Athene noctua), and the long-eared owl (Asio otus). These two species, living in semi-open habitats, were possibly the most abundant owls around the human settlements and surrounding cultivated fields of the Chalcolithic period. People must have been aware of the owl presence and possibly interacted with them. Why owls but no other animals have been the models may relate to the fact they are the most anthropomorphic of all animals, with large frontally-placed eyes in their enormous heads. In the iconography, owls are systematically represented, even today, with their two eyes staring at the observer, as opposed to the lateral view used for any other animal. Additionally, slate is one of the commonest surface rocks in southwestern Iberia, and it provides a blank canvas for engraving lines using pointed tools made of flint, quartz or copper. The way slates exfoliate makes easy to craft owl-looking plaques. To silhouette animals other than owls in a recognizable way would request extra carving abilities and specific tools. Plaque manufacture and design were simple and did not demand high skills nor intensive labor as demonstrated in replication experiments. Owl engravings could have been executed by youngsters, as they resemble owls painted today by elementary school students. This also suggests that schematic drawings are universal and timeless. We propose that the owl-like slate plaques are the remains of a set of objects used in both playful activities and in ritual ceremonies. The actual engraving of the plaques may have been part of the game. Owlish slate plaques were often perforated twice at the top. We interpret this as insertion points for actual bird feathers added to the plaques, right at the place where tufts emerge in live owls. The frontier among play and ritual is diffuse in liminal societies and there is no contradiction in playing with animal-like toys and, at some point, using them as offerings as part of community rituals related, for instance, to the colossal megalithic tombs so characteristic of the Copper Age.In the Copper Age, slate engraved plaques were produced massively in the southwestern corner of the Iberian Peninsula. Researchers have speculated about the function of these palm-sized stone objects for more than a century, although most have favored the idea that they represented goddesses, and served ritual purposes. The plaques are engraved with different designs of varying complexity. In some of them, the ones sporting two large frontal eyes, we clearly see owls modelled after two species present in the area: the little owl ( Early prehistorians held the hypothesis the plaques served a religious and/or symbolic purpose . A few years later, Correia8 interpreted the plaques as anthropomorphic idols representing a female goddess, as with the female divinities found all along the Mediterranean10. An alternative is later provided by Frankowski11, who argued that the plaques were not goddesses but representations of the dead themselves.The first discovered plaques were studied at the onset of systematic archaeology in the late nineteenth century in Portugal10 stated that the Iberian plaques were representations of a Mother Goddess, sometimes featured as an owl. Although the frontal view of an owl is utterly evident in many plaques  the slate plates of the Chalcolithic of Iberia and related objects with two frontal eyes are inspired in actual owls, and represent them either figuratively (the biomorphic ones) or in abstract form; (2) contrary to previous interpretations ascribing a highly ritualistic and deep significance to these objects (see references above), we suggest that the commonness of the material used and their basic and fast manufacture—as demonstrated by Lillios14, and therefore a fundamental part of Chalcolithic society19. Ignoring this fact may have led to identifying certain finds as symbolic or ritual when, quite simply, they are the trace of playful behaviors or learning activities. So far, the earlier set of toys may be the one found at the Siberian Upper Paleolithic site of Mal’ta, which included human, bird and other animal figurines20. Also in Siberia a human figurine and several animals, have been found at baby graves, dated 4500 years BP in the Bronze Age21. Whether or not the Chalcolithic slate plaques can be considered toys will remain speculative, but it is a fact that in ethnographic examples worldwide, many artisan practices, including pottery22, are learnt in childhood.This approach assumes that the owl-like objects may be the archaeological trace of playful and learning activities carried out by youngsters, who were possibly the largest demographic groupLast, we aimed to identify the owls depicted in the plaques to species. There were and still are few species of owls in Iberia and they differ in some recognizable ways. We will use these traits to make a proposal regarding the owl species depicted in at least the most figurative of all plaques. Identifying owl species can help to understand the circumstances and motivations for their depiction, as the ecology of the different species tells us about the context in which interaction with humans occurred, and their connections to the attributions and potential symbolism associated to them. In addition to this, we review other evidences that support the notion that owls were used as models in the Chalcolithic, acknowledging at the same time that numerous plaques cannot be directly ascribed to owls and may deserve alternative explanations.23, and specifically concerning pictures and drawings of slate plaques and other artifacts. In a few instances, the authors obtained photographs of some plaques from public collections, such as Museo Arqueológico Nacional, Madrid (MAN) and Museo Arqueológico Provincial de Badajoz. Information on the general shape, head/body proportions, eye coloration and plumage characteristics of owl species have been obtained from the Collins Bird Guide24, arguably one of the most authoritative books available for the identification of European bird species25. Curiously enough, the owls (Order Strigiformes) depicted in the aforementioned guide, and actually in every bird guide, are the only species depicted frontally, with the two eyes staring at the reader. It seems that humans, from the very first representation of owls in parietal art , reserve for the owls, and just the owls among birds, a frontal representation24 to reinforce and make unmistakable their owl being. We have placed particular importance to an aspect overlooked by previous researchers: the holes on the “heads” of the “2D” flat plaques, and the lack of them on the idols carved on “3D” substrates such as bones, and thus having a volume. We hypothesize that two holes make no sense to pass a string, as previously proposed despite their heavy weight (more than 300 g on average2), and the fact that they typically have no use-wear traces, as remarked by Lillios2 and Thomas et al.3. Instead, we suggest that the holes were used to insert actual feathers to simulate ear tufts  and the eagle owl (Bubo bubo). To outline the silhouette of owl tufts on thin slate plaques would have been very difficult, as they would easily break. The use of actual feathers would solve the problem. At the base of the feather, the rachis, or central shaft, gives way to the hollow tubular calamus . The calamus, or quill, with a rounded section of about 2–3 mm (diameter) in an (medium sized) owl’s flight feather, is the part that may be easily inserted in the hole(s) of the plaque. It may be twisted so that the remaining of the feather stays up and aligned with the plaque. Current and historic ethnographic evidence indicates that different cultures have carved figurines, often anthropomorphic, in which actual feathers have been inserted on top of the head. To gain further support for this interpretation, we have tested design types against number of holes, assuming that the most owl-like plaques should be the ones having two holes, whereas the less-owl-like plaques should tend to have just one or none. We have restricted the statistics to testing the number of holes in relation to the degree of owliness in a sample of plaques contained in the catalogue created and maintained by Lillios27. We generated a dataset of 100 plaques, including zoomorphic and non-zoomorphic in similar proportions, and recorded presence/absence of six morphologic traits present in actual owls . The presence of each one of these characters was considered as one point (1) in our owl score, with its absence counting as zero (0). This allowed us to assign a total owl score for each plaque, ranging from 0 to 6. We related this score with the number of perforations through a Kruskal–Wallis test.Our sources are mainly bibliographic, both concerning iconography of owls through the ages28). We assume that the differences in the craftsmanship of the plaques may well result from differences in the technical skill of the engravers associated to age and experience, as we see today in owl drawings depicted by children of different ages. With this in mind, we extracted owl images using Google29. We specifically searched for images drawn by children 4 to 13 years-old. We selected 100 drawings in which the age of the children was specified, and generated a score for each image based on the presence/absence of the same six owl traits  that we described above to evaluate the owliness of the slate plaques. Every recognizable trait adds one point, allowing us to obtain a complexity score for each drawing. We related the score with the age of the children through a Spearman correlation.To gain support to our hypothesis that the slate plaques and some related objects were children dolls or toys of little ritual value that may have been crafted for young people and not necessarily by experienced artisans of the community, we establish a comparison with owls as seen by contemporary children, taking therefore an actualistic approach  categorized as zoomorphic or biomorphicely Fig. .29.As additional support for our owl interpretation, we have used Google Lens, an image recognition technology developed by Google that uses visual analysis, on the picture of the Valencina slate plaque in Fig. 24. These species greatly differ in size  to the Eurasian eagle owl . Only two of those owl species have remarkable ear tufts , whereas the scops and the short-eared owl (Asio flammeus) have just a hint of tufts and often they are hidden altogether. The remaining species have no tufts at all. Two species have dark eyes , whereas the remaining species have bright yellow to orange irides contrasting with the black pupil at the center. Even though all species possess highly cryptic plumage patterns , different plumage patterns may be ascertained. Both eagle and long-eared owl, for instance, have dark strikes in the frontal plumage with lateral spikes. In the most figurative plaques we interpret the eyes has having a central pupil and a differently colored iris. The lines protruding from the iris, described by some scholars as sun rays3, we interpret as the highly modified feathers of the owl’s facial disk, that are disposed radially around the eye  and the long-eared owl (Asio otus), were the model for a majority of the owl-like plaques. For the plaques with dual perforations, and if they were used for inserting actual feathers, we favor the idea that long-eared owls were the most probable model . Slate exfoliates easily in sheets and it does not need to be mined underground nor carved with special tools, or put in an oven as with metal melting or pottery firing22. Slate sheets are practically blank canvases ready to be used if the intention is to engrave them. In addition, when the stone is carved, it has the peculiarity of alternating its natural black color with the white of the lines that have been engraved, a characteristic that facilitates the imitation of the cryptic plumage of the owls. As with Thomas et al.34, we expected differences in the craftsmanship of the plaques, that may well result from differences in the technical skill of the engravers associated to age and experience, as demonstrated for prehispanic pottery makers in the American Southwest22. Our actualistic approach, in which we resorted to a comparison with owl drawings by contemporary children of different ages clearly reveals two facts  owl34, who conducted replication experiments of Chalcolithic plaques with university students, considered slate as an “easily worked material, malleable and highly responsive to engraving tools of different hardnesses”. The engraving of the plaques varies in execution, including some rough models with few contorted lines . Owls have been represented frontally in parietal panels by different peoples and epochs worldwide, including Australia, southern Africa, the New World, or the Levant art of eastern and southern Iberia in the Chalcolithic35. Owls are so entwined in human culture, that, if asked to do so, both urban and rural children attending elementary school anywhere in the world would represent one easily and recognizably as a large-headed bird looking at the observer with two big eyes36. In this respect, owls are—and possibly were in a distant past—as iconic and familiar as a cow or a horse. Of all bird families, the owls and the diurnal birds of prey are perhaps the only ones that every human will recognize as culturally significant37. A survey of the bird shapes most preferred by humans, revealed that the shorter the neck and the bigger the eyes the most attractive resulted the species, with owls ranking among the most attractive bird types38.Owls are unique among birds due to their large heads and frontally placed eyes7. Despite its simplicity, their artistry was also praised and readily compared to other crafts produced in other parts of the Old World, from the Iberian Peninsula to Mesopotamia10. Gimbutas10 visually described the plaques as “owl goddesses on a trapezoidal plaque of schist”, also carved on bones or in pottery. After Gimbutas, only the goddess interpretation remained, and the owl model was forgotten.Since the discovery of the first engraved slate plaques, they were ascribed a profound symbolic and ritual purpose2 described experiments aimed at replicating engraved plaques using tools at the disposal of the ancient engravers. Later, Thomas et al.34 also reported the process of replicating plaques by university students with no previous artistic background. A modern experimenter would produce a finished plaque in about 3.5 h. This is important to remark, as it contrasts in terms of labour and complexity with other technological achievements by the same Chalcolithic peoples. Polished tools, such as handaxes, had been in place for millennia, and the beaker ceramics characteristic of the Iberian Chalcolithic included similar zigzagging patterns. However, the latter involved more manufacturing steps and the mastering of firing techniques. In other words, coeval everyday life objects predated the technology and styles of the plaques. In addition, the Chalcolithic culture is characterized by the erection of megaliths, composed by large and heavy stones weighing sometimes more than 150 tons. Some of these large stones were moved across relatively large distances33. It would have taken the coordinated participation of numerous people along protracted periods of time to build a single tholos, for instance. We establish this comparison to suggest that perhaps the social role, complexity, and value of the slate plaques may have been misunderstood. Only local materials were needed to procure the plaque itself , to grind and polish it  and to engrave it 3. In fact, there is no clear evidence of exchange or commerce with the plaques, as it was the case with luxurious items whose raw materials originated in distant places33.Lillios14, contributing to strengthening the bonds among group members. Considering the abundant raw material used in their making, plus how fast they could have been produced, and the importance of stone carving in other social contexts, such as the construction of megalithic tombs, we suggest their social value may have been different than that of other pieces crafted concurrently but using precious materials or rare rocks including gold, elephant ivory or rock crystal, which originated in distant areas sometimes hundreds of kilometers away from the site of discovery39. In the latter case, as with the exquisitely crafted rock crystal arrowheads found inside the Montelirio tholos, of the Valencina Copper Age settlement, the participation of highly skilled and experienced artisans is inferred. Curiously enough, researchers remarked in surprise that no slate plaques or “idols” were found among the hundreds of luxurious items found at Montelirio, in a clearly symbolic and ritual context33.Object manufacture involves the choice of a material for its accessibility and physical characteristics. Also, the use of tools in the framework of learning and developing an activity, i.e. stone carving, fundamental in the structuring of megalithic societies, as well as a shared social framework in which play and the toys acquire their playful meaning, since it should not be forgotten that it is play that gives meaning to the toy3. To determine the motor skills and mental abilities needed to use a stone burin on slate is out of the scope of this investigation, but it may be experimented in the future with children of different ages, even if the cultural context today is completely different to the distant past. The children of stone carves grew in an environment where they had first the chance to learn by direct observation and imitation, and later by collaboration with experienced carvers in increasingly difficult tasks. It is already established that children aged 7–9 years have the ability to recognize patterns and styles, as well as to draw them using paintbrushes22. We leave open the question on the actual earlier age of child participation in the making of slate plaques, as we have no current ethnographic examples to compare to.Competent engravers, perhaps adults or adolescents, may have initiated novice children. Whether this learning process took place in the household or in other production contexts is unknown; as of today, only one possible plaque workshop has been found with plaques at different stages of production 40 that it cannot be discarded that some “idols” were in fact objects with just a recreational value, literally dolls that may have entertained both their makers and younger members of the community in playful activities or as learning activities41. We propose that some plaques may have been decorated with actual feathers. The use of feathers as ornaments is widespread in multiple ethnographic examples around the world, and has even been inferred even for Neanderthals predating the arrival of Homo sapiens in Europe42. The interaction of human-gatherers with birds of prey in particular is not one of hunting as food items, as evidenced by numerous ethnographic examples44, and includes the keeping of pets45. Some of the plaques were painted, and some of the idols may have been dressed with textiles40, reinforcing the idea that at least some plaques functioned as dolls. In multiple ethnographic examples in both the Old and the New Worlds, dolls are made for children by adults or, very often, by the children themselves45.We concur with Maicas21.The fact that many plaques have been found in funerary contexts may reflect that they were at some point used as a tribute to the deceased. Offering plaques would thus be part of a community ritual. This may also indicate that toy or doll offering may have been a way in which the younger members of the group participated in the funerary rituals practiced by adults. Given the highly mimetic character of games, toys (or pieces manufactured by youngsters in any case) could have been offered when such rituals were imitated by the children as part of some of their games. After all, everyday life objects such as pottery and polished tools have also been found in burial contexts. The slate plaques of the Chalcolithic predate one millennium the set of toys of the Siberian Bronze Age made after animal models including birds. They were supposed to be used by children, and were finally deposited at baby graves, as if they had to remain with their deceased ownersNyctea scandiaca) at the cave of Trois-Fréres23. At the cave of Bourrouilla , the skeletal remains of 53 snowy owls highly modified by humans were found in levels of the Upper Paleolithic46. The dark tent tradition, a ritual shared by indigenous peoples in circumpolar northern regions, is performed at night by a shaman emitting animal calls, including owls, and it may have been introduced into North America by Siberian peoples about 5000 years ago47.At this point, we can ask why owls may have been used a models for the first dolls/toys ever made in Europe. Special relationships with owls are evidenced even before the onset of the Neolithic. The two older examples of bird parietal art have been found in French caves: the tufted owl engraved at Chauvet, and a family of three snowy owls  where we see recreational and learning objects. Dissanayake61 pointed out how blurred are the boundaries between art, ritual and play. For this author, play is a social, voluntary and spontaneous activity. Its most notable characteristic is its metaphorical capacity, being a behavior based on exaggeration and imitation: one thing becomes another, being able to express itself in different ways. Imitation becomes a simulation where the referential limits of the initial phenomenon are dissolved. The initiation of learning skills to produce functional objects, which were often decorated, has attracted the attention of scholars62. P. Crown22 has pointed out, for instance, that learning to make pottery begins in childhood in pottery-producing societies worldwide. Southwestern Pueblo children began to learn to make pottery as early as age five16.Children´s object play, and the objects themselves, has been disregarded in the archaeological literature until recently56. Thus, ludic behavior can be both the consequence and the precursor of other more serious behaviors, among them, the aesthetic and the ritual, with whom he also shares his ability to manage stress58. These capacities and emotions were surely present in our ancestors59. We are exactly the same species, even if culturally we have greatly evolved in the last few millennia. From this point of view, we can define the owl-like objects as the product of a playful simulation of reality, being metaphorical objects that imitate and exaggerate a specifically summoned phenomenon, that may have to do with frequent encounters with actual owls, creatures of the night with salient anthropomorphic features. In their social role, these objects had to behave as active patients with respect to the agency exercised by another, that is, they were treated by their possessors as an "alter ego and a social other," as proposed by Gell63.Play offers the child an activity in which he or she can learn the possibilities of ontological mutability necessary for all mimetic behavior23, in turn due to their peculiar anthropomorphism that predispose us to pay attention to them. Additionally, applying Ockham’s Razor, our hypothesis is simpler that the alternative of resorting to a complex symbolic world with fertility goddesses represented by idols64, or heraldic mnemonic devices as proposed by Lillios2, of which there are no proofs anyway. Why the manufacture of stone toys was discontinued about 5000 years ago may have to do with the advent of new technologies. If stone toys were made at the end of the stone age, metal tools in subsequent periods surely made easier the carving of wood figurines, which would hardly leave any traces in the archaeological records. Similarly, skin or textile pieces would disintegrate quite rapidly. Therefore, owl-like objects made in stone provide perhaps one of the few glimpses to childhood behaviour in the archaeological record of ancient European societies55.We wish to remark that our hypothesis that the slate plaques of the Iberian Peninsula in the Chalcolithic were toys inspired on owls which may have had, at least originally, a recreational use, is based on the transcultural fascination of humans by owls since time immemorialSupplementary Information.


Although it is well established that cosmetic makeup enhances perceived facial attractiveness, few studies have examined whether facial makeup modulates neural responses to face images. This study investigated behavioral and attractiveness-related brain responses to self-applied makeup, focusing on the N170, early posterior negativity, P300, and late positive potential components of event-related brain potentials. A total of 77 Japanese women participated in two experiments . Experiment 1 assessed the effects of self-applied makeup on attractiveness-related event-related potential amplitudes using facial images during a makeup identification task in which makeup was directly relevant to task demands. Experiment 2 examined the effects of self-applied makeup using images of one’s own face and another female’s face when performing a gender classification task, where the presence of makeup had no explicit connection to facial gender classification. In both experiments, faces with makeup were rated as more attractive and elicited more negative early posterior negativity and more positive late positive potential components, regardless of the participant’s own face or another person’s face. These findings suggest that people are spontaneously motivated to pay visual attention to faces with makeup, which supports the idea that makeup adds reward value to the facial appearance of the human. Moreover, neural evidence empirically confirmed that the benefits of makeup are not just limited to how others see your face but also extend to how you see your own face. From ancient Greece to today, people, especially women, have used facial makeup to enhance their attractiveness . AccordiCurrent research has attempted to empirically assess several effects of facial makeup. Morikawa and colleagues have used a psychophysical method and have revealed that observers perceived the eyes as larger in a photograph of a female model who applied eyeliner, mascara, or eye shadow compared to the photograph of a female model without any eye makeup, even though actual eye sizes were completely identical , 7. ThesFrom the perspective of facial recognition, Ueda and Koyama  and TagaA series of previous studies have supported the positive effects of makeup on perceived facial attractiveness. Both male and female observers rated faces with makeup as more attractive than faces without makeup –18. ThisFurthermore, the effects of makeup are not limited to the perceptions of others’ faces and also affect the perception of one’s own face , 16. In The idea that facial makeup can influence the perception and recognition of faces has also been supported by studies using neural measures. Ueno et al.  used funRecent studies employing the event-related potential (ERP) technique have illustrated the effects of makeup on facial perception , 28, parTanaka  digitallTagai et al.  showed iAlthough previous ERP research on the effects of facial makeup focused on early visual ERP components, several studies have shown that not only early latency ERP components but also middle and late latency ERP components are involved in facial attractiveness. Among the early latency ERP components, several studies have identified N170 as being sensitive to facial attractiveness –36. HoweIn addition to N170, it has been reported that faces that scored higher in perceived attractiveness elicited middle and late latency ERP components, called early posterior negativity (EPN) and late positive complex (LPC) –42. EPN The primary goal of this study was to explore the effects of makeup on ERP components elicited in response to facial images. Of particular interest were attractiveness-related ERP components ranging from N170 indexing configural processing of faces to EPN, P300, and LPP reflecting automatic or motivated attentional allocation to affective meanings of faces. Since facial makeup often links to an enhanced experience of attractiveness –18, faceFurthermore, previous ERP research on facial makeup has focused on reactions to other individuals’ faces, and no ERP studies have explored the effect of makeup on the response to one’s own face. Previous research has found that makeup can not only modify how others see our facial appearance but also how we see our own faces , 16. AddIt is also important to determine whether the effects of makeup on ERP responses to faces are observed at an implicit level. In a prior study, IAT results suggested that makeup implicitly influences how individuals evaluate faces . FurtherPrevious ERP studies used facial images of individuals with professionally applied makeup or using a digital makeup simulator , 28. AltAlthough fMRI studies have demonstrated that makeup activates emotion-related and reward-related brain regions , previouBased on this background, we conducted two experiments to address whether makeup elicited a change in ERP responses to faces. We examined the face-evoked ERP responses to an individual’s own face with and without makeup in a face classification task where participants needed to look carefully at facial images. We studied this issue by employing different face classification tasks in Experiment 1 and 2. Experiment 1 examined whether viewing one’s own face with makeup changed the attractiveness-related ERP amplitudes in a task where makeup was directly related to the classification of faces, whereas makeup had no clear connection to face classification in Experiment 2. Experiment 2 also investigated whether the effects of makeup on attractiveness-related ERP amplitudes occurred both in response to one’s own face and in responses to another person’s face. In both Experiments 1 and 2, we asked participants to self-apply their facial makeup.In addition to the attractiveness-related ERP components, P200 was also assessed. P200 is a positive deflection that peaks approximately 200–250 ms after stimulus onset over occipitotemporal sites and has been recently reported to be a more valid and reliable neural index of self-face processing –61. A seThree hypotheses were tested in this study. First, we hypothesized that self-applied makeup enhanced perceived facial attractiveness. Second, we hypothesized that makeup led to a change in attractiveness-related ERP amplitudes, both with an individual’s own face and with another person’s face. It was expected that faces with makeup would elicit a larger negative deflection in the EPN time window and a larger positive deflection in the P300/LPP time window than faces without makeup. However, the mechanism by which facial attractiveness modifies the N170 amplitudes remains controversial. Nevertheless, relatively more findings supported more negative N170 amplitudes in response to facial attractiveness , 36, proThirty-four Japanese women participated in this experiment  = 2.87 years). They were recruited from the general public by a Japanese marketing company and were paid JPY 7,000 (approximately USD 67) for their participation. Prior to the experiment, an online survey was conducted to identify potential participants. The eligibility requirements for participating in the experiment were as follows: (a) aged between 25 and 35 years; (b) wearing facial makeup for at least five days each week; (c) having a normal or corrected-to-normal vision; (d) having normal color vision acuity; (e) no current neurological or psychiatric disorders; (f) no history of neurological or psychiatric disorders; (g) no shrapnel or other metal or electronic implants in the body; (h) no skin disorders; (i) no scars or tattoos on the face; (j) no eyelash extensions within three months prior to the experiment; and (k) right-handedness, as determined by the Flinders Handedness survey (FLANDERS) questionnaire score between +5 and +10 . AdditioWe recruited only women because they were expected to self-apply their facial makeup and be able to distinguish between faces with makeup and without makeup much easier than men, which was assumed to be necessary for participants to complete our experimental procedure. Previous studies have revealed that perceived facial attractiveness correlated with the actual age of subjects who were photographed . MoreoveParticipants viewed six color images of their own faces with and without facial makeup using three different head orientations . PhotogrThe participants were asked to arrive at the test room with the facial makeup that they would normally wear while going out with their friends on the weekend, including foundation, eye shadow, eyebrow color, cheek color, and lipstick or lip gloss. Additionally, participants were asked to avoid consuming caffeine 6 h before the experiment, avoid smoking 3 h before the experiment, and avoid alcohol on the day or before the day of the experiment.After greeting participants and obtaining written informed consent, the experimenters took portrait photographs of the participants with facial makeup using three different head orientations. Next, the participants completely removed their makeup using cleansers and were photographed again using the same three head orientations.After the photography session, participants sat in a dimly lit and sound-attenuated room at a distance of 60 cm from the computer screen. A chin rest was used to maintain the viewing distance and maintain a steady head position. The experiment consisted of a makeup identification task, with continuous EEG recording, and a face rating task, which is described in detail below. The total duration of the experiment was approximately 90 minutes.The stimuli were displayed at the center of a 27-inch LCD monitor  against an identical light gray background color [RGB ]. Each facial image subtended a visual angle of 11.89° horizontally and 11.89° vertically.During the makeup identification task, participants used button presses to classify six facial images as faces with makeup or without makeup as quickly and accurately as possible. Participants indicated their responses by pressing the left and right buttons on a Cedrus response pad  using the index fingers of both hands. The assignment of the response buttons (left/right–makeup/no makeup) was counterbalanced across participants. The task was divided into six trial blocks consisting of 40 face presentations per block, totaling 240 trials overall. Each picture was presented 40 times. Each trial started with a white square frame presented for jittered inter-stimulus intervals (ISIs), followed by the presentation of facial images for 1,000 ms . ISIs ofAfter completing the makeup identification task, the EEG scalp cap was removed and participants performed the face rating task. During this task, participants rated each individual image for facial attractiveness and 14 additional facial attributes using an 11-point Likert scale . The trial started with a white square frame, presented for 1,000 ms, followed by a facial image that was displayed until the participant responded. The facial attributes rated for each image are presented immediately above the image. Participants rated the six images in succession for each facial attribute before proceeding to the next facial attribute. The presentation orders of the facial images and facial attributes were completely randomized. The task was run using PsychoPy software .All procedures were reviewed and approved by the ethical committee of the Shiseido Global Innovation Center. Data were collected prior to the COVID-19 pandemic outbreak.EEG data were recorded with a 64-channel Geodesics Sensor Net using Net Station 5.4 software . Amplified EEG signals were digitized using an A/D converter at a 1,000 Hz sampling rate with a 24-bit resolution. Continuous EEG data were recorded with respect to the Cz reference. The impedances were adjusted to below 50 kΩ, following the manufacturer’s recommendations.EEG data were analyzed offline using the EEGLAB toolbox, version 14.1.2b  running Preprocessed EEG data were segmented into epochs, beginning at 200 ms prior to stimulus onset and continuing for 1,000 ms after stimulus presentation. For each trial, waveforms were baseline corrected by subtracting the mean amplitude during the time interval 200 ms prior to stimulus onset from each data point in the waveform. Trials corresponding to incorrect responses were excluded from the analysis. Epochs were discarded if the signal amplitude variations exceeded −200 to 200 μV or the joint log probabilities deviated by six SDs from the mean of the probability distribution for all trials at the single-channel level or three SDs at the all-channel level .To quantify N170, P200, EPN, P300, and LPP amplitudes, the time windows and two regions of interest (ROI) were chosen a priori based on previous research , 59–61. t-test to determine whether the differences between the two conditions (makeup vs. no makeup) for the same participant were not equal to zero. Three head orientations were combined in the analysis. The significance level (α) was set to 0.05 for all analyses. We used a two-tailed test and computed the effect size (Cohen’s within-subject dz) for the t-test. The dependent variables were: (a) attractiveness rating scores during the face rating task, (b) response accuracy during the makeup identification task, (c) median reaction times (RTs) associated with correct classifications during the makeup identification task, (d) mean N170 amplitudes, (e) mean P200 amplitudes, (f) mean EPN amplitudes, (g) mean P300 amplitudes, and (h) mean LPP amplitudes. The results for the other 14 attributes examined during the face rating task were not reported in this study. All statistical analyses were performed using R version 3.6.2 . Each facial image subtended a visual angle of 8° horizontally and 10° vertically.During the gender classification task, participants were asked to classify five facial images as either female or male via button press as quickly and accurately as possible. Participants pressed either the left or right button on a Cedrus response pad  with the index fingers of both hands. The assignment of response buttons  was counterbalanced across participants. The task was divided into 12 trial blocks consisting of 50 face presentations per block for a total of 600 trials overall, with each picture presenting for a total of 120 trials. Each trial started with a white rectangular frame presented for different ISIs, followed by the presentation of a facial image for 1,000 ms . The ISIAfter completing the gender classification task, the EEG scalp cap was removed, and participants continued the face rating task. We did not include male facial stimuli during the task. The procedures for this task were the same as those described for Experiment 1, except that the participants rated their perceived emotions in response to each stimulus using the Self-Assessment Manikin (SAM) scale  at the eAll procedures were reviewed and approved by the ethics committee of the Shiseido Global Innovation Center. Data were collected prior to the COVID-19 pandemic outbreak.The EEG data were recorded using an elastic cap and an ActiveTwo EEG acquisition system . A total of 32 Ag-AgCl active electrodes  were used according to the international 10–20 system. Two additional external electrodes were placed on the left and right mastoids. The amplified EEG signals were digitized by an A/D converter at a 2,048 Hz sampling rate with a 24-bit resolution. The voltage from each active electrode was measured online with respect to a common mode sense active electrode, producing a monopolar  channel. The electrode offset was maintained at less than ± 40 mV. We used a different EEG recording system than the one that was used in Experiment 1 because of laboratory constraints. However, both systems have been reliably used in many previous studies.The EEG preprocessing procedure and ERP quantification methods were the same as those described for Experiment 1, except for the predefined electrode sites. Here, we selected four electrodes  for the occipitotemporal ROI, and six electrodes  for the centroparietal ROI. The layout of the selected electrodes is presented in α) was set to 0.05, and we calculated the ηG2 effect sizes for all ANOVAs. Additionally, in order to facilitate comparisons between Experiments 1 and 2, the main effect for the makeup factor was assessed by calculating the mean and SDs averaged across self-face and another female’s face, and then its effect size was shown using Cohen’s within-subject dz. The dependent variables were the same eight variables described for Experiment 1 plus the SAM valence and arousal rating scores for a total of 10 variables.We performed a 2 × 2 repeated measures analysis of variance (ANOVA) on each dependent variable, with the facial identity  and makeup (makeup vs. no makeup) conditions as within-subject factors. The male face condition was not included in the analysis. The significance level  = 0.17, p = 0.680, ηG2 <0.001) and makeup  = 0.02, p = 0.898, ηG2 <0.001) or the interaction between facial identity and makeup  = 1.84, p = 0.182, ηG2 = 0.005). Cohen’s dz for the makeup factor was 0.020.A within-subject two-way ANOVA of the female vs. male classification accuracy yielded no significant effects of facial identity  = 11.86, p = 0.001, ηG2 = 0.011) and makeup  = 27.85, p <0.001, ηG2 = 0.004) but no significant effect of the interaction between facial identity and makeup  = 1.07, p = 0.306, ηG2 <0.001). Cohen’s dz for the makeup factor was 0.805. Participants classified faces as female more quickly in the makeup condition than in the no makeup condition. Furthermore, participants classified their own faces as female more quickly than other females’ faces.A within-subject two-way ANOVA of the female vs. male classification RTs yielded significant effects of facial identity  = 5.81, p = 0.020, ηG2 = 0.020) and makeup  = 91.58, p <0.001, ηG2 = 0.350) but no significant effect of the interaction between facial identity and makeup  = 0.36, p = 0.553, ηG2 <0.001). Cohen’s dz for the makeup factor was 1.459. Participants rated faces with makeup as more attractive than faces without makeup. Additionally, participants rated other females’ faces as more attractive than their own faces.A within-subject two-way ANOVA of attractiveness ratings showed significant effects of facial identity  = 76.08, p <0.001, ηG2 = 0.327). However, no significant effects were found for facial identity  = 3.01, p = 0.090, ηG2 = 0.020) or the interaction between facial identity and makeup  = 3.45, p = 0.070, ηG2 = 0.008). Cohen’s dz for the makeup factor was 1.330. Participants rated faces with makeup as more pleasant than faces without makeup.A within-subject two-way ANOVA of the SAM ratings for emotional valence showed a significant main effect of makeup  = 7.32, p = 0.010, ηG2 = 0.042). However, no significant effects were found for makeup  = 3.50, p = 0.069, ηG2 = 0.020) or the interaction between facial identity and makeup  = 0.02, p = 0.895, ηG2 <0.001). Cohen’s dz for the makeup factor was 0.285. One’s own face was more emotionally arousing than other females’ faces. Both faces with makeup and without makeup were found to be mildly arousing.A within-subject two-way ANOVA of the SAM ratings for emotional arousal identified a significant main effect of facial identity  was rejected, and an average of 1.86 independent components  was removed as ocular components. None of the 10 channels located in the two ROIs were rejected by any participant. After rejecting bad epochs, not <84% of original trials remained for ERP averaging per participant for each condition .F = 2.20, p = 0.145, ηG2 <0.001), a significant effect of facial identity  = 6.68, p = 0.013, ηG2 = 0.001), and a significant interaction between facial identity and makeup  = 5.19, p = 0.028, ηG2 = 0.001). The analysis of simple main effects confirmed that another female’s face with makeup elicited more negative N170 amplitudes than that without makeup  = 6.12, p = 0.018, ηG2 = 0.002). Additionally, viewing one’s own face without makeup evoked more negative N170 amplitudes than viewing another female’s face without makeup  = 13.08, p <0.001, ηG2 = 0.003). The mean N170 amplitudes for each condition were as follows: makeup × one’s own face , no makeup × one’s own face , makeup × another female’s face , and no makeup × another female’s face . The mean N170 amplitudes for the faces with and without makeup pooled across self-face and another female’s face were −0.36 μV (SD = 3.12 μV) and −0.27 μV (SD = 3.13 μV), respectively. Cohen’s dz for the makeup factor was 0.226.A within-subject two-way ANOVA of the mean N170 amplitudes revealed no significant effect of makeup  = 52.27, p <0.001, ηG2 = 0.017) but no significant main effect of makeup  = 0.23, p = 0.636, ηG2 <0.001) as well as no significant interaction between facial identity and makeup  = 0.006, p = 0.941, ηG2 <0.001). Less positive P200 amplitudes were observed for one’s own face than another female’s face . The mean P200 amplitudes for the faces with and without makeup pooled across self-face and another female’s face were 3.99 μV (SD = 3.89 μV) and 3.95 μV (SD = 3.90 μV), respectively. Cohen’s dz for the makeup factor was 0.073.A within-subject two-way ANOVA of the mean P200 amplitudes revealed a significant main effect of facial identity  = 65.57, p <0.001, ηG2 = 0.033) and makeup  = 4.48, p = 0.040, ηG2 = 0.001) but no significant interaction between facial identity and makeup  = 0.006, p = 0.938, ηG2 <0.001). One’s own face evoked more negative EPN amplitudes than another female’s face, and faces with makeup evoked more negative EPN amplitudes than faces without makeup . The mean EPN amplitudes for the faces with and without makeup pooled across self-face and another female’s face were 1.64 μV (SD = 3.43 μV) and 1.79 μV (SD = 3.48 μV), respectively. Cohen’s dz for the makeup factor was 0.323.A within-subject two-way ANOVA of the mean EPN amplitudes indicated significant effects of facial identity  = 128.33, p <0.001, ηG2 = 0.077) and makeup  = 4.63, p = 0.037, ηG2 = 0.001) but no significant interaction between facial identity and makeup  = 0.001, p = 0.973, ηG2 <0.001). One’s own face evoked more positive P300 amplitudes than another female’s face, and faces with makeup evoked more positive P300 amplitudes than faces without makeup . The mean P300 amplitudes for the faces with and without makeup pooled across self-face and another female’s face were 3.13 μV (SD = 1.39 μV) and 3.03 μV (SD = 1.40 μV), respectively. Cohen’s dz for the makeup factor was 0.328.A within-subject two-way ANOVA of the mean P300 amplitudes indicated significant effects of facial identity  = 71.41, p <0.001, ηG2 = 0.140) and makeup  = 4.34, p = 0.043, ηG2 = 0.004) but no significant interaction between facial identity and makeup  = 0.27, p = 0.605, ηG2 <0.001). One’s own face evoked more positive LPP amplitudes than another female’s face, and faces with makeup evoked more positive LPP amplitudes than faces without makeup . The mean LPP amplitudes for the faces with and without makeup pooled across self-face and another female’s face were 1.79 μV (SD = 0.94 μV) and 1.66 μV (SD = 0.92 μV), respectively. Cohen’s dz for the makeup factor was 0.318.A within-subject two-way ANOVA of the mean LPP amplitudes indicated significant effects of facial identity  of makeup on these ERP amplitudes ranged from 0.3 to 0.4, which can be regarded as small to medium according to Cohen’s convention .http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research) on papers that include identifying, or potentially identifying, information, the individual(s) or parent(s)/guardian(s) must be informed of the terms of the PLOS open-access (CC-BY) license and provide specific permission for publication of these details under the terms of this license. Please download the Consent Form for Publication in a PLOS Journal . The signed consent form should not be submitted with the manuscript, but should be securely filed in the individual's case notes. Please amend the methods section and ethics statement of the manuscript to explicitly state that the patient/participant has provided consent for publication: “The individual in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details”.As per the PLOS ONE policy . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. </font> The Reviewer #1: YesReviewer #2: Yes********** <font color="black"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes********** <font color="black"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The literature is reviewed in a clear and concise manner. However, the aim of the present study is vague. The authors examined whether makeup evokes greater attractiveness-related brain responses when presented with an individual’s own face than when presented with another person’s face . It is unclear whether their assumptions regarding changes in brain responses depend on makeup effects, facial attractiveness, or discrimination between self and others . The authors should clearly explain the relationship between task processes and ERP components.In Experiment 1, the authors found that participants identified their own faces without makeup faster than they did with makeup. Participants rated their own made-up faces more attractive than without makeup. Previous studies have indicated that people detect attractive faces automatically and keep looking at them . Are the present results consistent with previous findings? In addition, RTs derived from Experiment 2 were faster for participant’s own made-up faces than for their own faces without makeup. Thus, I wonder whether the behavioral results are reliable. It is well known that RT data do not follow a normal distribution. The authors should perform a logarithmic transformation of RT data (or use median RTs) and conduct the statistical analysis again.Interpretations of results from Experiment 1 are not consistent. The authors stated that when participants viewed their own faces with makeup, larger EPN and LPP waveforms were elicited than when they viewed their own faces without makeup . However, although the amplitude of the LPP component was greater with makeup, the amplitude of the EPN component was greater without makeup . Do the amplitudes of ERP components reflect differences in facial attractiveness or RTs?For Experiment 2, why did the authors analyze the P200 component? The Introduction offers no explanation of mental processes based on P200. It is very confusing. If the P200 component is important in recognizing one’s face, it should be analyzed in Experiment 1 as well.The authors used different EEG setups in Experiments 1 and 2. Locations of electrodes also differed between the two experiments. Thus, the results of Experiment 1 are not directly comparable to those of Experiment 2. The authors should explain the reasons for these different EEG arrangements.The authors reported significant differences in brain responses between experimental conditions, but the effect size in each case was very small. I am not sure that these results are robust. Are there any criteria regarding the effect size?My impression is that the manuscript lacks logical connections between the Introduction and the Discussion. First, what is the ecological relevance of distinguishing made-up faces of self from those of others? In daily life, we see other's faces, but we rarely see our own. I think that makeup can change one’s mood . Do such changes in mood affect brain responses? Makeup can also enhance the salience of facial features . The authors discuss effects of makeup on facial attractiveness and visual attention, but not the effect of salience on object detection. They should clearly explain how ERP components are associated with mental processes of face recognition.I cannot read the descriptions in the figures  because of low resolution. Please improve the quality of figures.Reviewer #2: On the whole, this study has strengths that are worth mentioning -- I have a few clarification points that I would like to see, however.First of all, the literature review is thorough, and the novelty of the research is clear in terms of how it expands on the literature. I appreciated the expansion of the research to focus on reactions to one's own face, to comparing one's own face with another's, and the exploration of whether the results would still occur under implicit conditions. Likewise, having the more realistic notion of makeup that has been self-applied has its benefits, although it also provided some downsides, which I'll expand on below. The description of the methodology used was thorough, and the sample size was sufficient for research of this type, and for the most part, I found the analysis to likewise be complete and comprehensible. The discussion was clear at highlighting the key findings, and was reasonably transparent in terms of methodological challenges (i.e having them apply their own makeup without strict consistency).There were a few things I would like to see added to the study:1.) The description of the makeup the participants were told to apply was 'makeup that they would normally wear while going out with their friends on the weekend.' . On page 19, ln. 413, the makeup worn is classified as 'ordinary makeup'. This is not day-to-day wear -- it is makeup explicitly worn only on the weekends for a night out. This, to a certain extent, could help explain some of the results in terms of reaction time, as participants would be more used to seeing their bare faces, or the makeup they may have worn while at work, as opposed to the rarer, dressier makeup they were asked to put on. The researchers should further clarify how the type of makeup could differ from daily wear, and not refer to it as 'ordinary' makeup, as that could be confusing.2.) While the effect sizes were appropriate to the type of test used, the authors should acknowledge effect size in their discussion of results, making sure to specify not only a significant finding, but clarifying when a result was a large or more modest effect to better contextualize the results -- as well as briefly identifying what is a typical effect size for studies using this methodology.3.) The authors report all cell means and standard deviations, but in Study 2, there are several main effect tests that are significant; providing the main effect means and standard deviations would be preferable in addition to cell means. ********** <font color="black"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Yes: Hirohito M. KondoReviewer #1: Yes: Caitlin A. J. PowellReviewer #2: https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  24 Apr 2022We thank the editor and reviewers for positively receiving our manuscript. We are grateful to receive constructive and useful suggestions. In the revised manuscript that we submit with this response letter, we have undertaken required changes to address all the comments made. All the changes in the revised manuscript  are in red text in the "Revised Article with Changes Highlighted” file. You can also find our point-by-point response to each comment in the “Response to reviewers” file.AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 7 Jul 2022 <div> PONE-D-21-33037R1 </div>  <div> Cosmetic makeup enhances facial attractiveness and affective neural responses </div>  <div> PLOS ONEDear Dr. Arai,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.I am very sorry for the long delay. I have found it seriously difficult to find expert reviewers.However, I think the paper still needs a minor revision at this stage.Given the long delay, I suggest the authors revise their manuscript by solving the reviewer comments and then submit their last revision for acceptance.Please accompany the revised manuscript with a detailed letter explaining how you have responded to each of the reviewers' points, and where the changes appear in the revised manuscript.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Aug 21 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at  </div> A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. <div> If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.Please include the following items when submitting your revised manuscript:https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Vilfredo De PascalisAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):I am very sorry for the long delay. I have found it seriously difficult to find expert reviewers.However, I think the paper still needs a minor revision at this stage.Given the long delay, I suggest the authors revise their manuscript by solving the reviewer comments and then submit their last revision for acceptance.Please accompany the revised manuscript with a detailed letter explaining how you have responded to each of the reviewers' points, and where the changes appear in the revised manuscript.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color="black"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)********** <font color="black"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes********** <font color="black"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1: Yes********** <font color="black"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. </font> The Reviewer #1: Yes********** <font color="black"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes********** <font color="black"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The several descriptions remain difficult to understand. I pointed them out in my previous comments, but the authors did not update their manuscript. For example, in lines 397-399, the authors stated that “Viewing one’s own face with makeup on resulted in larger EPN waveforms than viewing one’s own face without makeup .” What does a larger waveform mean? That statement does not depend on whether the waveform indicates positive or negative deflection. The authors also stated that “Another key finding was that faces with makeup elicited larger EPN and LPP amplitudes regardless of whether the presence of makeup was explicitly relevant to the participant’s task" (lines 781-783). This is misleading. As described above, the amplitude of the EPN component was larger for the non-makeup condition. I found that the EPN component was negatively biased for the makeup condition than for non-makeup condition, whereas the LPP component was positively biased for the makeup condition than for non-makeup condition. The authors should check their statements in all the sections and reword them appropriately.Technical comment: the authors should enlarge the font size in the figures.********** <font color="black"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Yes: Hirohito M. KondoReviewer #1: **********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. </div> While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  18 Jul 2022We thank the editor and reviewers for positively receiving our manuscript. We are grateful to receive constructive and useful suggestions. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. Thank you for your consideration. I look forward to hearing from you.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 29 Jul 2022Cosmetic makeup enhances facial attractiveness and affective neural responsesPONE-D-21-33037R2Dear Dr. Arai,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Vilfredo De PascalisAcademic EditorPLOS ONEAdditional Editor Comments :First of all, I am sorry for the long-lasting time required to revise this manuscript. I have had difficulties finding expert reviewers serving for this manuscript and thank the authors for their patience.I see that the current revised version is quite improved after addressing the insightful reviewer' suggestions. The authors have addressed all the necessary changes following the reviewers’ suggestions. Thus, I think the manuscript can be accepted for publication at this stage.Reviewers' comments: 5 Aug 2022PONE-D-21-33037R2 Cosmetic makeup enhances facial attractiveness and affective neural responses Dear Dr. Arai:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProf. Vilfredo De Pascalis Academic EditorPLOS ONE


Introduction: Cerebrospinal fluid (CSF) has been implicated in amyotrophic lateral sclerosis (ALS) due to its ability to spread inflammatory proteins throughout the nervous system. We hypothesized that filtration of the CSF could remove pathogenic proteins and prevent them from altering motor phenotypes in a mouse model.Methods: We filtered the CSF from 11 ALS patients via 100 kilodaltons (kD) molecular weight cut-off filters. We used mass spectrometry-based discovery proteomics workflows to compare protein abundances before and after filtration. To test the effects of CSF filtration on motor function, we injected groups of mice with saline, filtered ALS-CSF, or unfiltered ALS-CSF (n=12 per group) and assessed motor function via pole descent and open field tests.Results: We identified proteins implicated in ALS pathogenesis and showed that these were removed in significant amounts in our workflow. Key filtered proteins included complement proteins, chitinases, serine protease inhibitors, and neuro-inflammatory proteins such as amyloid precursor protein, chromogranin A, and glial fibrillary acidic protein. Compared to the filtered ALS-CSF mice, unfiltered ALS-CSF mice took longer to descend a pole  and explored less on an open field .Conclusions:We demonstrated the ability to filter proteins from the CSF of ALS patients and identified potentially pathologic proteins that were reduced in quantity. Additionally, we demonstrated the ability of unfiltered ALS-CSF to induce motor deficits in mice on the pole descent and open field tests and showed that filtration could prevent this deficit. Given the lack of effective treatments for ALS, this could be a novel solution for patients suffering from this deadly and irreversible condition. Studies have explored the role of the cerebrospinal fluid (CSF) in amyotrophic lateral sclerosis (ALS). The CSF proteome in ALS patients contains increased pro-inflammatory proteins and molecules compared to normal -3, possiWe hypothesized that CSF filtration could remove pathogenic proteins and reduce motor phenotypes in ALS. Filtration was explored in the 1990s using a method called liquorpheresis to filter and reintroduce CSF into the thecal sac with a syringe pump ,8. SmallCryptococcus neoformans from rabbits in vivo [One newer method to filter CSF is Neurapheresis™, which extracts CSF from the lumbar subarachnoid space using a dual-lumen catheter, filters pathogenic material, and reintroduces CSF into the midthoracic region . Neuraph in vivo  and VX2  in vivo  and has  in vivo .Neurapheresis has been demonstrated as a therapeutic option for subarachnoid hemorrhage with minimal adverse effects in patients , thus coCSF samplesTen ALS patient CSF samples were procured from the Department of Veteran Affairs, Biorepository Brain Bank Biobank (VABBB), Boston, Massachusetts, United States, approved by Duke University Institutional Review Board , and one sample was collected from an ALS patient at Duke University Medical Center, approved by Duke University Institutional Review Board , via lumbar puncture with informed consent in accordance with the Duke University Medical Center Institutional Review Board. Demographic information, including patient age and sex of the samples, are shown in Table CSF filtrationCSF fractions from 11 samples were used in the proteomics study, of which 10 were filtered. The control was not filtered. Biorepository samples were stored at -80°C, thawed on ice, and then filtered according to the manufacturer’s instructions via 100kD ultra-centrifugation filters . The Duke patient sample was maintained at room temperature after collection and filtered using Neurapheresis with the modification that a single 100kD tangential flow filter was used instead of the dual filter design used in clinical trials. This sample was used in the mouse movement experiments. Processed samples were stored at -80°C until analysis.Mass spectrometry proteomicsoC with 6µL, 50mM dithiothreitol, then incubated in the dark for 30 minutes with 4µL 250mM iodoacetamide. Alkylation was quenched with 6µL dithiothreitol. Samples were incubated at 37oC for four hours with 14µL trypsin (2µg). Digestion was quenched with 10µL 5% formic acid.The volume necessary to reach 50µg total protein was calculated from the bicinchoninic acid assay  result ooC. Mobile phases were water containing 2% acetonitrile, 0.1% formic acid (mobile phase A), and acetonitrile containing 20% water and 0.1% formic acid (mobile phase B(MPB)). MPB was held at 5% for two minutes, increased to 25% over 47 minutes, increased to 40% over eight minutes, increased to 95% in one minute, and held at 95% for 16 minutes. Mass spectrometer parameters were: 1.9kV positive mode spray voltage; ion transfer tube temperature, 275°C; master scan cycle time, 1.5 seconds; m/z scan range, 375 to 1,600 at 120K resolution; 300% normalized automatic gain control; 120ms maximum full scan injection time; radio frequency lens, 40%; 15K mass resolving power for data-dependent scans; 1.5m/z isolation window; 30% normalized higher-energy collisional dissociation; 100% normalized Automatic Gain Control (AGC) Target; 21ms maximum injection time; and dynamic exclusion applied for 20-second periods.Peptide digests were analyzed with an Easy-nLC™ 1200 system  interfaced with an Orbitrap Exploris™ 480 Mass Spectrometer (Thermo Fisher Scientific Inc.). A quantity of 2µL was injected on an Acclaim™ PepMap™ (Thermo Fisher Scientific Inc.) 100 C18 LC trap column  in line with an EASY-Spray™ (Thermo Fisher Scientific Inc.) analytical column  at 45Homo sapiens protein database (Taxon 9606) obtained from UniProtKB/Swiss-Prot  and calculate protein abundances. SEQUEST-HT search node used the following parameters: tryptic cleavage at arginine and lysine, maximum of three missed cleavage sites; minimum peptide length of six amino acids; 5ppm precursor mass tolerance; 0.02Da fragment mass tolerance; maximum of three equal and four total dynamic modifications, which were oxidation of methionine and deamidation of asparagine and glutamine; dynamic protein terminus modifications of acetyl addition or methionine loss; and static carbamidomethylation of cysteine. Peptides were validated by the Percolator node with q-value set to 0.05 and strict false discovery rate set to 0.01. Protein abundances were calculated using precursor ion intensities and the summed abundances of precursors without normalization or scaling.Proteome Discoverer 2.4.305 (Thermo Fisher Scientific Inc.) was used to interrogate raw data against a Mouse injections and experimentsA total of 36 mice were divided into groups of 12 for unfiltered CSF, filtered CSF, and control , respectively. A quantity of 5ul CSF was injected into the Cisterna Magna (ICM) every six days for three injections per mouse. For pain management, buprenorphine was injected subcutaneously at 0.5mg/Kg before ICM . Mice weMice were tested in the open field and the ability to dismount a vertical pole. Pole tests were performed on days 1, 1.25, and 10 after the final injection. The open field test was performed on days 1 and 10 post-injection. For the pole test , the timStatistical analysisData were analyzed and plotted using R 4.1.1   and GrapMean time to descend in the pole test and mean distance traveled in the open field were calculated for each group at each time point. Two-way multiple comparisons ANOVA was used to any significant differences between experimental groups set at alpha=0.05, with post-hoc analysis done with multiple comparisons testing at alpha=0.05.Proteomics. Key proteins were grouped by functionality: there were 29 complement proteins, four serine protease inhibitors (SERPINs), three chitinases, 11 neurofilaments, tau proteins, and neuroendocrine proteins, and 11 other immune proteins.A total of 1,648 proteins were identified in the ALS-CSF samples (shown in Appendices). Of these, 58 were identified as key proteins for further analysis. ,6,23-27.1All proteins had lower average abundances when filtered, but 13 complement proteins, two SERPINs, one chitinase, three “neuro-proteins”, and four other immunological proteins were statistically significantly less abundant when filtered. Comparisons between the groups are shown in Figure Mouse motor testsThe pole descent test assessed maneuverability, coordination, and fine motor control . Mice weThe open field test assessed gross motor activity , and it We analyzed the proteome of the CSF from patients with ALS, characterized the changes in composition after filtration, and tested the effect of ALS-CSF on mouse motor activity. Proteomics identified over 1,600 proteins in the CSF, of which 58 were selected as key proteins implicated in the disease ,6,23-27.Key proteinsThe complement proteins mark targets for phagocytosis and elimination . ExcessiIn-vivo studies have directly linked chromogranin A (CHGA) to ALS, demonstrating it promotes secretion of SOD1, leading to neuron degeneration ,44. GliaC-reactive protein (CRP) may contribute to damage in Alzheimer’s , and a sS100-A9 mRNA levels were increased at end-stage ALS but deleting it in a mouse model had no impact on motor neuron survival and slightly accelerated symptoms . In one Mouse motor testsThe open field measures gross locomotor activity and has been previously studied in the context of ALS ,66. On dThe pole descent test has been tested as a measure of Parkinson’s disease ,68, but LimitationsOne key limitation of this study is the underlying assumption that the alteration in mouse behavior might be due to key proteins that we identified here; however, other filtered cytokines and molecules could also play a role that we have not identified in this study. Additionally, by using mass spectrometry, we are restricted to analyzing relative changes in the identified proteins. We would have had to utilize methods such as enzyme-linked immunosorbent assay (ELISA) to identify specific quantities and concentrations of proteins, but we believe that our methodology allows for adequate analysis of changes in the protein makeup of the CSF samples. Our samples are also inherently biased as we were only able to procure samples from male patients; thus, we should expand our future studies to include female patient samples. A limitation of our mouse studies is that we did not conduct studies that would allow us to monitor the recovery of the mice injected with ALS-CSF. CSF is recycled multiple times daily , thus thWe analyzed the changes in the proteome of ALS-CSF before and after filtration by molecular weight and demonstrated reductions in numerous proteins implicated in ALS pathophysiology and various biomarkers. Additionally, we demonstrated the ability of the unfiltered ALS-CSF to induce gross motor deficits in mice using pole descent and open field tests and showed that filtration could prevent this deficit. Clinical trials will be needed to test the potential of Neurapheresis as a treatment for ALS. It has been shown to be a safe and effective treatment for other neurological conditions, and with the lack of effective and affordable treatments for ALS at the present time, this could be a novel solution for patients suffering from this deadly and irreversible condition. Future studies include the implementation of repetitive filtration in mice with ALS to determine the volume and frequency of CSF filtration that could serve a therapeutic purpose.


Participants were ≥65 years without a diagnosis of dementia and presented for primary elective hip or knee arthroplasty. Plasma samples collected pre- and postoperatively were grouped as either control  or delirium . Widespread changes in plasma metabolite levels occurred following surgery. The only metabolites significantly differing between corresponding control and delirium samples were ornithine and spermine. In delirium cases, ornithine was 17.6% higher preoperatively, and spermine was 12.0% higher postoperatively. Changes were not associated with various perioperative factors. In binary logistic regression modeling, these two metabolites did not confer a significantly increased risk of delirium. These findings support the hypothesis that disturbed polyamine metabolism is an underlying factor in delirium that warrants further investigation.The mechanisms underlying the occurrence of postoperative delirium development are unclear and measurement of plasma metabolites may improve understanding of its causes. Participants ( Delirium is a clinical syndrome characterized by disturbances in attention, awareness, and cognition . DeliriuMass spectrometry methods for profiling metabolites have been used extensively to investigate disease processes and discover novel disease biomarkers ,12. Key Only a small number of studies to date have explored changes in metabolites associated with delirium. Previous work from our group has shown arginine metabolism is altered preoperatively in CSF from delirium patients . Other sParticipants were sampled from an observational cohort study, the methods for which are published . In brien = 28, aged: 76.2 ± 5.7) and age and sex-matched controls  underwent metabolomic analysis [Plasma samples from participants developing delirium , years in education, Charlson Comorbidity Index (CCI), estimated IQ, Bristol activities of daily living scale (BALD) score, Vertical Visual Analog Pain Scores, and American Society Anesthesiologists (ASA) physical status ,26,27. NPerioperative anesthetic and surgical data were collected and considered as potential confounders as below. Data available included: premedications; duration of surgery; intraoperative blood loss; intraoperative intravenous fluid volumes administered; use of peripheral nerve block/plexus blockade; general anesthetic and intraoperative sedation; perioperative analgesics and antiemetics; intraoperative tachycardia and/or hypotension; prophylactic antibiotics administered.Venous blood was sampled from fasted participants into lithium heparin bottles preoperatively at the time of anesthetic administration and postoperatively on Day 1. Samples were transported to the laboratory on wet ice within 12 h of collection, where they were centrifuged at 4 °C at 3750 rpm for 10 min and 500 mL aliquots of the supernatant pipetted  into 1.5 mL polypropylene tubes  and stored at −80 °C.Quantitative mass spectrometry-based metabolomic profiling was carried out by the Biocrates Absolute IDQ p180 . All plasma samples were prepared according to the manufacturer’s instructions and analyzed on a triple-quadrupole mass spectrometer . Metabolites (amino acids and biogenic amines) were derivatized using phenylisothiocyanate (PITC) in the presence of isotopically labeled internal standards, separated using a UPLC  system with a reverse-phase column  and quantified using a triple-quadrupole mass spectrometer  operating in the multiple reaction monitoring (MRM) mode. All the remaining metabolites  were quantified using the same mass spectrometer without column separation by the flow injection analysis (FIA) operating in MRM mode. Metabolite concentrations were calculated and expressed as micromole (μM). The mean of the coefficient of variation (CV) for the 184 metabolites in repeated quality controls was 0.12, and 85% of the metabolites had a CV of <0.15. Following these analyses, 138 metabolites were selected above limit of detection (LOD) values.g for 5 min with no brake and 500 mcl aliquots of the supernatant pipetted  into 1.5 mL polypropylene tubes  and stored at −80 °C. Spinal anesthesia was carried out, fasting, in the sitting or lateral positions using 25-gauge  Whitacre-type spinal needles with graduated metal introducers (Vygon). Once CSF was obtained, a 5 mL syringe  was attached to the spinal needle, and up to 5 mL of CSF was withdrawn by the anesthetist. The CSF was immediately transferred to a 30 mL sterile polypropylene universal container (Unisurge), which was placed on wet ice in an insulated container. All samples were transported to the laboratory within 12 h of collection. CSF samples were centrifuged at 4 °C at 3000 rpm/1811× CSF samples were transported to the Leonard Wolfson Biomarker Laboratory, University College London on dry ice. There, CSF Aβ42, t-tau, and p-tau concentrations were measured by a single, trained, technician, blinded to the clinical data, using the INNOTEST β-amyloid (1–42), hTau Ag, and Phospho-tau (181 P) ELISA kits, respectively , run on a FLUO star Omega BMG LABTECH instrument (chemiluminescent plate reader) according to the manufacturer’s protocols. Samples were thawed at 21 °C in an air-conditioned lab and vortexed before use. CSF biomarker values were determined using Omega software  and a 4-parameter curve fit. Intra- and interassay CVs, respectively, were 2.0% and 3.9% for Aβ42, 3.3% and 8.1% for T-tau, and 1.6% and 5.1% for P-tau. Longitudinal stability in the measurements is further monitored in the laboratory by participation in the Alzheimer’s Association Quality Control program.MetaboAnalyst software  was used for the data analysis. Data were normalized to the median and autoscaled before using PLS-DA scores and VIP scores. A normality test was carried out by SPSS . To compare the difference between pre and post-op metabolites, the paired Student’s t-test for parametric data and the Wilcoxon Mann-Whitney test for nonparametric data were performed. False discovery rates  were calculated based on Benjamini–Hochberg to determine if metabolites were statistically different between the two groups . For multiple comparisons on metabolites over 4 groups , a one-way ANOVA test for parametric data and Kruskal–Wallis test for nonparametric data were conducted. PLS-DA was carried out to highlight significant metabolites, which explain the maximum amount of variation between the groups. A VIP plot was created to identify the top 15 metabolites responsible for the observed separation between groups. t-test, χ2 analysis, and Mann-Whitney U test to compare the difference between control and delirium groups. To investigate whether perioperative changes in metabolites could be due to perioperative factors, correlation analyses were undertaken between perioperative variables and % change of metabolites, and the q-value from Benjamini–Hochberg was confirmed.Binary logistic regression was carried out to investigate risk factors for delirium. Statistical tests that we used were independent Baseline characteristics of the selected participants with and without delirium are displayed in Changes related to surgery: A total of 121 metabolites out of 138 significantly differed between pre-op and post-op samples  clearly differentiated pre-op and post-op plasma samples . Seven metabolites were commonly influential in both models , and their characteristics are shown in p-value, 0.005; adjusted results: odds ratio for delirium, 0.994; 95% (CI), 0.989–0.999; p-value, 0.027). Estimated IQ and MMSE show a significant difference for p-value in unadjusted results , 0.872–0.999; p-value, 0.047; MMSE: odd ratio for delirium, 0.711; 95% (CI), 0.528–0.958; p-value, 0.025). Neither spermine nor ornithine reached statistical significance. Although, the adjusted result for spermine was just outside the p-value threshold , 0.997–1.076; p-value, 0.071).To investigate potential predictor variables for delirium, binary logistic regression analysis was undertaken. Univariate analyses of perioperative variables between delirium and control groups are displayed in In this investigation, plasma from a nested case–control cohort of postoperative delirium underwent broad metabolite profiling and showed two metabolites, spermine and ornithine, were significantly elevated in delirium cases. The physiological context for the observations was either hip or knee arthroplasty, and the surgical procedures had an overriding impact on the plasma metabolome. The occurrence of delirium influenced the overall metabolome to a minor extent, and furthermore, those changes linked to delirium appear to be highly specific. In blood samples taken preoperatively, ornithine was almost 17.6% higher in persons prone to delirium. Furthermore, a metabolically related metabolite, spermine, was almost 12.0% higher postoperatively in cases of delirium.The fact that ornithine differed preoperatively, before the ensuing metabolic turbulence of surgery, indicates a possibility that this metabolite is a prognostic biomarker and/or risk factor for delirium. Although logistic regression modeling did not determine ornithine to confer additional risk in this nested cohort, sampling of larger cohorts is needed to definitively determine this risk. Ornithine overall was lowered by the surgical insult, but then this was also the case for the vast majority  of measured metabolites. This postsurgical lowering was only significant in delirium cases and not in controls, which is intriguing since ornithine is a precursor for polyamine formation, which includes spermine . Postoperative spermine was also not determined to be a significant risk factor here, but the odds ratio had a p-value of 0.07, indicating that with increased statistical power it possibly could be. The delirium-specific rise in spermine following surgery raises the question of whether the activity of the rate-limiting enzyme, ornithine decarboxylase (ODC) is augmented in individuals prone to delirium. The translation and activity of ODC are regulated by various factors, which include changes in polyamine concentrations . ODC conThere is a neurological context for the dysregulation of polyamines. Most pertinently, we have recently demonstrated using preoperatively collected cerebrospinal fluid (CSF) from this same cohort that spermidine, putrescine, and glutamine were significantly higher in delirium patients compared with controls . It shoup = 0.027). Aβ is a pathological feature of AD, and CSF Aβ is used for specific diagnosis of AD. Lower CSF Aβ42 normally signifies greater aggregation of Aβ in amyloid plaques in the brain [The aggregation of Aβ and phosphorylated tau protein are considered hallmarks of AD. The present investigation did not find evidence of CSF t-tau or p-tau associated with delirium; however, the data from the entire cohort did find a relationship , so the he brain . This sthe brain ,42. Furthe brain . It is nhe brain .The delirium and control groups in this case–control study were matched only for age and sex and differed in a number of other significant ways. Despite the relatively small sample size of this nested case–control study, which will have limited the statistical power, there are a number of key strengths to this investigation. Firstly, the collection of preoperative and postoperative blood samples is relatively unique; secondly, the detailed clinical characterization and measurement of CSF biomarkers; thirdly, the collection of perioperative information helps exclude potential confounding from the surgical and anesthetic procedures; and finally, the acquisition of annotated and quantitative metabolomic data provides a relatively broad understanding of the impact on metabolism. There are also some minor limitations. The nested case–control used available blood samples from participants matched only for age and sex who did and did not develop delirium following elective arthroplasty. A very small number of participants lacked perioperative variable data . The scaIn summary, metabolomic approaches are providing new insights underlying delirium occurrence, allowing new hypotheses to be formed. Once again, the findings here indicate that polyamine metabolism either contributes to the development of delirium or is a part of underlying neurodegeneration or vulnerability. Clearly, a comprehensive and focused examination of the entire arginine–ornithine biochemical pathway is now warranted. This should include the measurement of metabolites from various tissue compartments involved in the urea cycle, polyamine biosynthesis (including their acetylated forms), as well as interconnecting metabolites such as agmatine and glutamine. The most logical way forward should be to establish a bespoke targeted quantitative LC-MS/MS methodology for measuring all the metabolites in the wider biochemical pathway. Then, this methodology could be applied to samples acquired from a range of different postoperative delirium cohorts.