Transmembrane Channel-Like (Tmc) Subunits Contribute to Frequency Sensitivity in the Zebrafish Utricle.

Information about dynamic head motion is conveyed by a central "striolar" zone of vestibular hair cells and afferent neurons in the inner ear. How vestibular hair cells are tuned to transduce dynamic stimuli at the molecular level is not well understood. Here we take advantage of the differential expression pattern of tmc1, tmc2a, and tmc2b, which encode channel subunits of the mechanotransduction complex in zebrafish vestibular hair cells. To test the role of various combinations of Tmc subunits in transducing dynamic head movements, we measured reflexive eye movements induced by high-frequency stimuli in single versus double tmc mutants. We found that Tmc2a function correlates with the broadest range of frequency sensitivity, whereas Tmc2b mainly contributes to lower-frequency responses. Tmc1, which is largely excluded from the striolar zone, plays a minor role in sensing lower-frequency stimuli. Our study suggests that the Tmc subunits impart functional differences to the mechanotransduction of dynamic stimuli.Significance Statement Information about dynamic head movements is transmitted by sensory receptors, known as hair cells, in the labyrinth of the inner ear. The sensitivity of hair cells to fast or slow movements of the head differs according to cell type. Whether the mechanotransduction complex that converts mechanical stimuli into electrical signals in hair cells participates in conveying frequency information is not clear. Here we find that the transmembrane channel-like 1/2 genes, which encode a central component of the complex, are differentially expressed in the utricle and contribute to frequency sensitivity in zebrafish.

Differential expression of mechanotransduction complex genes in auditory/vestibular hair cells in zebrafish.

Ciliated sensory cells such as photo- and olfactory receptors employ multiple types of opsins or hundreds of unique olfactory G-protein coupled receptors to respond to various wavelengths of light or odorants. With respect to hearing and balance, the mechanotransduction machinery involves fewer variants; however, emerging evidence suggests that specialization occurs at the molecular level. To address how the mechanotransduction complex varies in the inner ear, we characterized the expression of paralogous genes that encode components required for mechanotransduction in zebrafish hair cells using RNA-FISH and bioinformatic analysis. Our data indicate striking zonal differences in the expression of two components of the mechanotransduction complex which are known to physically interact, the transmembrane channel-like 1 and 2 (tmc1/2) family members and the calcium and integrin binding 2 and 3 (cib2/3) paralogues. tmc1, tmc2b, and cib3 are largely expressed in peripheral or extrastriolar hair cells, whereas tmc2a and cib2 are enriched in central or striolar hair cells. In addition, a gene implicated in deaf-blindness, ush1c, is highly enriched in a subset of extrastriolar hair cells. These results indicate that specific combinations of these components may optimize responses to mechanical stimuli in subtypes of sensory receptors within the inner ear.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase.

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.

Group-Based Emotions Over Time: Dynamics of Experience and Regulation.

People's emotions toward their ingroups and salient outgroups often change over time as a result of changing circumstances or intentional self-regulation. To investigate such dynamics, two studies assessed participants' perceived past, present, and ideal levels of group-based emotions toward ingroups and outgroups, for several different types of groups. Consistent with predictions, participants ideally wanted to feel more positive and less negative emotions toward the ingroup compared to their present levels. However, contrary to predictions, ideal emotions toward competitive outgroups were more positive than negative. Several effects over time suggested the successful regulation of emotion: Ideal levels of positive ingroup emotion predicted group-related behavioral intentions as well as emotions reported at a later time, over and above present levels. This work puts group-based emotions in a subjective temporal context and opens new directions for theory-driven investigation and new possibilities for interventions.

Disruption of tmc1/2a/2b Genes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells.

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibular-induced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.SIGNIFICANCE STATEMENT We assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants revealed the presence of two distinct cell types, an upper layer of teardrop-shaped cells that rely on Tmc2a, and a lower layer of gourd-shaped cells that rely on Tmc1/2b.

Positive Emotions, More Than Anxiety or Other Negative Emotions, Predict Willingness to Interact With Robots.

Like early work on human intergroup interaction, previous research on people's willingness to interact with robots has focused mainly on effects of anxiety. However, existing findings suggest that other negative emotions as well as some positive emotions also have effects. This article systematically examines the roles of positive and negative emotions in predicting willingness to interact with robots, using an integrative analysis of data across five studies that use diverse interaction conditions and several types of robots. We hypothesize and find that positive emotions account for more variance than negative emotions. Practically, the findings suggest new strategies for interventions, aimed at increasing positive emotions to increase willingness to engage in intergroup interaction. No existing work has examined whether positive emotions are stronger predictors than negative emotions for willingness for human intergroup interaction, an important topic for future research.

Ndr kinases regulate retinal interneuron proliferation and homeostasis.

Ndr2/Stk38l encodes a protein kinase associated with the Hippo tumor suppressor pathway and is mutated in a naturally-occurring canine early retinal degeneration (erd). To elucidate the retinal functions of Ndr2 and its paralog Ndr1/Stk38, we generated Ndr1 and Ndr2 single knockout mice. Although retinal lamination appeared normal in these mice, Ndr deletion caused a subset of Pax6-positive amacrine cells to proliferate in differentiated retinas, while concurrently decreasing the number of GABAergic, HuD and Pax6-positive amacrine cells. Retinal transcriptome analyses revealed that Ndr2 deletion increased expression of neuronal stress genes and decreased expression of synaptic organization genes. Consistent with the latter, Ndr deletion dramatically reduced levels of Aak1, an Ndr substrate that regulates vesicle trafficking. Our findings indicate that Ndr kinases are important regulators of amacrine and photoreceptor cells and suggest that Ndr kinases inhibit the proliferation of a subset of terminally differentiated cells and modulate interneuron synapse function via Aak1.

Optimization of the metabolic stability of a fluorinated cannabinoid receptor subtype 2 (CB2) ligand designed for PET studies.

The central CB2 receptor represents a promising target for the treatment of neuroinflammatory diseases as CB2 activation mediates anti-inflammatory effects. Recently, the F-18 labeled PET radiotracer [18F]7a was reported, which shows high CB2 affinity and high selectivity over the CB1 subtype but low metabolic stability due to hydrolysis of the amide group. Based on these findings twelve bioisosteres of 7a were synthesized containing a non-hydrolysable functional group instead of the amide group. The secondary amine 23a (Ki = 7.9 nM) and the ketone 26a (Ki = 8.6 nM) displayed high CB2 affinity and CB2:CB1 selectivity in in vitro radioligand binding studies. Incubation of 7a, 23a and 26a with mouse liver microsomes and LC-quadrupole-MS analysis revealed a slightly higher metabolic stability of secondary amine 23a, but a remarkably higher stability of ketone 26a in comparison to amide 7a. Furthermore, a logD7.4 value of 5.56 ±â€¯0.08 was determined for ketone 26a by micro shake-flask method and LC-MS quantification.

Specific emotions as mediators of the effect of intergroup contact on prejudice: findings across multiple participant and target groups.

Emotions are increasingly being recognised as important aspects of prejudice and intergroup behaviour. Specifically, emotional mediators play a key role in the process by which intergroup contact reduces prejudice towards outgroups. However, which particular emotions are most important for prejudice reduction, as well as the consistency and generality of emotion-prejudice relations across different in-group-out-group relations, remain uncertain. To address these issues, in Study 1 we examined six distinct positive and negative emotions as mediators of the contact-prejudice relations using representative samples of U.S. White, Black, and Asian American respondents (N = 639). Admiration and anger (but not other emotions) were significant mediators of the effects of previous contact on prejudice, consistently across different perceiver and target ethnic groups. Study 2 examined the same relations with student participants and gay men as the out-group. Admiration and disgust mediated the effect of past contact on attitude. The findings confirm that not only negative emotions (anger or disgust, based on the specific types of threat perceived to be posed by an out-group), but also positive, status- and esteem-related emotions (admiration) mediate effects of contact on prejudice, robustly across several different respondent and target groups.

Representation and Incorporation of Close Others' Responses: The RICOR Model of Social Influence.

We propose a new model of social influence, which can occur spontaneously and in the absence of typically assumed motives. We assume that perceivers routinely construct representations of other people's experiences and responses (beliefs, attitudes, emotions, and behaviors), when observing others' responses or simulating the responses of unobserved others. Like representations made accessible by priming, these representations may then influence the process that generates perceivers' own responses, without intention or awareness, especially when there is a strong social connection to the other. We describe evidence for the basic properties and important moderators of this process, which distinguish it from other mechanisms such as informational, normative, or social identity influence. The model offers new perspectives on the role of others' values in producing cultural differences, the persistence and power of stereotypes, the adaptive reasons for being influenced by others' responses, and the impact of others' views about the self.

The dominance behavioural system: A multidimensional transdiagnostic approach.

OBJECTIVES: The dominance behavioural system (DBS) is multi-faceted, and various aspects of this system have links with a range of psychopathologies. Different scales have been used across psychopathologies though, and no single measure captures the core dimensions of the DBS. Our goal was to assess the importance of multiple DBS dimensions across psychopathologies. METHODS: Undergraduates (N = 612) completed a broad set of dominance items (many from pre-existing measures) and measures of lifetime depression, anxiety, and manic symptoms. RESULTS: Factor analysis suggested six distinct DBS factors: Authentic Pride, Hubristic Pride, Cooperation, Power/Influence, Discomfort with Leadership, and Ruthless Ambition. Depressive and anxious symptoms related to notably similar DBS profiles, characterized by lower authentic pride, lower perceived power, and greater willingness to endorse hubris. In contrast, hypomanic tendencies related to heightened pride, and an emphasis on the pursuit of power despite interpersonal costs. CONCLUSIONS: With a multi-faceted approach, the DBS appears to be relevant for understanding multiple forms of psychopathology. Although limited by the reliance on self-report questionnaires, this is the first transdiagnostic study to consider these multiple facets of the DBS. PRACTITIONER POINTS: The dominance system involves multiple separable dimensions. Manic tendencies appear tied to experiencing a heightened sense of pride and being willing to use more aggressive behavioural strategies to pursue dominance. Anxious and depressive tendencies appear particularly tied to low levels of subjective power, and more willingness to describe oneself as having hubris. Pride, the subjective sense of achieving power, and behavioural approaches to achieving power appear to be important dimensions of the dominance system for understanding psychopathology.

Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris.

Recombinant human mast cell chymase (rhChymase) was expressed in secreted form as an active enzyme in the SuperMan5 strain of GlycoSwitch® Pichia pastoris, which is engineered to produce proteins with (Man)5(GlcNAc)2 Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDS-PAGE as a single band of 30 kDa and treatment with peptide N-glycosidase F decreased this to 25 kDa, consistent with the established properties of native human chymase (hChymase). Polyclonal antibodies against hChymase detected rhChymase by Western blot. Active site titration with Eglin C, a potent chymase inhibitor, quantified the concentration of purified active enzyme. Kinetic analyses with succinyl-Ala-Ala-Pro-Phe (suc-AAPF) p-nitroanilide and thiobenzyl ester synthetic substrates showed that heparin significantly reduced KM, whereas heparin effects on kcat were minor. Pure rhChymase with Asn-linked glycans closely resembles hChymase. This bioengineering approach avoided hyperglycosylation and provides a source of active rhChymase for other studies as well as a foundation for production of recombinant enzyme with human glycosylation patterns.

Evil acts and malicious gossip: a multiagent model of the effects of gossip in socially distributed person perception.

Although person perception is central to virtually all human social behavior, it is ordinarily studied in isolated individual perceivers. Conceptualizing it as a socially distributed process opens up a variety of novel issues, which have been addressed in scattered literatures mostly outside of social psychology. This article examines some of these issues using a series of multiagent models. Perceivers can use gossip (information from others about social targets) to improve their ability to detect targets who perform rare negative behaviors. The model suggests that they can simultaneously protect themselves against being influenced by malicious gossip intended to defame specific targets. They can balance these potentially conflicting goals by using specific strategies including disregarding gossip that differs from a personally obtained impression. Multiagent modeling demonstrates the outcomes produced by different combinations of assumptions about gossip, and suggests directions for further research and theoretical development.

Human enteropeptidase light chain: bioengineering of recombinants and kinetic investigations of structure and function.

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK∼ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (kcat /KM = 6.83 × 106 M⁻¹ sec⁻¹) and DDDDR (kcat /KM = 1.89 × 107 M⁻¹ sec⁻¹) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge-charge interactions in the extended substrate binding pocket.

Integrating advice and experience: learning and decision making with social and nonsocial cues.

When making decisions, people typically gather information from both social and nonsocial sources, such as advice from others and direct experience. This research adapted a cognitive learning paradigm to examine the process by which people learn what sources of information are credible. When participants relied on advice alone to make decisions, their learning of source reliability proceeded in a manner analogous to traditional cue learning processes and replicated the established learning phenomena. However, when advice and nonsocial cues were encountered together as an established phenomenon, blocking (ignoring redundant information) did not occur. Our results suggest that extant cognitive learning models can accommodate either advice or nonsocial cues in isolation. However, the combination of advice and nonsocial cues (a context more typically encountered in daily life) leads to different patterns of learning, in which mutually supportive information from different types of sources is not regarded as redundant and may be particularly compelling. For these situations, cognitive learning models still constitute a promising explanatory tool but one that must be expanded. As such, these findings have important implications for social psychological theory and for cognitive models of learning.

Implicit evaluation of familiar and novel concepts presented at low levels of conscious detectability.

In 2 experiments, implicit evaluation of novel and familiar concepts was assessed using a sequential priming procedure that enabled estimates of evaluative priming effects at low levels of detectability. In Experiment 1, the novel concepts referenced common names, and in Experiment 2 they referenced nonsense words. Whereas familiar concepts yielded priming effects at low levels of detectability in both experiments, novel concepts did not elicit any priming effect. Implicit evaluation of novel concepts has been documented in related research but under conditions that differ from those investigated here. The present results identify important limiting conditions associated with the implicit evaluation effect.

Contextualizing person perception: distributed social cognition.

Research on person perception typically emphasizes cognitive processes of information selection and interpretation within the individual perceiver and the nature of the resulting mental representations. The authors focus instead on the ways person perception processes create, and are influenced by, the patterns of impressions that are socially constructed, transmitted, and filtered through social networks. As the socially situated cognition perspective (E. R. Smith & G. R. Semin, 2004) suggests, it is necessary to supplement consideration of intra-individual cognitive processes with an examination of the social context. The authors describe a theoretical model of processes of distributed social cognition that takes account of 3 levels: the individual perceiver, the interacting dyad, and the social network in which they are embedded. The authors' model assumes that perceivers elicit or create as well as interpret impression-relevant information in dyadic interaction and that perceivers obtain information from 3rd-party sources who are linked to perceivers and targets in social networks. The authors also present results of a multiagent simulation of a subset of these processes. Implications of the theoretical model are discussed, for the possibility of correcting biases in person perception and for the nature of underlying mental representations of persons.