Critics have argued that human-controlled avatar interactions fail to facilitate the kinds of expressivity and social understanding afforded by our physical bodies. We identify three claims meant to justify the supposed expressive limits of avatar interactions compared to our physical interactions. First, “ The concept of an ‘avatar’ derives its meaning from Sanskrit, literally referring to an earthly embodiment or incarnation of an otherworldly deity. As such, the concept has often been adopted to mean a concrete embodiment of something abstract. However, with the rise of digital technologies such as video games, ‘avatar’ is now typically understood to refer to a virtual image representing a particular person or entity in a digital space, often controlled by a human. In this way, the meaning of the concept has notably shifted from ‘something abstract made bodily’ to ‘something bodily made abstract.’If we view avatars as an abstraction from or a stand in for a bodily human, it seems intuitive to suppose that any social interactions between human-controlled avatars will be similarly abstract, and, consequently, that interacting with another human-controlled avatar is removed or uprooted from our more concrete, everyday encounters with other people. To put it another way, when we meet one another mediated through avatars, we might expect that the way we encounter one another is significantly different to how we encounter one another when we are physically face-to-face.While there has been significant work done on how people experience using avatars in virtual worlds, e.g., in terms of embodiment, presence, immersion, identification, self-identity, online friendships, and community , Daniel Stern calls attention to the way in which we perceive the dynamic style of people’s movements and that perceiving vitality is crucial for our understanding of others. We suggest that the LoL players’ reports give evidence that they perceive the other players’ avatars as having vitality and that being sensitive to vitality gives them important insight into the other human players’ feelings, intentions, and experiences. Crucially, this expressive vitality is not  explicitly inputted into the avatar by the player but arises through play. Moreover, the reports also put the Decoding Claim under pressure, as the players report seeing expressivity in the movements and actions of the avatars, rather than having to ‘figure out’ what the other players are feeling and experiencing.In this paper, we provide an in-depth analysis of how a set of skilled players of the popular, competitive game League of Legends (LoL) experience and interact with other players’ avatars in-game. Drawing from interviews with these expert practitioners, we find evidence to loosen the Limited Expressivity Claim and resist the Inputted Expressivity Claim. We suggest that these two claims overlook a key form of expressivity of human  bodies—“vitality.” In his book photorealistic as they are not, e.g., seeking to accurately mirror the subtle, expressive nuances of the user’s physical face or body. In other words, our analysis is not aimed at the increasingly photorealistic avatars that have been emerging over the past few years, especially in consumer VR tech , argues that we do not simply perceive others in terms of what they do but the how of their actions and movements, their manner and style . The same type of gesture can have a different style when expressed by different people in different circumstances; for instance, we might perceive someone waving shyly, exuberantly, weakly, tentatively. What Stern is drawing attention to here is the dynamics of our expressivity, what he calls “forms of vitality.”By focusing on individual gestures, what falls into the background is a more holistic way of experiencing the other’s bodily expressivity. Stern, in his book Stern claims that nearly all actions are marked by a vitality contour—a distinctive style of movement—and that our experience of vitality is “fundamental and primary” 2010, 25). He describes vitality as a gestalt that emerges from five interlinked dynamics: “movement, time, force, space, and intention/directionality” . Vitality is not an emotion but the tone of the dynamic flow of action and patterns of movement. Importantly, Stern does not see vitality as some kind of additional flair or detail that actions might have. Rather, he argues for the primacy of vitality: “The experience of vitality is inherent in the act of movement” . Stern suggests that it is vitality that marks the aliveness and dynamism of human action, stating that “[w]ithout manifestations of vitality, the world would be bereft of much of its interest, and human interactions would be digital rather than analogic, whatever that might be like” .010, 25. An individual’s vitality is not fixed. The same action can be performed with different styles depending, for instance, on the context, their mood, their well-being; we might wave openly to a friend and furtively to a crush. Moreover, the vitality of an action can change over time. Joel Krueger  illustraTheory Theory or Simulation Theory account of social cognition and still admit the perception of vitality as an important form of bodily expressivity that aids our understanding of others.Stern’s notion of vitality captures an important way in which bodily movement is expressive and emphasises the place vitality plays in our experience of others. Important for our purposes, recognizing our ability to perceive vitality broadens the range of behavior that we might perceive as expressive of another’s subjectivity and experience  bodies do  but the way in which players express themselves through their avatars. Hubert Dreyfus . This we call the “Inputted Expressivity Claim.”Note that we can, of course, deliberately perform bodily expressive gestures. One might wave exaggeratedly to get someone’s attention, make a show of nodding along to someone’s talk to indicate attentiveness, and mask one’s annoyance with a smile. To clarify, the claim made by Dreyfus and Scriven is not that we never consciously decide how to move our physical bodies in expressive ways but that our physical bodies are also sites of expressivity where smiles, frowns, hesitations, nose wrinkles, widened and narrowed eyes often play out without deliberation but when it comes to avatars we are limited to only expressivity that is explicitly inputted and performed by the player. As such, to the extent that we want to allow that avatars’ movements can be expressive of the player’s intentions and emotions, this expressivity is still notably different from the lively immediacy of bodily expressivity.directly pick…up” emotions, moods, and intentions of others when we encounter them face-to-face, and perceive fleeting smiles, furrowed brows, and sceptical looks as immediately meaningful, when we encounter someone’s avatar, we must “figure out what the [avatarial] gesture means”  game built around various genres of high fantasy, steampunk, and sci-fi. The game’s standard competitive format pits two teams of five players against each other, each player in control of an avatar with a unique set of abilities (known in-game as a “champion”),The avatars themselves are fantastical, ranging from yeti-looking creatures and minotaurs to tiny furry beings known as “yordles.”From a gaming perspective, movement in LoL is paradigmatic of many video games. Players move their champion around in-game primarily via clicks of their mouse. By right-clicking somewhere in the game world, their avatar will move to the clicked location unless stopped, e.g., by other in-game forces such as other players’ avatars, or by being redirected, e.g., by giving the avatar a new command through the click of their mouse. During performance, players will often execute several hundreds of movement commands every minute, primarily in order to adjust their avatar’s current position, as well as to deploy its abilities. In general, the only kinds of avatar actions available to the players are the mouse-directed relocation commands of the avatar (the avatar’s standard movement), as well as the aiming and use of the avatar’s abilities, which typically occur through a combination of keyboard and mouse use. While so-called “emotes” exist in LoL, which can cause one’s avatar to dance or laugh, these are rarely used once a game begins.In terms of communication, players can write to each other using the in-game text-based chat, even across the two teams if they wish, as well as via an in-game “pinging” system, used to immediately draw teammates’ attention to specific areas or events on the game map. In organized and competitive LoL, players typically communicate verbally with their own team using headsets.The data used in this paper are part of a larger set generated in the years 2018–2019 by the first author in connection with a research project on the phenomenology of organized competitive videogaming—also known as ‘esports’  encountering player avatars vs. bot avatars, (ii) perceiving the vitality of other players’ avatars, and (iii) using and manipulating vitality in-game. Based on this analysis, we move away from characterising avatars as essentially lacking expressivity and instead highlight the sensitivity that practitioners have to the forms of vitality that players express through their avatars and the role this plays in social understanding and performance in-game.Interviewer: [can one] provide a meaningful difference in the way one experiences a bot compared to the way one experiences a human player given that one does not have direct access to the other actual human? ...almost human-like, but they do not move in quite the same manner [Lyn’s emphasis] .Lyn: That’s the thing. Because when I compare this, then I think back on the actual bots you can play against. They move…When examining avatarial expressivity, a good place to start is comparing the players’ experiences of human-controlled avatars with their experiences of computer-controlled avatars or ‘bots.’ If we suspect that avatars lack the kind of expressivity that physical bodies have, then we might also suppose that players would be unable to differentiate between human-controlled avatars and bots. In LoL (and similar games of the same genre), the standard bots do move and act in a manner where a complete LoL novice might, at first, be somewhat hard-pressed to tell the difference between the bot and a human player. An enemy bot seemingly reacts to the activity of the player in relatively responsive ways, withdrawing when in danger and pushing forward when the player is in danger. However, with just a little practice, the differences between a human and a bot become obvious. During an interview, the distinction between bots compared to human-controlled avatars is especially emphasized by LoL professional Lyn:could present in the very same manner as a human-controlled player elicits the following response: “I would be very surprised, I would be befuddled…” .For Lyn, there is something subtly but qualitatively different between encountering a human player and a bot. Indeed, the very idea that a bot Being able to differentiate a bot from a player impacts how a player engages with an opposing avatar. LoL professional Lucas specifically elaborates on this, describing that beating a standard bot in LoL involves using one’s avatar in a calculative manner—for instance, by making mistakes on purpose to try and gauge the bot’s predetermined responses . In contrast, when facing skilled players, Lyn describes how these are normally experienced as more unpredictable, moving in complex and irregular patterns .in the interaction”, Lyn explains, emphasizing that, despite their complexity and unpredictability, she does not experience these bots as making any real decisions, but rather as her causing the bots to act in certain ways based on her own avatarial input [Lyn’s emphasis] .7One might surmise, then, that the difference between fighting bots and humans can be chalked up to the complexity and unpredictability of beating other human players compared to bots. While unpredictability is a central feature of human avatarial interactions, focusing on this alone as the central distinctive feature contradicts the LoL informants’ descriptions of fighting more challenging computer-controlled opponents available. As an example, in LoL, players can battle extremely capable bots – so-called ‘Doom Bots’, computer-controlled avatars that spontaneously and unpredictably acquire high-powered and novel abilities throughout a match. Yet, even in this case, “…there is little humanity causing bots to respond to her own actions, the practitioners do not appear to experience other player-controlled avatars as moving around and acting in an automatic, predestined manner. In drawing attention to the idea of humanity in the interaction, Lyn appears to point to not simply what bots or player-controlled avatars are doing. Rather, she highlights the way in which player-controlled avatars are not simply responsive to actions but are experienced as an agent in the interaction, as some kind of reciprocal other.Based on these descriptions, we can appreciate that human-controlled avatarial movements and actions can be characterized by something beyond the unpredictability and skilfulness of the interactors themselves—that there seems to be something distinctly human about fighting human-controlled avatars. In contrast to Lyn’s descriptions of merely Let us now turn to how practitioners describe encountering human-controlled avatars in more detail. In LoL, opposing players' avatars will often ‘jitter’ rapidly around each other in a manner as a product of their hundreds of rapid but precise clicks per minute on their gaming mouse to relocate their avatar in the virtual space. An important reason for jittering like this, according to the informants, is to make themselves a more unpredictable and difficult target.When several human-controlled avatars are jittering near each other, they come to constitute what Lyn describes as an in-game “dance” —a dynamical back-and-forth of trying to step close enough to or angle your position in an optimal way to hit and pressure your opponent, while dodging and weaving to avoid potential, retaliatory attacks. The resulting interaction will likely look like a fast-paced and pixelated mess to a LoL-outsider, increasing in complexity the more avatars are jittering around near each other simultaneously. In-game, from a strategic standpoint, jittering is an effective way to move against human opponents, but, importantly for our purposes, these interactions between human players are also described as animated by the practitioners in ways that bot interactions are not .seeing it. The way you become conscious of the human player is in the way they perform and the way they move [Loke’s emphasis] .You would. It is something you just… experience, right? It’s this thing once more about We might then be tempted to think that the distinction between human-controlled avatars and bots rests on the presence of jittering; that jittering is what makes human-controlled avatars human-like in the way bots are not. However, bots can also jitter. Is there, then, a single distinctive manner in which human-controlled avatars move that make them, as Lyn describes it, “human-like”? The answer to this seems to be no. Practitioners are quick to emphasise that human-controlled avatars do not all move in one uniform manner. For instance, even when different human players control graphically identical avatars, they each move and act in different manners. When asked if he would experience the same identical avatar played by different human players across different rounds in a match distinctly, Loke explains:manner that human-controlled avatars move and act that not only distinguishes them from bots, but, as Loke notes, also between individual players. That is to say, human-controlled avatars seem to have an expressive range that bots do not. Echoing Stern’s discussion of vitality forms, it is not just what human players are doing that is different, there is something about how they do it that is distinctive (even between one another). In addition, not only are the dynamics of the avatar central to Loke’s experience of the other avatar as a human player, but this experience is even described in terms of “seeing it.”In this way, the informants’ descriptions indicate that there seems to be something about the Depending on how quickly the avatar moves, you can see if the person is stressed or not. If they move rapidly with small clicks all over the place, this normally means the player is stressed. Whereas if it is this random, nice and easy clicking over all the time, then the person is calm and in control – and on top of the situation. So, there you can read it .Lyn likewise emphasizes the importance of seeing and reading others’ avatarial “body language” , stressing that, while she “knows it sounds dumb,” she can see how other players in-game are doing mentally based on how their avatar moves and acts , and later even adding that it is actually “really difficult to hide one’s intention about what one is doing…” . She elaborates when further describing the phenomenon of experiencing the body language of other avatars:players, rather than bots, then, seems to be underpinned by a sensitivity to the vitality contour expressed in the player’s playstyle. This suggests that the experience of another player’s autonomy and agency through their avatar is not simply linked to what they do but to the way in which their temperament, experiences, and emotions shine through in their movements and actions.Based on the flow of subtle stylistic differences in the patterns of movement between the jittering avatars, the informants notably use a wide repertoire of adjectives and adverbs to describe their immediate sense of how different human-controlled avatars move, including “pressured,” “brave,” “apathetic” or “frustrated,” “aggressive,” and “supportive” . Loke notes that when consistently outplaying an opponent, he will “sense their frustration” through their avatar  and Lyn explains that when cooperating with a familiar teammate, she will be able to see if her teammate’s movements and playstyle seem especially “nervous” that day, and even what “general mental state” the teammate enters the game in . Informants even describe being caught by surprise at how expressive an opponents’ movements can be, with LoL practitioner Leon noting that he will catch himself thinking “Wow, he is stressed—that guy!” . Note that the practitioners are not referring to discrete expressive gestures made by an avatar here  but to the dynamic patterns of movements and actions of the avatar. The “body language” they are referring to is the holistic way the whole avatar of the human player is moving in a temporally extended manner; it is the tone of the dynamic flow of action and patterns of movement; it is the vitality contour of the player’s avatar. Experiencing player-controlled avatars as other Moreover, while players often bring their own temperament and style of moving and acting to their gameplay, as Leif notes , these can change over time during a match depending on the in-game circumstances. In response to a highly pressured in-game situation, the avatarial style of a player with an aggressive playstyle might grow and appear more cautious during a round, and vice versa. That is to say, their vitality is not fixed. Likewise, any player’s general style, like Krueger’s guitar player, will also develop from their time as novice to proficient.has this vital aspect based on how the player is currently playing. Indeed, as we have seen, the player may be unaware that their stress is shining through in the way they are moving their avatar around. To put it another way, there is not a button or a menu for inputting vitality into these patterns of movement (cf. selecting a pre-programmed wave), the vitality emerges through the way the avatar dynamically moves and interacts with other avatars and the virtual world.Avatarial expressivity is notably not something the LoL players typically pay explicit attention to during performance – even when they are directly engaged with other players. As we have seen, the trainers describe “seeing” the others’ avatars as expressive. As noted by Loke, it is something that becomes an “intuitive feeling” ; a pre-reflective part of their experience of the other players’ in-game movements and actions. Nor, importantly, are these expressive forms of vitality typically directly inputted by the player into their avatarial actions. For instance, a player does not necessarily present as stressed because they are deliberately and intentionally inputting that expressive style into their avatar’s movement; rather, the movement of the avatar inherently Through the unfolding, even changing, expressive style and vitality of an avatar’s movements and actions, the humanity of the other player seems to become salient for the LoL players. Moreover, seeing human-controlled avatars as expressive appears to impart social understanding of the player ‘behind’ the avatar within the context of the game world. However, this is not based on the perception of the static expressivity of discrete facial expressions, but on the perception of forms of vitality of the player’s avatar’s movements and actions in the game. By moving away from scrutinizing how avatars ‘emote’ or how they fail to replicate offline bodies, to looking at how players experience avatarial unfolding, dynamic movement as expressive and personal in terms of vitality, we are able to appreciate an important aspect of avatar expressivity, and a significant part of how players perceive each other during performance, that has so far been overlooked.One possible interjection here is that whatever social understanding of an opposing player is imparted via avatarial expressivity merely amounts to instances of projection. That is to say, one might interject that players simply read their own, e.g., emotional states into the avatarial movements of other players. There are at least two reasons to be sceptical of this. For one, the practitioners by no means have to be undergoing the same emotional state that they perceive through the avatarial movements of other players. Second, the expressivity of one’s own and other players’ avatars, including the imparted social understanding, are not mere addendums to the experience of proficient LoL interactions. As we shall see, far from projecting, e.g., one’s own emotions onto other players’ avatars, the practitioners’ ability to accurately see and engage with an opponent’s state of mind can be integral to successful LoL performance.informs the way that the practitioners play. One informant, Balder, concludes a longer description of how he experiences movements in LoL by noting:that, that one makes his decisions along the way! [Balder’s emphasis] There is something in these… movements, where you can read a whole lot. I guess that is how it is. I never thought about it like that, but that is really how it is. Right, right—that’s what one does! And it is actually from The practitioners do not simply report being able to see the expressive forms of vitality in relation to human-controlled avatars. Being able to see these expressive forms of vitality The way I often do it is… I usually—and this is one of my secrets—I usually play insanely aggressively right from the beginning in order to see “What do they do if I play insanely aggressively?” Players not only report acting upon the expressive movements and actions of others’ avatars but also that they sometimes use their own expressive styles in a strategic manner. For instance, a player can exaggerate a particular playstyle in order to draw out or learn something about the style and even temperament of the opponent.Adding to this, players sometimes use avatarial expressivity as ways to deceive their opponents. As an example, Balder later notes that “[i]f you are well ahead, but [the opponent] keeps playing very defensively, then you can underplay how strong you really are” . Manipulating one’s own avatarial style, such as moving, and acting meekly, as Lyn similarly describes, can work to draw an overly cautious opponent in . Such a move not only involves a sensitivity to the other player’s expressive style but also involves deliberately performing a particular form of vitality, knowing that the other players will perceive one’s expressivity in a particular manner. In this regard, it is precisely because the avatarial expressivity of other LoL players ordinarily can impart accurate instances of social understanding, that being able to at times distort this imparted social understanding can be integral to successful performance, as it can be in offline, physical competitions too.seeing and of using avatarial expressivity, other in-game actions can also play a role, such as attacking and using abilities. While the practitioners do not describe any explicit experienced differences in expressivity between, e.g., avatarial movements and avatarial attacks and ability use, their descriptions do point to an expressive relation between these during performance. That is to say, attacking actions can factor into avatarial expressivity, with Lyn noting that, depending on whether or when an opponent attacks her when she moves about in a” tough” or “confident” manner, “flexing her muscles”, this can tell her a lot about the person she is fighting . In addition, factors such as how far away an avatar is able to hit an opponent (its attack range), as well as its unique set of available abilities, can further factor into what an avatar’s movements express. For example, when and how a player controlling a melee-ranged avatar’s movements appears aggressive or cautious (whether deliberate or not) can be very different from when and how the movements of a longer-ranged avatar might do so—something a capable player remains sensitive to throughout their performance. Expressive style, then, is shaped by the overall in-game situation.Adding further nuance to this, both in the context of Recognising that players see and use expressivity as part of their in-game performance also sheds further light on a classic strategic move used in many forms of gaming: tilting. In games like LoL, tilting refers to the way that players either themselves experience or attempt to elicit a strong emotional reaction from an opponent that obstructs and disrupts their performance. In this context, if an opponent’s style quickly changes from confident to frustrated or frenetic in response to pressure in-game, players can tell that this opponent is further susceptible to becoming tilted, for instance by ganging up on them repeatedly throughout a match (even if the team has to invest a lot of time and resources to do so) in order to render their play ineffective long-term.The expressive avatarial activity is not only something that the players see but is something they actively use and act upon as competitors. Being sensitive to the expressivity of the other players is not just an interesting detail about how avatars can be experienced in-game but can be imperative to successful performance. Where players deliberately obfuscate or manipulate the way they move to convey a particular expressive style, these tactics work because the player not only can see their opponent’s style but presumes that their opponent can also see their own expressive vital style.As we have seen, there has been much scepticism about the extent to which avatars can, if at all, be experienced as expressing a player’s thoughts, feelings, and actions. As detailed above, this scepticism is often expressed in three related, but separable, claims: the Limited Expressivity Claim, the Inputted Expressivity Claim, and the Decoding Claim. We now reconsider each of these claims in light of our analysis of the LoL players’ reports about how they experience, and make use of, the expressive vitality of avatars.8The Limited Expressivity Claim argues that avatars simply do not have a sophisticated enough expressive repertoire to allow us to perceive the other player’s thoughts and feelings through their avatar. When scrutinising the options that players have to express themselves in-game, focus is usually placed upon how players can: (i) design the appearance of their avatar; (ii) input pre-programmed ‘emote’ functions, such as waving, blowing a kiss, bowing, that their avatar performs; and (iii) use in-game chat functions to type and talk to one another over headsets. This often leads researchers to highlight the difference between encountering avatarial bodies in-game compared to face-to-face encounters in the real world.vitality of avatars in-game.Recalling the quote from Stern from earlier, we might suppose that due to the limited expressivity of avatars, in-game interactions are precisely experienced as “digital rather than analogic”—i.e., as lacking the aliveness and dynamism of human action. This quote may even suggest that interactions that take place on digital platforms are incomparable with the actions and movements of physical bodies. On the contrary, however, our analysis of the practitioners’ experiences lends support to the idea that vitality as a form of expressivity is present in these digital spaces. The practitioners describe how they experience the dynamic movements, actions and interactions of avatars as expressive. This expressivity is not found in discrete facial expressions or gestures but refers to the holistic style that a human-controlled avatar has. This motivates our claim that players perceive the between players.The informants describe a rich array of expressive styles that human-controlled avatars have. Player-controlled avatars can move and act hesitantly to aggressively, calmly to erratically, frustratedly to nervously. Seeing an avatar moving around in a frenetic way, for instance, might reveal that the player is stressed out. The informants do not simply describe the human-controlled avatars as expressive but describe how this tells them something about the player ‘behind’ the avatar. Indeed, as the informants highlight, seeing the vital style with which another player’s avatar is moving gives them crucial information about how that player is doing, what emotional state they are in, what kind of player they are, and this informs how they engage with and interact with that particular player. Notably, players also describe how this expressivity of human-controlled avatars can dynamically shift over the course of a game as a player’s style moves, for instance, from calm to aggressive to stressed. Indeed, the vitality of another player not only seems to be perceived by other players but can emerge through on-going interactions Based on our analysis, the LoL players’ accounts arguably add further nuance to our understanding of vitality by emphasizing that, despite not resembling offline bodily interactions, human-controlled avatarial interactions can still have a wide range of vitality contours and thus be expressive in an analogue way even in digital contexts. In other words, the practitioners’ descriptions emphasize that what makes an interaction analogue rather than digital is not dependent on whether this interaction in fact takes place in a digital space, and, consequently, that Stern’s framework of vitality contours can help illuminate virtual interactions too.almost human but not quite. There are two ways in which we might interpret the participants’ descriptions of bots as ‘lacking humanity’ within the framework of vitality. On the one hand, we might conclude that the bots simply lack expressive vitality. Indeed, Stern precisely speculates that when interactions lack vitality, we would experience them as digital rather than analogic  confidence, competence, and even spotlight oneself as a target for tilting, it can be presumed that often this vitality is precisely not something that the players are deliberately or consciously inputting into their avatars. Rather it is something that emerges through the what to do  but not how they do it . Indeed, the vitality described by the informants often sounds like the kind of unconscious body language which it is suggested that avatars cannot possess.Taking vitality as a mode of expression into account rebuts the claim that expressivity is something that must necessarily be cognitively, laboriously, or deliberately inputted by the player. Players might be commanding their avatars perform vitality; that it is not always automatic and pre-reflective. At first glance, this appears to support the Inputted Expressivity Claim that the player must decide to input this expressivity into the avatar. However, just as using our physical bodies to perform expressive behaviour does not eliminate the possibility of pre-reflective bodily expressivity, the deliberate performance of vital styles with an avatar does not refute the occurrence of pre-reflective avatar vitality. It is also interesting to note that in order for the deception to work, the opponent players must perceive the nervous or hesitant vitality as giving them access to the player’s real emotional state and skill level. We might suppose, then, that the very success of performing vitality, rests on vitality typically being something that is not deliberately inputted by the player.In Section What, then, about the Decoding Claim? This is the claim that when we interact with people in the physical world, we do not need to engage in cognitively onerous processes of working out what bodily expressions mean but we see bodily expressivity as immediately meaningful and salient. In contrast, writers such as Dreyfus claim that when it comes to avatars, we have to explicitly decode any expressive signals given by avatars and work out what this might reveal about the player.see, and are often surprised by, the human players’ emotions, intentions and skilfulness in the way their avatar performs and moves. We do not, then, find descriptions that suggest that the players must engage in some kind of cognitively onerous activity of ‘figuring out’ the expressive actions of the other avatars, nor descriptions of having to make inferences about what the other players are experiencing.How the informants’ descriptions of perceiving and using the expressive movements of avatars relate to the Decoding Claim is not entirely clear. It is notable that a number of the informants at times describe being able to ‘read’ the body language of the avatars. This may sound like support for the idea that players must decode what the expressive movements of the avatar might reveal about the avatar’s player. However, it is also notable that the informants do not describe the avatars as signalling or performing vital styles. Rather they describe how they as expressions of the player. It is not that they perceive the avatar as signalling stress, rather, they seem to perceive the player as stressed. Indeed, in light of the participants’ descriptions, the claim that players must necessarily cognitively figure out what the other is doing just seems overly laborious—particularly when we consider the fast-pace at which players must perceive, adapt and respond to the expressivity of their opponents and teammates in the heat of battle.As such, the reports do not appear to suggest that the players are adopting a bifurcated approach of seeing a signal and then cognitively decoding it. Indeed, they report seeing the avatarial movements smart. In order for our social perception to become smart, experience and familiarity are important. We are not born with a pre-programmed understanding of all bodily expressivity. Our social perception is informed and shaped by our experience, it develops over time and (hopefully) becomes more sophisticated as we grow. We also talk of certain people being more socially sensitive and perceptive—able to pick up on subtle bodily cues and respond to them appropriately. Our social perception is also culturally informed and embedded , the informants’ descriptions might not resonate with a novice gamer sitting down with LoL for the first time. To put it another way, the requisite know-how might be shared among certain communities, e.g., sufficiently experienced gamers, and not others. In this context, appreciating the saliency and relevance of vitality contours of avatarial interactions of LoL has been made possible precisely by turning to the perspectives of experienced, familiar informants. In other words, the LoL informants’ descriptions underscore the continuous importance of looking to actual, smart perceivers’ experiences of avatarial interactions when making claims about the limits of avatarial expressivity.Based on reports from expert LoL players, we have argued that discussions about the expressivity of avatars have overlooked ways in which the vitality contours of avatars can be perceived and used by skilled players and that perceiving vitality can give rise to social understanding of the player ‘behind’ the avatar. We have further argued that this evidence also suggests that vitality is not necessarily inputted into the avatarial body but emerges through gameplay, and that this vitality can be perceived in the movement of the avatar, rather than through onerous cognitive inference. Furthermore, the ability to perceive avatarial expressivity in this way, we have argued, will depend on the person’s history and familiarity with the relevant avatar movements and interactions.interactions, we suspect the literature on participatory sense-making (De Jaegher & Di Paulo, Following our analytic strategy laid out in Section A second, related question concerns potential differences between being a player and being a spectator when it comes to seeing avatarial expressivity. More specifically, it remains an open question to what extent avatarial engagement and interaction impacts or is necessary for seeing other human-controlled avatars as expressive. With the rise of competitive, organized video gaming as spectator sports, what part, if any, does vitality play in the spectating experience of avatars competing? Based on the results of our analysis, we suspect that familiarity with the game will plausibly factor into this, too. While one informant did touch upon the difference between participating and spectating, we were unable to draw any final conclusions on this question.Second Life and Gather Town, their precise place in the myriad of different avatar interactions that exist today remains an open question. On the one hand, if a game like LoL, with its bird-eye perspective, lack of photorealistic avatars, and interactions often distinct from offline, physical forms of interaction, can still facilitate salient expressivity relating to other people’s mental states, it might well be that other forms of digital interaction with stronger similarity to human-like perspectives and traits can afford even more salient degrees of such expressivity. On the other hand, in the spirit of looking beyond avatars’ ability to mimic our physical bodies, we also find it particularly interesting to consider avatar-based platforms where interacting humans control not only a single, delineated avatar that they move around. Consider the fast-paced strategy game StarCraft II. Here, competing players are in control of entire military complexes, constantly undergoing construction and expansion, as well as vast clusters of armies moving in unison—all of which takes place at interactive rates much higher than LoL (Lowood, Third, while we suspect vitality contours will play a role in games beyond LoL, as well as other avatar-based social platforms such as  Lowood, . As note


Brugada (BrS) and early repolarization syndromes (ERS), the so-called J wave syndromes (JWS), are associated with life-threatening ventricular arrhythmias. Pharmacologic approaches to therapy are currently limited. In this study, we examine the effects of ARumenamide-787 (AR-787) to suppress the electrocardiographic and arrhythmic manifestations of JWS and hypothermia.Na and IKr in HEK-293 cells stably expressing the α- and β1-subunits of the cardiac (NaV1.5) sodium channel and hERG channel, respectively. In addition, we studied its effect on Ito, INa and ICa in dissociated canine ventricular myocytes along with action potentials and ECG from coronary-perfused right (RV) and left (LV) ventricular wedge preparations. The Ito agonist, NS5806 (5–10 μM), ICa blocker, verapamil (2.5 μM), and INa blocker, ajmaline (2.5 μM), were used to mimic the genetic defects associated with JWS and to induce the electrocardiographic and arrhythmic manifestations of JWS  in canine ventricular wedge preparations.We studied the effects of AR-787 on Ito) and enhancement of the sodium channel current (INa), with lesser effects to inhibit IKr and augment calcium channel current (ICa). AR-787 diminished the electrocardiographic J wave and prevented and/or suppressed all arrhythmic activity in canine RV and LV experimental models of BrS, ERS and hypothermia.AR-787  exerted pleiotropic effects on cardiac ion channels. The predominant effect was inhibition of the transient outward current (IOur findings point to AR-787 as promising candidate for the pharmacologic treatment of JWS and hypothermia. The J wave syndromes (JWS) are characterized by distinctive J waves and/or ST segment elevation in specific ECG-leads and are associated with risk for development of polymorphic ventricular tachycardia and fibrillation (pVT and VF) leading to sudden cardiac death (SCD). The two principal forms of JWS are Brugada syndrome (BrS) and early repolarization syndrome (ERS) .First line treatment for high-risk patients suffering from JWS is an implantable cardioverter defibrillator (ICD), an approach that is problematic in young infants for whom a pharmacologic alternative would be preferable. Pharmacological therapy is also desirable for individuals experiencing frequent appropriate ICD shocks . Thus, tKCND3-encoded KV4.3 transient outward potassium current (Ito) plays a pivotal role in the pathophysiology of both syndromes  of 500–2,000 ms. A detailed description of the arterially perfused ventricular wedge preparation has been previously published ) was purchased from MolPort SIA  in powder form. It was dissolved in 100% DMSO to create a stock solution of 50 mM and diluted with external solution to the desired final concentration. DMSO content was always 0.1% or less. Verapamil and NS5806 were purchased from Sigma-Aldrich . Stock solutions of all drugs were prepared in 100% DMSO. Drug-response relationships were determined by introducing increasing concentrations of the pharmacological agent to different groups of cells. Thus, each cell was exposed to a single concentration of AR-787.t-test. Data are shown as mean ± SEM. Statistical significance was considered at p<0.05.Statistical analysis was performed using one-way repeated measures analysis of variance (ANOVA) or a two-factor completely randomized design ANOVA followed by a post hoc Tukey test. Our statistical model was a full factorial in which all the factors were allowed to interact together. Statistical differences in voltage clamp analysis were evaluated using Student’s paired single-sided AR-787, and other compounds of the same class, are under IP protection  and Lankenau Institute for Medical Research + Simon Fraser University provisional patent application submitted October 1, 2021.Fig 1 illustrates the molecular structure of Arumenamide-787 (AR-787) and the effect of AR-787 on Ito, ICa and action potential morphology recorded from enzymatically dissociated canine RV epicardial myocytes [to. At +20 mV, Ito density was 21.79 +/- 3.06 pA/pF in control. At concentrations of 1, 10 and 50 μM, AR-787 reduced Ito density by 34% (to 14.34+/-2.42 pA/pF), 68% (to 7.00+/-2.80 pA/pF) and 85% (to 3.23+/-2.20), respectively . Action potential duration at 90% repolarization (APD90) did not change significantly (403.0±10.3 vs. 390.6±8.3 ms). The IC50 for inhibition of Ito is estimated to be approximately 8 μM. AR-787 (10 μM) increased ICa peak density from -5.13+/-0.77 pA/pF to -6.95+/-1.88 pA/pF at 0 mV . The amplitude of the developing IKr was enhanced between -60 to -20 mV and reduced above 0 mV. These effects resulted in a very minor net increase of IKr at voltages associated with action potential repolarization consistent with the observation that APD and QT interval were largely unaffected.Quinidine, the drug currently recommended for use in JWS, in addition to blocking INa was measured in HEK cells co-transfected with SCN5A+SCN1B. AR-787 (10 μM) significantly enhanced sodium current by approximately 4- and 7-fold at -50 and -40 mV, respectively . However, the drug had no effect on the voltage-dependence of activation and steady-state fast inactivation . The early and late components of the persistent sodium current (%) underlying phases 1–2 of the cardiac AP were unaltered by AR-787 (10 μM). The kinetics of fast inactivation were significantly accelerated by approximately 1.4-folds by AR-787 (10 μM) at -20 mV and 0 mV (Table 1).The effect of AR-787 on Ito and/or to reduced levels of INa or ICa. Table 2 shows the effects of the Ito agonist NS5806  and of AR-787  on action potential and ECG parameters . The introduction of 5–10 μM NS5806 to the coronary perfusate increased the magnitude of phase 1, which accentuated the Epi AP notch and electrocardiographic J wave, thus leading to the development of the JWS phenotype. AR-787 (10 μM) readily reversed these effects in all cases.In another experimental series, we used canine ventricular RV and LV wedge preparations to generate experimental models of JWS by pharmacologically mimicking the effects of the associated genetic defects leading to an increase of IFig 4 shows the effect of 1, 10 and 50 μM AR-787 to suppress the electrocardiographic (ECG) manifestations of JWS in models of ERS and BrS generated by exposing LV (Panels A and B) and RV (Panel C) wedge preparations to 10 μM of NS5806. From top to bottom, all columns (Col) in each panel show simultaneously recorded transmembrane action potentials from the M and Epi regions together with a pseudo-ECG. Fig 4 shows recordings obtained under control conditions, 20–40 min of exposure to 10 μM NS5806 and following further addition of 1–50 μM AR-787 to the coronary perfusate.Col 2 of Fig 4A, 4B and 4C). Fig 4C shows loss of the RV Epi action potential dome after 40 min of exposure to the Ito agonist NS5806 (10 μM). AR-787 caused a concentration-dependent diminution in the manifestation of the AP notch and associated ECG J wave. Although not shown in the figure, it is noteworthy that several episodes of Phase 2 reentry, manifesting as closely coupled extrasystoles, developed following 40 min of exposure to NS5806. AR-787 (50 μM) was effective in fully restoring the AP dome and in totally suppressing all arrhythmic activity.The addition of NS5806 to the coronary perfusate dramatically augmented the Epi AP notch resulting in the accentuation of the J wave in the ECG, but without the development of arrhythmias  following NS5806 (5–10 μM) on AP and ECG parameters  recorded from LV wedge preparations (n = 3). Addition of AR-787 (10 μM) to the coronary perfusate readily reversed the effect of NS5806 to augment phase 1 magnitude of the epicardial AP (Epi Ph1 magnitude) as well as the dramatic increase in J wave amplitude. QT interval was unaffected by either drug.Fig 6 shows the effect of AR-787 to suppress ECG and arrhythmic manifestations in a wedge model of BrS in which the disease phenotype was provoked using NS5806 to mimic the gain of function of Ito and ajmaline to mimic the loss of function of INa caused by genetic defects associated with BrS. The Ito agonist greatly amplified the J wave in the ECG causing the typical coved type Brugada sign. The addition of ajmaline led to loss of the action potential dome in the epicardium but not in the M region, thus creating transmural dispersion of repolarization, which led to the development of phase 2 reentry and pVT . The addition of AR-787  restored the action potential dome in the epicardial action potentials via its effect to inhibit Ito and enhance INa, thus restoring homogeneity of repolarization throughout the preparation, resulting in suppression of all arrhythmic activity. Washout of AR-787 resulted in reappearance of the BrS phenotype with restoration of a prominent J wave in the ECG due to loss of the epicardial AP dome .Fig 7 shows the effect of AR-787 to suppress the electrocardiographic and arrhythmic manifestations of JWS in a wedge model of ERS in which the disease phenotype was provoked using NS5806 to mimic the gain of function of Ito and verapamil to mimic loss of function of calcium channel current (ICa), both of which have been shown to be associated with genetic defects causing ERS. Exposure of the in a LV wedge to NS5806 and verapamil resulted in the development of a prominent J wave and a closely coupled extrasystole following loss of the epicardial action potential dome (Panel B), most likely due to phase 2 reentry, which subsequently triggered pVT (Panel C). AR-787 (10 μM) reduced Phase 1 magnitude, restored the AP dome and totally suppressed all arrhythmic activity (Panel D). These effects of the drug were observed within minutes of its introduction to the coronary perfusate. AR-787 was effective in suppressing all of the electrocardiographic and arrhythmic manifestations of JWS .Fig 8 shows the effect of AR-787 to suppress ECG and arrhythmic manifestations in a wedge model of ERS in which the disease phenotype was provoked using NS5806 to mimic the gain of function of Ito and ajmaline to mimic the loss of function of sodium channel currents caused by mutations in genes encoding the cardiac sodium channel. The Ito agonist greatly amplified the J wave in the ECG. The addition of ajmaline led to the development of a closely coupled extrasystole in epicardium, likely due to a phase 2 reentrant mechanism. The addition of AR-787 (10 μM) reduced the epicardial AP notch and the J wave in the ECG, leading to suppression of all arrhythmic activity .As we previously reported, therapeutic hypothermia (32°C) produces ECG and arrhythmic manifestation similar to those of BrS and ERS . In 2 LVTable 3 summarizes the effectiveness of AR-787 to suppress phase 2 reentry and pVT in the various models of BrS, ERS and hypothermia tested.to blocker and/or INa enhancer capable of mediating therapy for a variety of inherited cardiac arrhythmia syndromes is now in its third decade [to and enhance INa can effectively suppress the electrocardiographic and arrhythmic manifestations of JWS. Our findings point to AR-787 as a promising new agent for pharmacologic therapy of JWS due largely to its unique effect to block Ito and enhance INa, and thus prevent repolarization abnormalities underlying arrhythmogenesis in patients with JWS.The search for an ion-channel specific and cardio-selective Id decade ,3. The pto with agents such as quinidine and acacetin, augmenting inward calcium channel current (ICa) using drugs such as isoproterenol, as well as combined inhibition of Ito and augmentation of ICa using phosphodiesterase inhibitors such as cilostazol and milrinone in BrS and ERS [to, but also by enhancing INa and to a lesser extent ICa during the early phases of the action potential. The effect of AR-787 to enhance INa is similar to that previously described by us for lithospermate B, which suppresses arrhythmogenesis in experimental models of the J wave syndromes [Our study demonstrates the effect of AR-787 at concentrations as low as 10 μM to suppress the electrocardiographic and arrhythmic manifestations of both BrS and ERS as well as of hypothermia. Our findings add to the long list of studies providing support for the hypothesis that an inward shift in the balance of currents contributing to the early phases of the ventricular AP exerts ameliorative effects on the electrocardiographic and arrhythmic manifestations of JWS. Augmenting this inward depolarizing reserve reverses the effects of the outward shift in the balance of currents in the early phase of the epicardial action potential caused by the genetic defects underlying in JWS ,9–16. Us and ERS . AR-787 yndromes ,18. ThesKr, prolongs the QT interval and predisposes to development of life-threatening Torsade de Pointes (TdP) arrhythmias. High plasma levels of quinidine are required to avoid this side effect, but these high doses cause approximately 50% of patients to develop serious gastrointestinal side effects. AR-787 does not exhibit these undesirable effects. Cilostazol is not always effective, and the other pharmacological approaches mentioned have limitations as well. Thus, there is a critical need for additional safe and effective agents. Our findings point to AR-787 as a promising candidate in the armamentarium available for the treatment of JWS.In the clinic, ERS and BrS share similarities in their response to pharmacological therapy. Electrical storms (and the associated accentuated J waves) can be suppressed with β-adrenergic agonists –22. ChroWhile the pharmacological models that we employ may not precisely mimic the effect of the diverse genetic defects underlying the clinical syndromes, “more realistic” transgenic large mammal models do not exist. Our observations using the wedge models over 20 years ago suggested for the first time that an early repolarization pattern in the ECG, previously thought to be totally benign, can reveal the presence of a substrate for the development of malignant arrhythmias . These e


Prdm16 luciferase knockin reporter mouse model is generated, enabling high throughput monitoring of Prdm16 transcription. Single clonal analysis reveals high heterogeneity of Prdm16 expression in the inguinal white adipose tissue (iWAT) cells. Amongst all transcription factors, androgen receptor (Ar) shows the strongest negative correlation with Prdm16. A sex dimorphism for PRDM16 mRNA expression is present in human WAT, with female individuals exhibiting increased expression than males. Androgen‐AR signaling mobilization suppresses Prdm16 expression, accompanied by attenuated beiging in beige adipocytes, but not in brown adipose tissue. The suppressive effect of androgens on beiging is abolished upon overexpression of Prdm16. Cleavage under targets and tagmentation mapping reveals direct binding of AR within the intronic region of Prdm16 locus, whereas no direct binding is detected on Ucp1 and other browning‐related genes. Adipocyte‐selective deletion of Ar potentiates beige cell biogenesis whereas adipocyte‐specific overexpression of AR attenuates white adipose beiging. This study highlights an essential role of AR in negative regulation of PRDM16 in WAT and provides an explanation for the observed sex difference in adipose beiging.PRDM16 (PR domain containing protein 16) serves as a dominant activator of brown and beige adipocyte. However, mechanisms underlying the regulation of PRDM16 expression are incompletely understood. A  Prdm16, and thereby suppresses its expression and mitigates adipose browning. The study provides a new explanation for sex‐difference in adipose browning.Utilizing a PRDM16 reporter model, androgen receptor (AR) is identified as the strongest negative regulator of PRDM16, which displays a sex dimorphic expression in both human and mouse. Androgen‐AR complex binds at the intronic region of  Seminal work by Wu et al. identified thermogenic competent adipocytes scattered within white adipose tissue (WAT), termed beige adipocytes. Activation of brown and beige adipocytes improves body weight and adiposity, and confers additional metabolic benefits including insulin‐sensitization, improved lipid/glucose‐clearance, and anti‐atherosclerosis and anti‐cancer effects. Nevertheless, despite similar functionality, brown and beige adipocytes are of distinct cellular lineage and their mechanisms of action are also not identical.Obesity poses a grave threat to health, contributing to increased risks associated with type 2 diabetes, fatty liver, cardiovascular disease and even COVID‐19.1 In t In brown adipocytes, PRDM16 serves as a cell fate determinator to switch between skeletal myoblasts and brown adipocytes. Later studies found that transgenic over‐expression of PRDM16 robustly induces beige adipocyte development in white adipose depots. By comparison, deletion of PRDM16 in mature adipocytes leads to complete ablation of beige fat function, and severe insulin resistance, with modest impact on BAT. These findings establish a key function for PRDM16 in beige adipocyte biogenesis.PRDM16 (PR domain containing 16) was first identified as a master transcription regulator during a search for bona fide brown adipose identity factor.5 In b Intriguingly, PRDM16 also exerts metabolic‐protective functions in WAT beyond modulating adipose browning, including repression of adipose fibrosis and arborization of intra‐adipose sympathetic fibers.Mechanistically, PRDM16 forms complexes with various transcriptional cofactors in a promoter‐dependent context, acting bifunctionally to turn on a full set of brown‐selective genes while repressing white adipocyte‐selective genes.5, 9Prdm16 knock‐in reporter mouse model and single clonal analysis, we found high cellular heterogeneity in Prdm16 expression in stromal vascular cells (SVC) of mouse inguinal WAT (iWAT). Importantly, the androgen receptor (Ar) was identified as the transcription factor showing the strongest negative correlation with Prdm16. Consistently, a sex dimorphic pattern for PRDM16 expression was observed in both human and mouse WAT. AR exerts an inhibitory effect on Prdm16 transcription through its ligand‐dependent, genomic action by directly binding on the Prdm16 locus. Furthermore, adipocyte‐selective deletion or overexpression of AR in mice either prompted or inhibited brown remodeling in WAT but not in BAT. These data highlight a pivotal role of androgen‐AR signaling in regulating PRDM16 expression and beiging in WAT.Nevertheless, in contrast to the understanding on its physiological functions, much less is known as to how PRDM16 expression, especially at the transcriptional level, is regulated. Using an in‐house generated luciferase‐based 22.1Prdm16 knock‐in allele (C57BL/6J‐Prdm16‐T2A‐Luc) was generated (termed PRDM16‐Luc KI mice). By CRISPR/Cas9‐mediated homologous recombination, open reading frame encoding T2A peptide and firefly luciferase (Luc) was positioned in frame before the stop codon of murine Prdm16 gene  faithfully and robustly reports endogenous Prdm16 transcription both in vivo and in vitro.Luciferase expression in PRDM16‐Luc KI reporter mice was examined by quantitation of luciferase activity in interscapular BAT Figure , in vivoR Figure . Furtherd Figure , consistd Figure . Collect2.2FigurePrdm16 expression among iWAT SVCs.To better understand the regulatory mechanism underpinning PRDM16 transcription in WAT, we used PRDM16‐Luc KI reporter mice to quantitate PRDM16 expression at single cell resolution. To this end, SVCs in iWAT from PRDM16‐Luc KI mice were isolated, immortalized, followed by limiting dilution, and eventually 64 mono‐clones were obtained Figure2A. The Prdm16High) and lowest (Prdm16Low) luciferase activities were selected for further analysis  were identified  respectively. In both cell types, a trend of increase of PRDM16 in female subjects was found . Following this treatment regime luciferase activity in WATs (including iWAT and eWAT), but not in BAT, was suppressed as compared to those receiving the vehicle solution  beige adipocytes  after DHT treatment, which was attributed to both coupled and uncoupled respiration Figure . This is14 Col2.5FigurePrdm16 and Ucp1 expression were largely abolished  were used in our study  was identified as a brown adipose‐enriched factor and recruits PPARγ to all of its brown‐adipose selective binding sites including Prdm16 and Ucp1 and thus maintains the brown‐specific characteristics. Using clonal analysis, we found AR as the most potent hit within the negative regulators of PRDM16 transcription. Notably, compared to PPARγ/EBF2, which are the more general modulators of adipose browning, the negative regulation directly elicited by AR is highly selective since a number of other brown and beige‐genes including Ucp1, Dio2, Tnfrsf9, and Tmem26 are not the direct target of AR, as evidenced by our CUT&Tag analysis. Recently a study reported the general inhibitory effect of androgens on WAT thermogenic capacity and the downregulation of browning marker genes including Prdm16, Ucp1, and Dio2. Considering the role of PRDM16 at the apex of WAT brown remodeling, the suppressive effect of androgens on WAT browning is likely secondary to the direct transcriptional blockade of PRDM16. Indeed, our study demonstrated that overexpression of PRDM16 overrides the inhibition of Ucp1 expression and suppression of oxygen consumption induced by DHT in beige adipocytes. Therefore to our knowledge this is the first study to uncover the selective and negative regulation of PRDM16 at the transcriptional level.PRDM16 plays a key role in brown remodeling in both BAT and WAT.7, 8 Hyperandrogenemia predicts a worse metabolic outcome in PCOS patients. Amelioration of hyperandrogenism, either using antiandrogen or oral contraceptive pill, improves abdominal adiposity and metabolic disorders. Androgen administration in obese postmenopausal women or female rhesus macaques causes a significant gain in visceral fat and accelerates adipose tissue dysfunction. Although, in males obesity seems more commonly associated with hypogonadism, this is likely because that in males testosterone is aromatized into 17β‐estradiol (E2) for energy homeostasis. Indeed, while orchidectomized male rodents treated with either testosterone or E2 remain lean, those treated with the non‐aromatizable androgen DHT develop obesity and glucose intolerance. In the same vein, a large scale interventional study on male subjects reported that the percentage of body fat increases in the group receiving testosterone daily together with aromatase inhibitor (to inhibit the aromatization of testosterone to estradiol), indicating that activation of AR favors body fat accumulation while much of the beneficial effects observed in androgen therapy in male is mediated by aromatized estradiol. Despite these clinical observations, the mode of action of androgen in WAT is still obscure.Androgens play an important role in regulation of body fat distribution and function. Androgen excess, more commonly seen in female  and prepubertal children, is significantly correlated with body mass index, abdominal obesity, insulin resistance, metabolic derangements, and subclinical cardiovascular disease.21 Hyp In adult human, whether beige adipocytes in human is sex‐dependent awaits future investigations, but fluorodeoxyglucose‐PET/CT scan‐based studies, which cannot differentiate between beige and brown adipocytes, reveal sex as an independent determinant of BAT activity with women having more often detectable BAT. Different from mice, in human omental WAT possesses the browning capacity, coincided with the recent sn‐Seq result that beige‐like adipocytes were detected in visceral WAT but not in subcutaneous WAT in human. Our study uncovers a higher level of PRDM16 in omental WAT of female individuals and this thus intriguingly raises the possibility that this gender difference on PRDM16 partially explains the higher incidence of visceral obesity in males and post‐menopausal females. Furthermore, considering the involvement of PRDM16 beyond controlling UCP1 and beiging, such as adipose fibrogenesis and sympathetic innervation, further studies will have to determine whether the androgen/AR‐mediated suppression of PRDM16 underlies those biological processes in WAT.Our work offers a new foundation for understanding the physiological function and working mechanism of androgens and AR in WAT. Sex differences in beige cell activity has been observed in animal models.27 In Ar and Prdm16 were also negatively correlated, but not in human. The exact mechanism for such discrepancy is unclear. It is likely attributed to the fact that in human AR mRNA expression is comparable between two sexes while in mice Ar is expressed at a higher level in male than in female. This suggests the mechanism regulating the mRNA expression of AR is different in human and mouse which awaits future investigation.Our results showed in both humans and mice, PRDM16 exhibit sex dimorphism, demonstrating that AR activity is negatively associated with PRDM16 expression in both species. Furthermore in mice the expressions of Ar gene using aP2‐Cre has been reported to exhibit normal weight and adiposity, but this mice strain also displays a partial deletion of Ar in the brain, which confounded the study. In the current study, a more stringent adipocyte‐selective Ar KO mice carrying AdipoQ promoter‐driven Cre recombinase were used. Together with adipocyte‐selective AR over‐expressing mice, we were at a better position to interrogate the function of androgen and AR in adipose tissue. It is interesting to find that the regulation of androgen on PRDM16 is depot specific. Ablation of AR signaling augments PRDM16 expression and beige biogenesis in iWAT and even eWAT. In contrast, the suppressive action of AR on PRDM16 is invalid in BAT, since neither supplementation of AR agonist, adipocyte‐selective deletion nor overexpression of AR, alters PRDM16 expression in mouse BAT. The unresponsiveness of brown adipocytes to androgen in PRDM16 suppression seems to be intrinsic and is downstream of AR since overexpression of AR in BAT still failed to downregulate PRDM16 expression in mice. The mechanism is currently unclear. PRDM16 expression is much higher in BAT compared to WAT, suggesting that the mechanisms regulating PRDM16 expression in these fat depots are distinct. In BAT, the action of those positive regulators of PRDM16 dominates, which is too strong to be overridden by AR, considering that the expression of AR is low in BAT. Nonetheless, it is still worthwhile to map the binding sites of AR on PRDM16 in BAT in future, which will offer additional insights on the actions of androgens in BAT.Cre‐loxP‐mediated deletion of the diposity,29 butIn sum, because of the global rise in overweight, obesity, and metabolic syndromes, it is imperative to improve our understanding on the energy utilization in WAT, especially visceral WAT. The current revelation of androgen in negative modulation of PRDM16 would potentially provide a good therapeutic window for anti‐obese and anti‐metabolic disease drugs targeting PRDM16.4Prdm16 gene and replacing it with T2A peptide and firefly luciferase. Ar flox/flox mice were generated by floxing exon 2 of mouse Ar gene through homologous recombination. Transgenic mice with Cre recombinase driven by the adipoQ promoter (AdipoQ‐Cre transgenic mice) were crossed with the Ar flox/flox mice to generate adipocyte‐selective knockout of Ar mice (AKO), which selectively abolished Ar gene expression within adipocytes. CRISPR/Cas9 technology was used to insert a CAG‐LoxP‐STOP‐LoxP‐AR expression cassette at the Rosa26 gene locus of wild‐type C57BL/6J mice (AR KI mice). AdipoQ‐Cre transgenic mice were mated with AR KI mice in which the expression of human AR sequence was turned on by Cre‐mediated deletion of a STOP cassette between the CAG promoter and human AR coding sequence (AR OE). All above‐mentioned transgenic mice were on a C57BL/6J background. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, and Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (21‐051‐MIS). Mice were housed in a specific‐pathogen‐free environment  with free access to food and water. 8‐week‐old male mice were used for experiments unless specified otherwise. For cold exposure, mice were housed at 6 °C, and those housed at 22 °C were used as the control. For DHT supplementation, mice were orally given equal volume of DHT (10 mg kg−1 d−1) or vehicle (oil) for 7 days.PRDM16‐Luc KI mice (KI) were generated by deleting the stop codon of mouse n = 52, female n = 64) undergoing laparoscopic cholecystectomy for gallstone and gallbladder polyps between 2019 and 2021 at the research institute of general surgery from Jinling Hospital. Participants were excluded if they had 1) acute infection or systemic inflammatory disease or 2) severe organ dysfunction of liver, kidney, and heart. The study protocol was approved by the Ethics Committee of the Jinling Hospital (2019NZKY‐008‐03) and all participants were provided with the informed written consent prior to the procedure. The omental adipose tissue was dissected and flash frozen in liquid nitrogen.The study enrolled 116 patients . The mice and the adipose tissues were harvested 15 min later and the bioluminescence imaging was performed with an IVIS Imaging Device (Xenogen Corp.). The results were quantified by Living Image software (PerkinElmer). Luciferase activity of cells and tissues was measured using the Steady‐Glo Luciferase Assay (Promega) according to the manufacturer's instructions. The signal was measured with EnSpire Alpha 2390 (PerkinElmer) and normalized with protein concentrations.Mice were intraperitoneally injected with D‐luciferin  with 10% fetal bovine serum (HyClone). Cells were immortalized by infecting the retrovirus expressing SV40 large T antigen gene. Limiting dilution method was used to establish 64 clonal cell lines.SVCs were obtained by dissecting iWAT from PRDM16‐Luc KI mice. The dissected iWAT was rinsed in PBS, minced, and digested for 40 min at 37 °C in 0.1% w/v Collagenase Type I (Sigma‐Aldrich) with D‐Hanks buffer. Digested tissues were filtered through a 250 µm nylon mesh and centrifuged at 800 × Rps18 and RPS18 were used as the reference genes for mouse and human samples, respectively.Total RNA was extracted with Trelief RNAprep FastPure Tissue&Cell Kit (Tsingke). First‐strand cDNA was synthesized using the TransScript Uni All‐in‐One First‐Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech) with 1 µg of RNA as the template for each reaction. mRNA levels were quantified under optimized conditions with Hieff qPCR SYBR Green Master Mix (Yeasen) according to the manufacturer's instructions. β‐actin , and GAPDH . HRP‐linked anti‐rabbit IgG  was used as the secondary antibody. The signals were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).Cells and adipose tissues were lysed in RIPA Lysis (Servicebio) containing PMSF (Beyotime). Lysates were resolved by 10% SDS‐PAGE and transferred onto a PVDF membrane, blotted with antibodies to PRDM16 (Abcam), AR (Abcam), UCP1 (Abcam), luciferase (Abcam), m), FCCP (5 µm), rotenone (3 µm), antimycin (5 µm). 2 mg of adipose tissues were equilibrated at 37 °C in carbonate free condition for 1 h before measurement.The OCR of cultured adipocytes and adipose tissues was measured using Seahorse XF Cell Mito Stress Test Kit (Agilent) and analyzed by the XFe24 Seahorse bioanalyzer (Agilent). Adipocytes were treated with DHT (Meilunbio) or DMSO for 24 h and then equilibrated in carbonate free medium and incubator for 1 h. After measuring basal levels of OCR, the following drugs were sequentially loaded to each well: oligomycin  were transfected into SVCs via electroporation using NuclofZctor II . The cells were incubated for 24 h and screened in selection media containing puromycin  for 3 days. Dox (1.5 µg mL−1) was used for inducing PRDM16 and AR overexpression.To generate Prdm16 knocking down was performed following a previous publication and the construct was purchased from Addgene (#15505). The siRNA sequences used were as follows: scramble shRNA: 5′‐GCGGAGAAAGUGGAUUUAU‐3, Prdm16‐shRNA: 5′‐GAAGAGCGUGAGUACAAAU‐3′. After electroporation into the immortalized adipose SVCs, the cells were selected for positive transfectants with G418 for 5 days. The G418‐resistant SVCs were differentiated to beige adipocytes in vitro.shRNA‐mediated blication5 and p‐value < 0.05) of results was used to identify genes strongly up‐ or down‐regulated by CINPs using unlogged data and with a false discovery rate < 0.05. Fold‐change was calculated with the average transcript levels compared to control values that were in turn log2‐transformed and calculated for Spearman correlation coefficients between treatments. All query sequences were queried against commonly used databases by BLASTx search to identify homologues.RNA‐seq was performed by Shanghai Majorbio Bio‐pharm Technology Co., Ltd. using Illumina HiSeq X10 (Illumina). Significance analysis  and control IgG  were incubated with the samples on a rotating platform overnight at 4 °C. Goat anti‐rabbit IgG H&L (Abcam) diluted in Dig‐Wash buffer was incubated with the sample at room temperature for 1 h. After washing with Dig‐Wash buffer, pA‐Tn5 adapter complex was added with gentle vortexing and incubated at room temperature for 1 h, and washed with Wash buffer. The samples were resuspended in Tagmentation buffer, incubated at 37 °C for 1 h and STOP buffer was added and incubated at 55 °C for 30 min and 70 °C for 20 min. DNA was extracted according to the protocols. The library DNA was purified and amplified for sequencing with the Illumina NovaSeq 6000. To amplify libraries, 15 µL of DNA was mixed with 25 µL of 2× CAM and 5 µL of a universal i5 and a uniquely barcoded i7 primer, using a different barcode for each sample. The sample was placed in a Thermocycler (Bio‐Rad) with a heated lid using the following cycling conditions: 72 °C for 3 min; 95 °C for 3 min; 14 cycles of 98 °C for 10 s and 60 °C for 5 s; final extension at 72 °C for 1 min and hold at 4 °C. Post‐PCR clean‐up was performed by adding VAHTS DNA Clean Beads.CUT&Tag analysis was performed with Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme) following the manufacturer's instructions. Cells were harvested, counted (10 000 cells), and centrifuged for 3 min at 600 × m, Meilunbio) or DMSO for 24 h. The adipocytes were washed with PBS, fixed in 4% formaldehyde for 10 min, permeabilized in PBST (PBS +0.1% TritonX‐100) for 10 min, blocked in 5% BSA for 30 min on ice, and incubated with Anti‐UCP1 antibody (Abcam) at 4 °C overnight. The 2nd day, after washing for three times, the adipocytes were incubated with fluorescently labeled goat anti‐rabbit IgG (Thermo Fisher Scientific) for 1 h, followed by incubation with BODIPY 493/503 (Thermo Fisher Scientific) plus Hoechst 33342 (Merck) for 20 min. Laser scanning microscopy 800 (ZEISS) with oil‐immersion objective lens was used for confocal imaging.Adipose tissues were fixed in 4% formaldehyde overnight. After paraffin embedding and sectioning at 5 µm, the sections were stained with hematoxylin and eosin (Beyotime) according to standard protocols. For immunohistochemistry, the sections were blocked with 5% BSA in PBS for 1 h, and incubated with anti‐PRDM16 antibody (R&D systems) at 4 °C overnight. The results were imaged with a light microscopy Motic BA600 (Motic Germany GmbH) and a slice scanner Pannoramic MIDI II (3DHISTECH). PRDM16‐Luc SVCs were differentiated into adipocytes and treated with DHT . For animal and cell‐based studies, statistical analysis was performed with GraphPad Prism 7. After calculating normality by D'Agostino–Pearson omnibus test, two‐sided unpaired The authors declare no conflict of interest.S.Z., T.N., and L.L. contributed equally to this work. H.X.H., D.W., T.N., and S.Z. designed the study. S.Z., T.N., L.L., P.G., Y.Z., W.S., Z.L., Q.L., Y.Q., W.W., and M.X. performed the experiments. S.Z., T.N., L.L., and P.G. analyzed the data. Q.Lo. performed the bioinformatics analyses. H.X.H., D.W., S.Z., T.N., K.L., and C.C. wrote and edited the manuscript and all authors reviewed and approved the manuscript. H.X.H., D.W., and T.N. supervised the study.Supporting InformationClick here for additional data file.Supplemental Table 1Click here for additional data file.Supplemental Table 2Click here for additional data file.Supplemental Table 3Click here for additional data file.


The knowledge, attitude, and practice (KAP) of Chinese patients with allergic rhinitis (AR) on AR is poorly known. This study investigated the KAP towards AR in patients with this disease and explored the factors associated with KAP.This cross-sectional study enrolled patients with AR in Zhangjiagang Hospital of Traditional Chinese Medicine between October 2022 and March 2023.P = 0.037), AR history of > 6 years , and know their own allergens  were independently associated with the sufficient knowledge. AR history of ≥ 6 years , and liking sports  were independently associated with the positive attitude. The knowledge scores , attitude scores , age 36–45 , employed , and liking sports  were independently associated with the proactive practice.This study included 656 valid questionnaires. Most participants were 26–35 years old (36.13%) and were female (55.18%). The knowledge, attitude, and practice scores were 5.70 ± 2.88 (possible range: 0–12), 29.51 ± 3.52 (possible range: 9–45), and 34.13 ± 7.55 (possible range: 9–45), indicating poor knowledge, unfavorable attitudes, and proactive practice. AR history of 3–5 years (adjusted odds ratio (adjOR) = 1.62, 95% confidence interval (CI): 1.03–2.54, Patients with AR have poor knowledge and unfavorable attitudes but good practice toward AR. Continuous quality teaching interventions and education on patients for AR were recommended.The online version contains supplementary material available at 10.1186/s12889-023-16607-6. Allergic rhinitis (AR) is a common type I hypersensitivity response of the upper respiratory tract to seasonal and perennial aeroallergens , 2. AssoKnowledge, attitudes, and practice (KAP) surveys allow the identification of misconceptions that could be targeted in the future to improve the subject’s performance , 10. ProTherefore, this study aimed to evaluate the KAP towards AR in patients with AR and the factors associated with KAP.This cross-sectional study enrolled patients with AR in Zhangjiagang Hospital of Traditional Chinese Medicine, Suzhou, Jiangsu, between October 2022 and March 2023. The inclusion criteria were (1) 18–80 years old, (2) diagnosed with AR, and (3) volunteered to participate in the study and able to understand the questionnaire. The study was approved by the ethics committee of Zhangjiagang Traditional Chinese Medicine Hospital , and informed consent was obtained from the participants.The questionnaire was designed based on previous studies , 17 and The final questionnaire included demographic data , knowledge dimension, attitude dimension, and practice dimension. The knowledge dimension included 12 questions, with 1 point for correct answers and 0 points for incorrect or unclear answers. The score range was 0–12 points. The attitude dimension included 9 questions using a five-point Likert scale. Questions involved in positive attitude were assigned positive values from strongly agree (5 points) to strongly disagree (1 point). Questions involved in negative attitudes (items A1 and A4) were assigned negative values. The score range was 9–45 points. The practice dimension included 9 questions, using a five-point Likert scale, from always (5 points) to never (1 point). The score range was 9–45 points.Sufficient knowledge, positive attitude, and proactive practice were defined as scores > 70% in each dimension .The e-questionnaires were established using the Sojump platform in China, which generated the QR code. The participants logged in and filled out the questionnaire by scanning the QR code sent by WeChat. In order to ensure the quality and completeness of the questionnaires, only one submission per IP address was allowed, and all items were mandatory. The researchers and trained research assistants assisted in explaining the aim of the study and answered the questions reported by the participants without intervening in the answering process. The researchers examined all questionnaires for completeness, internal coherence, and reasonableness, and questionnaires with repeated options were considered invalid.P-values < 0.05 in univariable analysis were included in the multiple logistic regression analysis. Two-sided P-values < 0.05 were considered statistically significant.SPSS 22.0  was used for statistical analysis. The continuous variables  were presented as means ± standard deviation (SD). The continuous variables were tested for normal distribution. Those with a normal distribution were tested using Student’s t-test (two groups) or ANOVA (more than two groups). Those with a non-normal distribution were tested using the Mann-Whitney U-test (two groups) or Kruskal-Wallis analysis of variance (more than two groups). The demographic data and distribution of knowledge, attitude, and practice dimensions were presented as n (%). Correlations between KAP dimensions were assessed using Pearson’s test. Multiple logistic regression analyses were performed to identify the factors associated with KAP. The variables with In this study, 664 questionnaires were collected, but eight were excluded because all options were repeated. Therefore, 656 valid questionnaires were included in the analysis. As shown in Table P < 0.001), urban residency (P < 0.001), higher education (P < 0.001), with higher income (P = 0.026), with longer AR duration (P < 0.001), passed allergen tests (P < 0.001), known allergens (P < 0.001), and liking sports (P = 0.002) (Table The mean knowledge score was 5.70 ± 2.88 (possible range: 0–12), indicating poor knowledge. Higher knowledge scores were observed in younger participants (P = 0.004) (Table The mean attitude score was 29.51 ± 3.52 (possible range: 9–45), indicating unfavorable attitudes. Higher attitude scores were observed in participants who liked sports (P = 0.001), urban residents (P = 0.001), with non-long-term stable jobs (P = 0.002), passed allergen tests (P < 0.001), known allergens (P < 0.001), and liking sports (P < 0.001) (Table The mean practice score was 34.13 ± 7.55 (possible range: 9–45), indicating good practice. Higher attitude scores were observed in younger participants  and practice  scores. The attitude scores were correlated with the practice scores  (Table The knowledge scores are correlated with the attitude (P = 0.037), AR history of ≥ 6 years , and know their own allergens  were independently associated with the sufficient knowledge  and liking sports  were independently associated with the positive attitude (Table AR history of P = 0.001), attitude scores , age 36–45 , employed , and liking sports  were independently associated with the proactive practice , and the KMO was 0.894. Finally, the correlations between KAP dimensions were weak, but statistically significant, indicating that there not due to chance alone. Still, it suggests that several factors are acting together to influence the KAP dimensions.This study had limitations. The participants were from a single hospital deserving a limited area in China, limiting the generalizability of the results, especially in the context where the allergens can vary with geography and climate. Considering the prevalence of AR, the sample size was small. As for all cross-sectional KAP surveys, the data represent a specific point in time , 10. No In conclusion, patients with AR in the Suzhou area have poor knowledge and unfavorable attitudes but good practices toward AR. Continuous quality teaching interventions and education on patients for AR were recommended.Additional file 1: Supplementary Table S1. Distribution of knowledge dimension.Additional file 2: Supplementary Figure S1. Distribution of the attitude dimension. Supplementary Figure S2. Distribution of the practice dimension.