Do Chinese viewers watch e-sports games for a different reason? Motivations, attitude, and team identification in predicting e-sports online spectatorship.

Introduction: Understanding factors that predict the intention of e-sports game online spectatorship has drawn a great deal of scholarly attention. Prior work on this topic has primarily focused on explaining the mediation mechanism between the spectator motivations and behavioral intention, such as attitude and subjective well-being, while overlooking the specific role of team identification and satisfaction with past experience, which were understudied in the previous studies but also prominent in the context of e-sports spectatorship. Besides, previous research was mainly conducted in a Western context; therefore, little was known about the online e-sports audience in China and their motivations to view e-sports games on live-streaming platforms. Methods: The current study thus aims to examine if and how these factors are related to the intention of watching e-sports games online by hierarchical regression and structural equation modeling. Results: Results from a survey of 452 Chinese e-sports online audiences suggested that three motivations (skill improvement, entertainment, and friends bonding), attitude towards e-sports game online spectatorship, and satisfaction with past experience were positively related to watching intention. These motivations and satisfaction also positively influenced attitude, while socialization opportunity (one of the motivations) negatively influenced attitude. Furthermore, team identification negatively moderated the relationship between satisfaction with past experience and behavioral intention. Also, attitude mediated the association between motivations, satisfaction, and behavioral intention. Discussion: In general, our study identifies the motivations that relate to Chinese e-sports viewers' attitude as well as their watching intention, and underscores the role of satisfaction with past experience, attitude, and team identification in the context of e-sports. These findings contribute to deriving a holistic view of e-sports game online spectatorship.

Split luciferase-based biosensors for characterizing EED binders.

The EED (embryonic ectoderm development) subunit of the Polycomb repressive complex 2 (PRC2) plays an important role in the feed forward regulation of the PRC2 enzymatic activity. We recently identified a new class of allosteric PRC2 inhibitors that bind to the H3K27me3 pocket of EED. Multiple assays were developed and used to identify and characterize this type of PRC2 inhibitors. One of them is a genetically encoded EED biosensor based on the EED[G255D] mutant and the split firefly luciferase. This EED biosensor can detect the compound binding in the transfected cells and in the in vitro biochemical assays. Compared to other commonly used cellular assays, the EED biosensor assay has the advantage of shorter compound incubation with cells. The in vitro EED biosensor is much more sensitive than other label-free biophysical assays (e.g. DSF, ITC). Based on the crystal structure, the DSF data as well as the biosensor assay data, it's most likely that compound-induced increase in the luciferase activity of the EED[G255D] biosensor results from the decreased non-productive interactions between the EED subdomain and other subdomains within the biosensor construct. This new insight of the mechanism might help to broaden the use of the split luciferase based biosensors.

An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED.

Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.

Carbon nanotube-assisted optical activation of TGF-ß signalling by near-infrared light.

Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and signalling cascades at precise times and spaces using light, many of these light-sensitive proteins are activated by ultraviolent or visible light, which has limited tissue penetration. Here, we report a single-walled carbon nanotube (SWCNT)-assisted approach that enables near-infrared light-triggered activation of transforming growth factor ß (TGF-ß) signal transduction, an important signalling pathway in embryonic development and cancer progression. The protein complex of TGF-ß and its latency-associated peptide is conjugated onto SWCNTs, where TGF-ß is inactive. Upon near-infrared irradiation, TGF-ß is released through the photothermal effect of SWCNTs and becomes active. The released TGF-ß activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-ß signalling in living mice.

GARP regulates the bioavailability and activation of TGFß.

Glycoprotein-A repetitions predominant protein (GARP) associates with latent transforming growth factor-ß (proTGFß) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFß activation and the structural basis of coassociation remain unknown. We find that Cys-192 and Cys-331 of GARP disulfide link to the TGFß1 prodomain and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFß1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFß-binding protein for binding to proTGFß1. Association between GARP and proTGFß1 prevents the secretion of TGFß1. Integrin α(V)ß(6) and to a lesser extent α(V)ß(8) are able to activate TGFß from the GARP-proTGFß1 complex. Activation requires the RGD motif of latent TGFß, disulfide linkage between GARP and latent TGFß, and membrane association of GARP. Our results show that GARP is a latent TGFß-binding protein that functions in regulating the bioavailability and activation of TGFß.

Latent TGF-ß structure and activation.

Transforming growth factor (TGF)-ß is stored in the extracellular matrix as a latent complex with its prodomain. Activation of TGF-ß1 requires the binding of α(v) integrin to an RGD sequence in the prodomain and exertion of force on this domain, which is held in the extracellular matrix by latent TGF-ß binding proteins. Crystals of dimeric porcine proTGF-ß1 reveal a ring-shaped complex, a novel fold for the prodomain, and show how the prodomain shields the growth factor from recognition by receptors and alters its conformation. Complex formation between α(v)ß(6) integrin and the prodomain is insufficient for TGF-ß1 release. Force-dependent activation requires unfastening of a 'straitjacket' that encircles each growth-factor monomer at a position that can be locked by a disulphide bond. Sequences of all 33 TGF-ß family members indicate a similar prodomain fold. The structure provides insights into the regulation of a family of growth and differentiation factors of fundamental importance in morphogenesis and homeostasis.

A structural hypothesis for the transition between bent and extended conformations of the leukocyte beta2 integrins.

Integrins mediate cell adhesion in response to activation signals that trigger conformational changes within their ectodomain. It is thought that a compact bent conformation of the molecule represents its physiological low affinity state and extended conformations its active state. We have determined the structure of two integrin fragments of the beta2 subunit. The first structure, consisting of the plexin-semaphorin-integrin domain, hybrid, integrin-epidermal growth factor 1 (I-EGF1), and I-EGF2 domains (PHE2), showed an L-shaped conformation with the bend located between the I-EGF1 and I-EGF2 domains. The second structure, which includes, in addition, the I-EGF3 domain, showed an extended conformation. The major reorientation of I-EGF2 with respect to the other domains in the two structures is accompanied by a change of torsion angle of the disulfide bond between Cys(461)-Cys(492) by 180 degrees and the conversion of a short alpha-helix (residues Ser(468)-Cys(475)) into a flexible coil. Based on the PHE2 structure, we introduced a disulfide bond between the plexin-semaphorin-integrin domain and I-EGF2 domains in the beta2 subunit. The resultant alphaLbeta2 integrin (leukocyte function-associated antigen-1) variant was locked in a bent state and could not be detected with the monoclonal antibody KIM127 in Mg(2+)/EGTA. However, it retained the binding activity to ICAM-1. These results provide a structural hypothesis for our understanding of the transition between the resting and active states of leukocyte function-associated antigen-1.

Mutation of a conserved asparagine in the I-like domain promotes constitutively active integrins alphaLbeta2 and alphaIIbbeta3.

The leukocyte beta2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of beta2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the beta2 inserted (I)-like domain. This mutation converted alphaLbeta2 from a resting into a high affinity conformer because alphaLbeta2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by alphaLbeta2N329S because of its reactivity with the beta2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation in beta3 generated constitutively active alphaIIbbeta3 that adhered to fibrinogen. This Asn is conserved in all human beta subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of the alpha7 helix and the beta6-alpha7 loop in the I-like domain.

The crystal structure of the plexin-semaphorin-integrin domain/hybrid domain/I-EGF1 segment from the human integrin beta2 subunit at 1.8-A resolution.

Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.