), lymphovascular invasion, tumour margins, degree of lymphoid infiltrate at the tumour-host interface and within the tumour, degree of desmoplasia/hyalinization at the tumour-host interface and within the tumour, vascular density at the tumour-host interface, cell margins and calcification. Histological subtyping was done according to the 4th World Health Organization Classification of Tumours of the Breast . The ElsImmunohistochemical staining for ER, PR, HER2 and AR were performed by investigators on representative paraffin embedded tissue in all selected cases. CK5/6, EGFR, 34ßE12 and CK14 were evaluated in the cases found to be triple negative . Relevant negative and positive control slides were included with each batch of slides stained with immunohistochemical markers.The antibodies, dilution and the method used and interpretation of IHC stained sections are shown in Table The tumours were broadly categorized into luminal, HER2 and triple negative breast carcinoma (TNBC) groups based on ER, PR and HER2 status. A proportion of the luminal breast carcinomas could have been further subclassified as luminal B, based on positivity for HER2 . HoweverAll patients had been treated and managed according to standard protocols  with 119p value of < 0.05 was taken as significant. Univariate Cox regression analysis was used to analyze the prognostic impact (OS and DFS) of AR expression on the entire sample as a whole and also when stratified according to histological grade, ER status and menopausal status. Multivariate Cox regression analysis was performed to analyse factors affecting OS and DFS. Statistical Package for the Social Sciences SPSS  was used for data analysis.Proportions and 95% confidence intervals were calculated to estimate prevalence. The relationship between clinicopathological parameters, IHC molecular subtype and the AR expression was analyzed using Student T test and chi-square tests. A 2–95% CI 25.7–27.7 kg/m2). Eleven (7.8%) had a family history of breast carcinoma.The characteristics of the study sample are summarized in Table Positive AR expression Fig.  was seenp > 0.05) (Table p = 0.003) and positivity for ER (p = 0.001) and PR (p = 0.001) were associated with AR positivity. AR-negativity was significantly associated with necrosis > 50% (p = 0.031) and a moderate to extensive lymphocytic infiltrate at the tumour margin (p = 0.025)  Table . Of the 5) Table .Table 3p = 0.016) and AR-positivity was associated with the luminal subtype. Basal triple negative breast carcinomas showed an association with AR negativity.AR was expressed across all IHC molecular subtypes, with 50.6% of luminal, 29.4% of HER2 and 21.6% of TNBC subtypes showing AR-positivity. The association of AR status and the IHC molecular subtype is shown in Table The mean duration of follow up was 46.70 (95% CI 46.495–46.905) months (3.89 years). One hundred and seven patients were alive and free of disease. There were 34 cases of early relapse. Twenty five patients died during this period. Of the nine that were living with disease, six had developed metastases and three had local recurrences.Positive margin involvement was seen in ten cases (7.1%), of which eight were mastectomies and two were wide local excisions in which no subsequent mastectomy was performed. Three of the patients who died, had positive margin involvement at the time of primary surgery. All three had undergone mastectomy. The remaining seven patients were disease free at follow up.p = 0.351) and DFS (p = 0.840). Post-hoc calculations of the power of the study showed that the power of the study to detect a difference in DFS and OS between the two groups (AR+ vs AR-) with an alpha value of 0.05 was 4.6 and 11.2% respectively.Androgen receptor status showed no significant association with OS (p = 0.030)  tumours showed significantly better OS than postmenopausal women with ER-positive, AR-negative (ER+/AR-) tumours (30) Fig. . DFS didp = 0.035) and DFS (p = 0.022) of this subgroup was significantly different from the grade 1/2/AR-positive tumours. Additionally among grade 1/2 tumours, AR-positive tumours showed a significantly better OS (p = 0.042) than AR-negative tumours  and ER-negative, AR-negative (ER−/AR-) tumours showed significantly better DFS than ER-negative, AR-positive (ER−/AR+) tumours (p = 0.036)  or disease-free survival (p = 0.826) when the tumours were categorized by molecular subgroup.The AR status of the tumour had no impact on either overall (p = 0.015) and ER−/AR+ tumours had significantly worse DFS than ER−/AR- tumours (p = 0.014) . However, many studies including a metanalysis of 22 studies involving 10,004 women  found thThe prognostic effect of AR expression may vary according to the age, menopausal status, and hormonal status of the tumour. Although the mean age was 55.46 years and most participants were in the post-menopausal age group, their age ranged widely from 29 to 77 years. The cases included both ER positive and ER negative cancers. This clinical heterogeneity needs to be taken into consideration, when interpreting the results of the group as a whole. Subsequent subgroup analysis was done according to menopausal status, tumour grade and ER status.Postmenopausal women with ER+ tumours comprised a subgroup with limited clinical heterogeneity. We found that AR-positivity was associated with a better OS among postmenopausal women with ER-positive tumors. This same association was not seen among premenopausal women with ER-positive tumours. Androgen is thought to act as an anti-oestrogen in premenopausal women, but as an oestrogen agonist in postmenopausal women. Therefore it is possible that the prognostic implications of AR status may be dependent on the menopausal status. However the smaller numbers of women in the premenopausal group  is a limitation that must be considered when interpreting the findings. Hu et al. who studied postmenopausal women also found AR expression to be associated with a more favorable prognosis in ER-positive tumors . HoweverAmong grade1/2 tumours, AR positive tumours showed a better prognosis than AR negative tumours. However this may be due to the fact that low grade tumours were more likely to be ER positive. ER+/AR+ tumours showed better OS than ER+/AR- tumours and ER−/AR+ tumours showed worse DFS than ER−/AR- tumours on both univariate and multivariate analysis. Other studies too have found that breast cancers with discordant ER/AR expressions (ER+/AR- or ER−/AR+) demonstrated a worse prognosis in comparison to breast cancers with concordant expression (ER + AR+ or ER−/AR -) in multivariable models . As discThe implications of AR expression in HER2 amplified BCa and TNBC is uncertain. Some studies show no effect with AR-positivity , 34, somNone of the patients studied had been treated with AR agonists or antagonists. Therefore the predictive value of AR status on treatment was not studied. The small sample size in the current study is a limitation. The power of the study is low due to the small sample size. The effects being investigated are also small. These factors increase the likelihood of both false negative and false positive results. This has to be taken into consideration in the interpretation of the results. Additionally the effect of adjuvant therapy also needs to be considered when assessing the impact on prognosis.However despite these limitations, the finding that AR positivity was associated with a better prognosis in ER positive tumours, especially in postmenopausal women is in keeping with other studies. AR positivity was associated with poorer outcomes in ER negative tumours. These findings are supportive of the use of androgen agonists in ER+/AR+ tumours and AR antagonists in ER−/AR+ tumours , 11. LarThe histological and IHC molecular tumour characteristics of this BCa patient cohort from Sri Lanka, were mostly similar to the findings from other regions of the world including Europe and United States of America , 36, 37.Currently, where the androgen receptor (AR) is emerging as a new biomarker and a potential new therapeutic target in the treatment of BCa , the stu


Continuous ceramic fiber comprising silicon carbide–zirconium carbide (SiC–ZrC) binary phases was obtained through melt spinning, electron-beam curing and pyrolysis of a pre-ceramic precursor of polyzirconocenecarbosilanes (PZCS). After pyrolysis and heat treatment, ZrC particles with mean diameters of 15–20 nm were formed and homogeneously dispersed in a matrix of fine crystalline β-SiC with an average grain size of 6–10 nm. Concentration of Zr in the fiber varies from 14.88% to 17.45% by mass. Fibers consisting of near-stoichiometric ZrC and SiC with little free carbon can be obtained through pyrolysis decarbonization of the as-cured fiber in hydrogen from room temperature to 1000 °C, and subsequently heat treatment in argon up to 1600 °C for 1 h. High-temperature treatment of these amorphous inorganic fibers leads to crystallization of the binary phases of β-SiC and ZrC. The removal of free carbon under hydrogen results in more rapid growth of β-SiC and ZrC crystals, in which obvious aggregation of the dispersed ZrC particles among the continuous β-SiC matrix can be ascribed to a fast migration of Zr cation. Silicon carbide (SiC) fiber synthesized from polycarbosilane is one of the most important reinforcements for ceramic matrix composites (CMCs), which are now finding more and more applications to meet harsh environments of high temperature and air-oxidation such as turbo-engine blades in aerospace industry ,2,3,4,5.m, in K) [In general, SiC does not melt at any known temperature and its high decomposition temperature (approximately 2700 °C) makes it natural candidates for high temperature applications without the risk of creep failure under temperatures of 1200 °C ~0.5Tm, in K) ,11. HoweTm, in K m, in K) .2 with only 2.4% in mass. It was found that the incorporated ~50 nm TiB2 particles reside in triple point of SiC GBs which limits the sliding of SiC [Crystallization and strengthening of GBs in polycrystalline SiC can also be achieved via precipitation or introduction of non-soluble secondary phase with a higher melting point and modulus than SiC. Creep in SiC fiber can be retarded by introduction TiBg of SiC ,15. Thusg of SiC . For theg of SiC ,17. The g of SiC .2=SiHCH3) with a molecular Equation (1) [3 and n = 10–25. The polymer has an average molecular weight of 1080 g/mol, as determined by a gel-permeation chromatography (GPC) using toluene as the eluent and polystyrene as the calibration standard. The softening point of PZCS for melt spinning is around 120 °C and the ceramic yield in Ar at 1000 °C is 58%.Polyzirconocenecarbosilane (PZCS) was synthesized from dimethyldichlorosilane, zirconocene dichloride and metallic sodium in toluene and used as precursor for the fabrication of the SiC–ZrC composite fiber. The synthesis procedure and pyrolysis behavior of PZCS polymer have been reported in detail ,17, whiction (1) , herein 2 or Ar atmosphere, then heated to 1600 °C under Ar atmosphere and maintained at 1600 °C for 1 h. In above-mentioned two cases, a heating and cooling rate is 2 °C/min. For ease of description, the former was marked as H2–Ar process fiber and the latter was marked as Ar–Ar process fiber.PZCS about 40 g was charged into the spinning can and heated to the spinning temperature (135–140 °C) under a nitrogen atmosphere and then extruded through a single-hole spinneret with a diameter of 0.25 ± 0.05 mm. The PZCS green fibers were cured in a flow reactor in argon by electron-beam irradiation . The as-cured fibers were heated to 1000 °C under HThe elemental contents in the fibers were analyzed, in which the contents of Si and Zr were measured by ICP-OES in a Thermo Fisher ICAP6300 spectrometer , the contents of carbon and hydrogen were acquired by an Elementar Vario EL determinator . The TC-436 N/O analyzer was used to determine the content of oxygen element .α radiation (λ = 0.15406 nm at 40 kV and 30 mA).The phase compositions in the pyrolysized fibers were identified by X-ray diffraction  at 2θ = 10°–90° with Cu K−1.Free carbon in the fibers was examined with a Raman micro-spectrometry , using the green line of a He-Ne laser (632.8 nm) as excitation source and scattering was measured in the first-order spectrum ranging 900–2000 cmThe microstructures and elemental concentrations of the particles in the fibers were characterized with scanning electron microscopy  and transmission electron microscopy  equipped with an X-ray energy dispersive spectrometer (EDS). The samples were sprayed with a carbon film and then observed with SEM.2, which happens via chemical reactions between Si-H with oxygen into Si-O-Si linkage and water [The used precursor PZCS is a thermoplastic polymer, which shows excellent spinnability around 150 °C, but the derived green fiber will undergo remelting and lose their fabric shape before thermosetting and pyrolysis into inorganic fiber. Therefore, curing or aging of this green fiber into thermosetting one is the first key step herding the following inorganic chemical transformation. It was well known that a traditional polycarbosilane can be cured by oxidation in hot air or oxidized gases such NOnd water ,14. Thisnd water .Surface and cross-section morphologies of the EB-cured PZCS fiber are shown in 2 or in Ar atmosphere. Pyrolysis of the PZCS in Ar finally leads to formation of ZrC, SiC and free carbon in the residual inorganic fibers after releasing of complicated gaseous species such as methane, hydrogen and silanes [2–Ar process or Ar–Ar process at the temperatures of 1200, 1400 or 1600 °C show minor differences from each other. 2–Ar process 1200, 1400 or 1600 °C for 1 h. In all three cases, the ceramic fibers show very dense and homogeneous microstructures without any visible cracks, voids or other flaws.The as-cured fibers are then transferred into a thermosetting state that does not undergo remelting during pyrolysis up to 1000 °C either in H silanes . The surThe backscattered electron (BSE) image mainly reflects the distribution of elements on the sample surface. The brighter the region, the higher the atomic number. BSE images of the fibers at 1200 °C b and 1402–Ar process at 1200, 1400 and 1600 °C for 1 h is shown in XRD analysis of the above ceramic fibers annealed by HXRD analysis of the other ceramic fibers by Ar–Ar process up to various temperatures of 1200, 1400 or 1600 °C is given in 2–Ar process. With the temperature up to 1400 °C or 1600 °C, the diffraction peak shapes of crystalline ZrC formed via the H2–Ar process become sharper than those formed via the Ar–Ar process. It is also very clear that the crystallinity of SiC formed via the H2–Ar process is better than that via Ar–Ar process when the heat treatment is up to 1600 °C. That is, the introduction of H2 atmosphere below 1000 °C has effect on the growth of ZrC and SiC grain sizes at 1600 °C, which is got to know via the following analysis.Compared the results shown in 2 atmosphere consist of Zr, Si, C and O elements. With the pyrolysis atmosphere changing from Ar to H2 below 1000 °C, the Si content increases from 43.82% to 51.95%, the Zr content from 14.88% to 17.10%, and the carbon content decreases by about 10%, which results in the decrease of the C/(Si + Zr) atomic ratio from 1.90 to 1.15. After the Ar–Ar process or H2–Ar process at 1600 °C, the contents Si and Zr slightly increase while the carbon content further decreases, which can be ascribed to carbothermal reduction of C and O elements. The C/(Si + Zr) atomic ratio in the fibers by H2–Ar process at 1600 °C is 1.11, which means the fibers consist of near-stoichiometric ZrC and SiC.−1 are recorded. The scattering peak at 1590 cm−1 is ascribed to the Eg2  mode of the graphene layers and usually labeled as G band (name after “graphite”), while the scattering peak at 1358 cm−1 is designated to the D band of pyrolytic carbon (named after “defect”). The ratio of intensities of D band and G band is larger than 1, which means a large amount of free carbon exists in ceramic fiber obtained in argon at 1600 °C. In the fibers obtained via the H2–Ar process, the intensities of both peaks at 1358 and 1590 cm−1 become very weak, which means free carbon in the SiC–ZrC ceramic fibers is almost removed by H2.It was known that pyrolysis of PZCS in Ar led to the formation of ZrC, SiC and free carbon in the resultant fiber . Then fr−1 ascribed to the β-SiC in 2–Ar process.Compared with the Roman spectra of the SiC–ZrC fibers obtained via the Ar–Ar process a, the pe2 atmosphere below 1000 °C. Benefiting from the decarbonization of H2, the production of free carbon in the ceramic fibers is reduced and the crystallinity of ZrC and SiC grain sizes is increased, as well as stoichiometric ZrC and SiC can be obtained.From the elemental analysis and Raman spectra, it is found that a larger amount of carbon can be removed from the as-cured fibers by the introduction of H2–Ar process consist of two clearly defined phases of SiC and ZrC while free carbon is hardly observed in 2–Ar process, the average crystalline grain size of ZrC is about 18 nm  in a matrix of β-SiC with smaller grain size (~8 nm). After pyrolysis in hydrogen below 1000 °C, a more rapid growth of ZrC and SiC crystalline grains occurred in Ar up to 1400 or 1600 °C. In the same ceramic fiber, the crystalline grain size of ZrC was larger than that of SiC and the aggregation of Zr became apparent at 1600 °C.