Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells.

The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

Improving Scale Equivalence by Increasing Access to Scale-Specific Information.

Measures of the same phenomenon should produce the same results; this principle is fundamental because it allows for replication-the basis of science. Unfortunately, measures of a psychological construct in one language can often measure something a bit different in another language (i.e., low "scale equivalence"). Historically, the problem was thought to stem from insufficient knowledge of best-practice translation procedures. Yet solutions based on this diagnosis and their widespread adoption have not resolved the issue. In this article, we suggest that an additional problem might be insufficient information about the measure being translated. If so, low scale equivalence is a problem that translators and cross-cultural psychologists cannot solve on their own. We explore the possibility that measure-specific translation guides be created by original scale builders for the most widely used measures of important psychological constructs. We describe why such guides are needed, when they are needed, what they might look like, their feasibility, and next steps, providing a complete example guide and test case in a supplement concerning the Primals Inventory. In this article, we seek to spark discussion on translation practices happening behind the scenes and how greater transparency can improve scale equivalence, in the spirit of open science.

Character strengths and fluid intelligence.

OBJECTIVE: Research on the associations between cognitive and noncognitive personality traits has widely neglected character strengths, that means positively and morally valued personality traits that constitute good character. METHOD: The present study aimed to bridge this gap by studying the associations between character strengths and fluid intelligence using different operationalizations of character strengths (including self- and informant-reports) and fluid intelligence in children, adolescents, and adults. RESULTS: The results, based on four samples (N = 193/290/330/324), suggested that morally valued personality traits are independent of fluid intelligence, with the exception of love of learning, which showed small but robust positive relationships with fluid intelligence across all samples. CONCLUSIONS: Nonetheless, we argue for further research on the associations with other cognitive abilities and interactions between character strengths and intelligence when examining their relationships with external criteria.

Development and Linguistic Cue Analysis of the State-Trait Cheerfulness Inventory-Short Form.

The present study derived a short form of the State-Trait Cheerfulness Inventory-Trait Version (STCI-T30) using an item response theory framework. Latent trait test-retest correlations and reliability across the latent continuum in the STCI-T30 remained high. Moreover, the STCI-T30 showed external validity with criterion variables (e.g., playfulness) and a short writing task completed by these participants was rated by unacquainted judges to infer the author's cheerfulness, seriousness, and bad-mood. Results suggested significant self-other and inter-judge agreement of cheerfulness, seriousness, and bad-mood and linguistic cues analysis suggested cheerfulness and bad-mood manifested through writing in tone, social processes, and affect.

Character Strengths: Person-Environment Fit and Relationships With Job and Life Satisfaction.

Several studies demonstrated the relevance of character strengths in the workplace. For example, it has been shown that they positively relate to performance and are strong predictors of job satisfaction. Furthermore, it was demonstrated that occupational groups differ in their average levels of character strengths. However, little is known about the effects of the congruence between a person's strengths profile with the average profile within an occupational group (environmental congruence) on well-being. In a nationally representative sample (N = 870) of employed adults, we analyzed data on character strengths (t1), and measures of job and life satisfaction at three different time points (t1-t3; separated by 1 year). We studied (1) whether employees in different occupational groups differ with regard to their levels and configurations of character strengths, (2) how levels and configurations of character strengths relate to concurrent and predictive job and life satisfaction, and (3) whether a fit between strengths of a person and the environment goes along with current and future job and life satisfaction. Results confirmed previous findings that small, but meaningful, differences in character strengths among employees in different occupational groups can be found and that character strengths positively relate to current and prospective job and life satisfaction. Furthermore, results suggested that a better person-environment fit goes along with higher job and life satisfaction. These results suggest character strengths and could play an important role in vocational and career counseling.

The Not so Good, the Bad and the Ugly: Differential Bacterial Adhesion and Invasion Mediated by Salmonella PagN Allelic Variants.

While advances in genomic sequencing have highlighted significant strain variability between and within Salmonella serovars, only a few protein variants have been directly related to evolutionary adaptation for survival, such as host specificity or differential virulence. The current study investigated whether allelic variation of the Salmonella adhesin/invasin PagN influences bacterial interaction with their receptors. The Salmonella enterica, subspecies enterica serovar Typhi (S. Typhi) allelic variant of PagN was found to bind significantly better to different enterocytes as well as to the extracellular matrix protein laminin than did the major Salmonella enterica, subspecies enterica serovar Typhimurium (S. Typhimurium) allele. The two alleles differed at amino acid residues 49 and 109 in two of the four predicted PagN surface loops, and residue substitution analysis revealed that a glutamic acid at residue 49 increased the adhesive and invasive properties of S. Typhi PagN. PagN sequence comparisons from 542 Salmonella strains for six representative S. enterica serovars and S. diarizonae further supported the role of glutamic acid at residues 49 and 109 in optimizing adhesion to cells and laminin, as well as for cell invasion. In summary, this study characterized unique residues in allelic variants of a virulence factor that participates in the colonization and invasive properties of different Salmonella stains, subspecies and serovars.

Drosophila melanogaster as a model for arbovirus infection of adult salivary glands.

Arboviruses are an emerging threat to public health. Arbovirus transmission to vertebrates hinges on dissemination from the arthropod gastrointestinal tract, and ultimately infection of the arthropod salivary glands. Therefore, salivary gland immunity impacts arbovirus transmission; however, these immune responses are poorly understood. Here, we describe the utility of Drosophila melanogaster as a salivary gland infection model. First, we describe the use of a salivary gland-specific driver to launch RNA interference or virus replicon transgenes. Next, we infect flies with an arbovirus panel and find multiple viruses that infect Drosophila salivary glands, albeit inefficiently. We find that this infection is not controlled by antiviral RNA silencing; thus, we silence a panel of immune genes in the salivary glands, but do not observe changes in infection. These data suggest that Drosophila may be used to study salivary gland infection, and that there are likely unexplored pathways controlling infection of this tissue.

Laughing away the pain: A narrative review of humour, sense of humour and pain.

BACKGROUND AND OBJECTIVE: The link between humour and sense of humour with pain has been a topic of research for decades. The purpose of the present article was to review the different studies that have been conducted to date on the association between humour and sense of humour with pain. DATABASES AND DATA TREATMENT: The literature search was conducted using the PubMed, Science Direct and ProQuest databases. Forty-one studies were reviewed, and the results are summarized and structured into three sections: experimental pain, chronic pain and pain in children. RESULTS: For experimental pain, the findings support the idea that humorous distractions, such as watching a comedy clip, increase pain tolerance, although most of the studies indicate that other non-humorous distractions produce similar effects. Regarding chronic pain, humour has been studied as a way of coping with pain and the emotional distress produced by chronic pain conditions. The results of correlational studies show significant associations between the use of humour and main variables such as anxiety and catastrophizing. Finally, concerning pain in children, similar findings to those described for the previous sections have been reported, with a notable presence of studies on clinic clown interventions, which promote emotional well-being among children and their parents, although their effectiveness in pain reduction is controversial. CONCLUSIONS: The study of the link between humour and pain is still on an early stage, and overcoming the limitations of previous studies is required to strengthen the promising results that have been observed up to date. SIGNIFICANCE: This review summarizes all main findings regarding humour, sense of humour and pain up until the first half of 2018 and offers a list of aspects to be considered in further studies regarding the link of humour and pain to contribute to a more systematic research.

Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier.

Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer's disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocin-induced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2AKITA) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes.

Assessing the Temperamental Basis of the Sense of Humor: Adaptation of the English Language Version of the State-Trait Cheerfulness Inventory Long and Standard Form.

The State-Trait Model of Cheerfulness assesses the temperamental basis of the sense of humor with the traits and respective states of cheerfulness, seriousness, and bad mood. Cheerfulness is a dominant factor in current measures of the sense of humor and explains both, the disposition to engaging in smiling and laughter, as well as humor behaviors, and trait seriousness and bad mood are antagonistic to the elicitation of amusement (albeit for different reasons). Several studies have shown the validity and reliability of the STCI questionnaire in German and other language versions (i.e., Spanish). In this study, the English language version with 106 items (STCI-T <106>) was translated, checked for its item and scale characteristics, and tested with a confirmatory factor analysis approach (N = 1101) to investigate the factorial validity of the STCI-T <106> scale. Results show good psychometric characteristics, good internal consistencies, and a fit to the postulated underlying structure of the STCI-T. Then, the standard form with 60 items (STCI-T <60>) was developed and the psychometric characteristics initially tested. In an independent sample (N = 169), the characteristics of the standard form were compared to the parent form and German equivalent. It showed good psychometric characteristics, internal consistencies, as well as a good self- and peer-report congruence. To conclude, the STCI-T <106> is the measure of choice for the assessment of the temperamental basis of the sense of humor and the separate facets of the traits, while the standard form (60 items) allows of an economic assessment of cheerfulness, seriousness, and bad mood, free of context-saturated items and humor preferences.

Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors.

Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers that are associated with diarrhea. The genome of EAEC strain 042, a diarrheal pathogen validated in a human challenge study, encodes multiple colonization factors. Notable among them are aggregative adherence fimbriae (AAF/II) and a secreted antiaggregation protein (Aap). Deletion of aap is known to increase adherence, autoaggregation, and biofilm formation, so it was proposed that Aap counteracts AAF/II-mediated interactions. We hypothesized that Aap sterically masks heat-resistant agglutinin 1 (Hra1), an integral outer membrane protein recently identified as an accessory colonization factor. We propose that this masking accounts for reduced in vivo colonization upon hra1 deletion and yet no colonization-associated phenotypes when hra1 is deleted in vitro. Using single and double mutants of hra1, aap, and the AAF/II structural protein gene aafA, we demonstrated that increased adherence in aap mutants occurs even when AAF/II proteins are genetically or chemically removed. Deletion of hra1 together with aap abolishes the hyperadherence phenotype, demonstrating that Aap indeed masks Hra1. The presence of all three colonization factors, however, is necessary for optimal colonization and for rapidly building stacked-brick patterns on slides and cultured monolayers, the signature EAEC phenotype. Altogether, our data demonstrate that Aap serves to mask nonstructural adhesins such as Hra1 and that optimal colonization by EAEC is mediated through interactions among multiple surface factors. IMPORTANCE Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers of the human intestine and can cause diarrhea. Compared to other E. coli pathogens, little is known about the genes and pathogenic mechanisms that differentiate EAEC from harmless commensal E. coli. EAEC bacteria attach via multiple proteins and structures, including long appendages produced by assembling molecules of AafA and a short surface protein called Hra1. EAEC also secretes an antiadherence protein (Aap; also known as dispersin) which remains loosely attached to the cell surface. This report shows that dispersin covers Hra1 such that the adhesive properties of EAEC seen in the laboratory are largely produced by AafA structures. When the bacteria colonize worms, dispersin is sloughed off, or otherwise removed, such that Hra1-mediated adherence occurs. All three factors are required for optimal colonization, as well as to produce the signature EAEC stacked-brick adherence pattern. Interplay among multiple colonization factors may be an essential feature of exceptional colonizers.

Structure and Relaxation in Solutions of Monoclonal Antibodies.

Reversible self-association of therapeutic antibodies is a key factor in high protein solution viscosities. In the present work, a coarse-grained computational model accounting for electrostatic, dispersion, and long-ranged hydrodynamic interactions of two model monoclonal antibodies is applied to understand the nature of self-association, predicting the solution microstructure and resulting transport properties of the solution. For the proteins investigated, the structure factor across a range of solution conditions shows quantitative agreement with neutron-scattering experiments. We observe a homogeneous, dynamical association of the antibodies with no evidence of phase separation. Calculations of self-diffusivity and viscosity from coarse-grained dynamic simulations show the appropriate trends with concentration but, respectively, over- and under-predict the experimentally measured values. By adding constraints to the self-associated clusters that rigidify them under flow, prediction of the transport properties is significantly improved with respect to experimental measurements. We hypothesize that these rigidity constraints are associated with missing degrees of freedom in the coarse-grained model resulting from patchy and heterogeneous interactions among coarse-grained domains. These results demonstrate how structural anisotropy and anisotropy of interactions generated by features at the 2-5 nm length scale in antibodies are sufficient to recover the dynamics and rheological properties of these important macromolecular solutions.

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans.

At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington's disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.

Assessing Dispositions Toward Ridicule and Laughter in the Workplace: Adapting and Validating the PhoPhiKat-9 Questionnaire.

The current paper addresses the measurement of three dispositions toward ridicule and laughter; i.e., gelotophobia (the fear of being laughed at), gelotophilia (the joy of being laughed at), and katagelasticism (the joy of laughing at others). These traits explain inter-individual differences in responses to humor, laughter, and social situations related to humorous encounters. First, an ultra-short form of the PhoPhiKat-45 (Ruch and Proyer, 2009) was adapted in two independent samples (Construction Sample N = 157; Replication Sample N = 1,774). Second, we tested the validity of the PhoPhiKat-9 in two further independent samples. Results showed that the psychometric properties of the ultra-short form were acceptable and the proposed factor structure could be replicated. In Validation Sample 1 (N = 246), we investigated the relation of the three traits to responses in a ridicule and teasing scenario questionnaire. The results replicated findings from earlier studies by showing that gelotophobes assigned the same emotions to friendly teasing and malicious ridicule (predominantly low joy, high fear, and shame). Gelotophilia was mainly predicted by relating joy to both, teasing and ridicule scenarios, while katagelasticism was predicted by assigning joy and contempt to ridicule scenarios. In Validation Sample 2 (N = 1,248), we investigated whether the fear of being laughed at is a vulnerability at the workplace: If friendly teasing and laughter of co-workers, superiors, or customers are misperceived as being malicious, individuals may feel less satisfied and more stressed. The results from a representative sample of Swiss employees showed that individuals with a fear of being laughed at are generally less satisfied with life and work and experience more work stress. Moreover, gelotophilia went along with positive evaluations of one's life and work, while katagelasticism was negatively related to work satisfaction and positively related to work stress. In order to establish good work practices and build procedures against workplace bullying, one needs to consider that individual differences impact on a person's perception of being bullied and assessing the three dispositions may give important insights into team processes.

Analysis of the brain mural cell transcriptome.

Pericytes, the mural cells of blood microvessels, regulate microvascular development and function and have been implicated in many brain diseases. However, due to a paucity of defining markers, pericyte identification and functional characterization remain ambiguous and data interpretation problematic. In mice carrying two transgenic reporters, Pdgfrb-eGFP and NG2-DsRed, we found that double-positive cells were vascular mural cells, while the single reporters marked additional, but non-overlapping, neuroglial cells. Double-positive cells were isolated by fluorescence-activated cell sorting (FACS) and analyzed by RNA sequencing. To reveal defining patterns of mural cell transcripts, we compared the RNA sequencing data with data from four previously published studies. The meta-analysis provided a conservative catalogue of 260 brain mural cell-enriched gene transcripts. We validated pericyte-specific expression of two novel markers, vitronectin (Vtn) and interferon-induced transmembrane protein 1 (Ifitm1), using fluorescent in situ hybridization and immunohistochemistry. We further analyzed signaling pathways and interaction networks of the pericyte-enriched genes in silico. This work provides novel insight into the molecular composition of brain mural cells. The reported gene catalogue facilitates identification of brain pericytes by providing numerous new candidate marker genes and is a rich source for new hypotheses for future studies of brain mural cell physiology and pathophysiology.

Self-association motifs in the enteroaggregative Escherichia coli heat-resistant agglutinin 1.

The heat-resistant agglutinin 1 (Hra1) is an integral outer membrane protein found in strains of Escherichia coli that are exceptional colonizers. Hra1 from enteroaggregative E. coli strain 042 is sufficient to confer adherence to human epithelial cells and to cause bacterial autoaggregation. Hra1 is closely related to the Tia invasin, which also confers adherence, but not autoaggregation. Here, we have demonstrated that Hra1 mediates autoaggregation by self-association and we hypothesize that at least some surface-exposed amino acid sequences that are present in Hra1, but absent in Tia, represent autoaggregation motifs. We inserted FLAG tags along the length of Hra1 and used immune-dot blots to verify that four in silico-predicted outer loops were indeed surface exposed. In Hra1 we swapped nine candidate motifs in three of these loops, ranging from one to ten amino acids in length, to the corresponding sequences in Tia. Three of the motifs were required for Hra1-mediated autoaggregation. The database was searched for other surface proteins containing these motifs; the GGXWRDDXK motif was also present in a surface-exposed region of Rck, a Salmonella enterica serotype Typhimurium complement resistance protein. Cloning and site-specific mutagenesis demonstrated that Rck can confer weak, GGXWRDDXK-dependent autoaggregation by self-association. Hra1 and Rck appear to form heterologous associations and GGXWRDDXK is required on both molecules for Hra1-Rck association. However, a GGYWRDDLKE peptide was not sufficient to interfere with Hra1-mediated autoaggregation. In the present study, three autoaggregation motifs in an integral outer membrane protein have been identified and it was demonstrated that at least one of them works in the context of a different cell surface.

Diarrhoeagenic Escherichia coli in mother-child Pairs in Ile-Ife, South Western Nigeria.

BACKGROUND: Diarrhoeagenic Escherichia coli (DEC) pathotypes are among the most common bacterial causes of morbidity and mortality in young children. These pathogens are not sought routinely and capacity for their detection is limited in Africa. We investigated the distribution and dissemination of DEC in 126 children paired with their mothers in a Nigerian community. METHODS: A total of 861 E. coli were isolated from 126 children with diarrhoea and their mothers. Antimicrobial susceptibility of each isolate was determined by Kirby-Bauer disc diffusion technique. All the isolates were screened for DEC markers by multiplex PCR. Genetic relatedness of DEC strains was determined by flagellin typing and Insertion element 3 (IS3)-based PCR. RESULTS: DEC were identified from 35.7% of individuals with the most common pathotype being shiga toxin-producing E. coli (42, 16.7%). Identical pathotypes were found in 13 (10.3%) of the mother-child pairs and in three of these strains from mothers and their children showed identical genetic signatures. Over 90% of DEC isolates were resistant to ampicillin, sulphonamide, tetracycline, streptomycin or trimethoprim, but only 9 (7.2%) were ciprofloxacin resistant CONCLUSION: The data suggest that healthy mothers are asymptomatic reservoirs of multiply-resistant strains that are pathogenic in their children and there are instances in which identical strains are found in mother-child pairs.

Gpr116 Receptor Regulates Distinctive Functions in Pneumocytes and Vascular Endothelium.

Despite its known expression in both the vascular endothelium and the lung epithelium, until recently the physiological role of the adhesion receptor Gpr116/ADGRF5 has remained elusive. We generated a new mouse model of constitutive Gpr116 inactivation, with a large genetic deletion encompassing exon 4 to exon 21 of the Gpr116 gene. This model allowed us to confirm recent results defining Gpr116 as necessary regulator of surfactant homeostasis. The loss of Gpr116 provokes an early accumulation of surfactant in the lungs, followed by a massive infiltration of macrophages, and eventually progresses into an emphysema-like pathology. Further analysis of this knockout model revealed cerebral vascular leakage, beginning at around 1.5 months of age. Additionally, endothelial-specific deletion of Gpr116 resulted in a significant increase of the brain vascular leakage. Mice devoid of Gpr116 developed an anatomically normal and largely functional vascular network, surprisingly exhibited an attenuated pathological retinal vascular response in a model of oxygen-induced retinopathy. These data suggest that Gpr116 modulates endothelial properties, a previously unappreciated function despite the pan-vascular expression of this receptor. Our results support the key pulmonary function of Gpr116 and describe a new role in the central nervous system vasculature.

Gelotophobia and the challenges of implementing laughter into virtual agents interactions.

This study investigated which features of AVATAR laughter are perceived threatening for individuals with a fear of being laughed at (gelotophobia), and individuals with no gelotophobia. Laughter samples were systematically varied (e.g., intensity, laughter pitch, and energy for the voice, intensity of facial actions of the face) in three modalities: animated facial expressions, synthesized auditory laughter vocalizations, and motion capture generated puppets displaying laughter body movements. In the online study 123 adults completed, the GELOPH <15 > (Ruch and Proyer, 2008a,b) and rated randomly presented videos of the three modalities for how malicious, how friendly, how real the laughter was (0 not at all to 8 extremely). Additionally, an open question asked which markers led to the perception of friendliness/maliciousness. The current study identified features in all modalities of laughter stimuli that were perceived as malicious in general, and some that were gelotophobia specific. For facial expressions of AVATARS, medium intensity laughs triggered highest maliciousness in the gelotophobes. In the auditory stimuli, the fundamental frequency modulations and the variation in intensity were indicative of maliciousness. In the body, backwards and forward movements and rocking vs. jerking movements distinguished the most malicious from the least malicious laugh. From the open answers, the shape and appearance of the lips curling induced feelings that the expression was malicious for non-gelotophobes and that the movement round the eyes, elicited the face to appear as friendly. This was opposite for gelotophobes. Gelotophobia savvy AVATARS should be of high intensity, containing lip and eye movements and be fast, non-repetitive voiced vocalization, variable and of short duration. It should not contain any features that indicate a down-regulation in the voice or body, or indicate voluntary/cognitive modulation.

Intense or malicious? The decoding of eyebrow-lowering frowning in laughter animations depends on the presentation mode.

Joyful laughter is the only laughter type that has received sufficient validation in terms of morphology (i.e., face, voice). Still, it is unclear whether joyful laughter involves one prototypical facial-morphological configuration (Duchenne Display and mouth opening) to be decoded as such, or whether qualitatively distinct facial markers occur at different stages of laughter intensity. It was proposed that intense laughter goes along with eyebrow-lowering frowning, but in decoding studies of pictures, these "frowns" were associated with perceived maliciousness rather than higher intensity. Thus, two studies were conducted to investigate the influence of the presentation mode (static, dynamic) and eyebrow-lowering frowning on the perception of laughter animations of different intensity. In Study 1, participants (N = 110) were randomly assigned to two presentation modes (static pictures vs. dynamic videos) to watch animations of Duchenne laughter and laughter with added eyebrow-lowering frowning. Ratings on the intensity, valence, and contagiousness of the laughter were completed. In Study 2, participants (N = 55) saw both animation types in both presentation modes sequentially. Results confirmed that the static presentation lead to eyebrow-lowering frowning in intense laughter being perceived as more malicious, less intense, less benevolent, and less contagious compared to the dynamic presentation. This was replicated for maliciousness in Study 2, although participants could potentially infer the "frown" as a natural element of the laugh, as they had seen the video and the picture. Thus, a dynamic presentation is necessary for detecting graduating intensity markers in the joyfully laughing face. While this study focused on the decoding, future studies should investigate the encoding of frowning in laughter. This is important, as tools assessing facially expressed joy might need to account for laughter intensity markers that differ from the Duchenne Display.