The role of phosphorylation in calmodulin-mediated gating of human AQP0.

Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.

Gender stereotypes and social perception of vocal confidence is mitigated by salience of socio-indexical cues to gender.

Introduction: Socio-indexical cues to gender and vocal affect often interact and sometimes lead listeners to make differential judgements of affective intent based on the gender of the speaker. Previous research suggests that rising intonation is a common cue that both women and men produce to communicate lack of confidence, but listeners are more sensitive to this cue when it is produced by women. Some speech perception theories assume that listeners will track conditional statistics of speech and language cues (e.g., frequency of the socio-indexical cues to gender and affect) in their listening and communication environments during speech perception. It is currently less clear if these conditional statistics will impact listener ratings when context varies (e.g., number of talkers). Methods: To test this, we presented listeners with vocal utterances from one female and one male-pitched voice (single talker condition) or many female/male-pitched voices (4 female voices; 4 female voices pitch-shifted to a male range) to examine how they impacted perceptions of talker confidence. Results: Results indicated that when one voice was evaluated, listeners defaulted to the gender stereotype that the female voice using rising intonation (a cue to lack of confidence) was less confident than the male-pitched voice (using the same cue). However, in the multi-talker condition, this effect went away and listeners equally rated the confidence of the female and male-pitched voices. Discussion: Findings support dual process theories of information processing, such that listeners may rely on heuristics when speech perception is devoid of context, but when there are no differentiating qualities across talkers (regardless of gender), listeners may be ideal adapters who focus on only the relevant cues.

How Does the Accuracy of Children's Number Representations Influence the Accuracy of Their Numerical Predictions?

Predictions begin with an extrapolation of the properties of their underlying representations to forecast a future state not presently in evidence. For numerical predictions, sets of numbers are summarized and the result forms the basis of and constrains numerical predictions. One open question is how the accuracy of underlying representations influences predictions, particularly numerical predictions. It is possible that inaccuracies in individual number representations are randomly distributed and averaged over during summarization (e.g., wisdom of crowds). It is also possible that inaccuracies are not random and lead to errors in predictions. We investigated this question by measuring the accuracy of individual number representations of 279 children ages 8-12 years, using a 0-1,000 number line, and numerical predictions, measured using a home run derby task. Consistent with prior research, our results from mixed random effects models evaluating percent absolute error (PAE; prediction error) demonstrated that third graders' representations of individual numbers were less accurate, characterized by overestimation errors, and were associated with overpredictions (i.e., predictions above the set mean). Older children had more accurate individual number representations and a slight tendency to underpredict (i.e., predictions below the set mean). The results suggest that large, systematic inaccuracies appear to skew predictions while small, random errors appear to be averaged over during summarization. These findings add to our understanding of summarization and its role in numerical predictions.

Gender stereotypes drive perceptual differences of vocal confidence.

One's ability to express confidence is critical to achieve one's goals in a social context-such as commanding respect from others, establishing higher social status, and persuading others. How individuals perceive confidence may be shaped by the socio-indexical cues produced by the speaker. In the current production/perception study, we asked four speakers (two cisgender women/men) to answer trivia questions under three speaking contexts: natural, overconfident, and underconfident (i.e., lack of confidence). An evaluation of the speakers' acoustics indicated that the speakers significantly varied their acoustic cues as a function of speaking context and that the women and men had significantly different acoustic cues. The speakers' answers to the trivia questions in the three contexts (natural, overconfident, underconfident) were then presented to listeners (N = 26) in a social judgment task using a computer mouse-tracking paradigm. Listeners were sensitive to the speakers' acoustic modulations of confidence and differentially interpreted these cues based on the perceived gender of the speaker, thereby impacting listeners' cognition and social decision making. We consider, then, how listeners' social judgments about confidence were impacted by gender stereotypes about women and men from social, heuristic-based processes.

Contextualized Knowledge Reduces Misconceived COVID-19 Health Decisions.

How do we resolve conflicting ideas about how to protect our health during a pandemic? Prior knowledge influences our decisions, potentially creating implicit cognitive conflict with new, correct information. COVID-19 provides a natural condition for investigating how an individual's health-specific knowledge (e.g., understanding mask efficacy) and their personal context (e.g., outbreak proximity) influence their protective health behavior endorsement, as information about the virus, its spread, and lethality has changed over time. Using a dual-process-model framework, we investigated the role cognitive conflict has on health decision-making. We used a computer mouse-tracking paradigm alongside geographical information systems (GIS) as a proxy for context. The results support a contextualized-deficit-model framework in which relevant knowledge and context-based factors help individuals override cognitive conflict to make more preventative health decisions. Findings from this study may provide evidence for a more effective way for experts to combat non-adherence due to conflicting health information.

Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli.

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol-1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol-1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

Biochemical, structural and dynamical studies reveal strong differences in the thermal-dependent allosteric behavior of two extremophilic lactate dehydrogenases.

In this work, we combined biochemical and structural investigations with molecular dynamics (MD) simulations to analyze the very different thermal-dependent allosteric behavior of two lactate dehydrogenases (LDH) from thermophilic bacteria. We found that the enzyme from Petrotoga mobilis (P. mob) necessitates an absolute requirement of the allosteric effector (fructose 1, 6-bisphosphate) to ensure functionality. In contrast, even without allosteric effector, the LDH from Thermus thermophilus (T. the) is functional when the temperature is raised. We report the crystal structure of P. mob LDH in the Apo state solved at 1.9 Å resolution. We used this structure and the one from T. the, obtained previously, as a starting point for MD simulations at various temperatures. We found clear differences between the thermal dynamics, which accounts for the behavior of the two enzymes. Our work demonstrates that, within an allosteric enzyme, some areas act as local gatekeepers of signal transmission, allowing the enzyme to populate either the T-inactive or the R-active states with different degrees of stringency.

Predictions of Miscommunication in Verbal Communication During Collaborative Joint Action.

Purpose The purpose of the current study was to examine the lexical and pragmatic factors that may contribute to turn-by-turn failures in communication (i.e., miscommunication) that arise regularly in interactive communication. Method Using a corpus from a collaborative dyadic building task, we investigated what differentiated successful from unsuccessful communication and potential factors associated with the choice to provide greater lexical information to a conversation partner. Results We found that more successful dyads' language tended to be associated with greater lexical density, lower ambiguity, and fewer questions. We also found participants were more lexically dense when accepting and integrating a partner's information (i.e., grounding) but were less lexically dense when responding to a question. Finally, an exploratory analysis suggested that dyads tended to spend more lexical effort when responding to an inquiry and used assent language accurately-that is, only when communication was successful. Conclusion Together, the results suggest that miscommunication both emerges and benefits from ambiguous and lexically dense utterances.

Cognitive Processing of Miscommunication in Interactive Listening: An Evaluation of Listener Indecision and Cognitive Effort.

Purpose The purpose of the current study was to evaluate the social and cognitive underpinnings of miscommunication during an interactive listening task. Method An eye and computer mouse-tracking visual-world paradigm was used to investigate how a listener's cognitive effort (local and global) and decision-making processes were affected by a speaker's use of ambiguity that led to a miscommunication. Results Experiments 1 and 2 found that an environmental cue that made a miscommunication more or less salient impacted listener language processing effort (eye-tracking). Experiment 2 also indicated that listeners may develop different processing heuristics dependent upon the speaker's use of ambiguity that led to a miscommunication, exerting a significant impact on cognition and decision making. We also found that perspective-taking effort and decision-making complexity metrics (computer mouse tracking) predict language processing effort, indicating that instances of miscommunication produced cognitive consequences of indecision, thinking, and cognitive pull. Conclusion Together, these results indicate that listeners behave both reciprocally and adaptively when miscommunications occur, but the way they respond is largely dependent upon the type of ambiguity and how often it is produced by the speaker.

Contrast Is in the Eye of the Beholder: Infelicitous Beat Gesture Increases Cognitive Load During Online Spoken Discourse Comprehension.

We investigated how two cues to contrast-beat gesture and contrastive pitch accenting-affect comprehenders' cognitive load during processing of spoken referring expressions. In two visual-world experiments, we orthogonally manipulated the presence of these cues and their felicity, or fit, with the local (sentence-level) referential context in critical referring expressions while comprehenders' task-evoked pupillary responses (TEPRs) were examined. In Experiment 1, beat gesture and contrastive accenting always matched the referential context of filler referring expressions and were therefore relatively felicitous on the global (experiment) level, whereas in Experiment 2, beat gesture and contrastive accenting never fit the referential context of filler referring expressions and were therefore infelicitous on the global level. The results revealed that both beat gesture and contrastive accenting increased comprehenders' cognitive load. For beat gesture, this increase in cognitive load was driven by both local and global infelicity. For contrastive accenting, this increase in cognitive load was unaffected when cues were globally felicitous but exacerbated when cues were globally infelicitous. Together, these results suggest that comprehenders' cognitive resources are taxed by processing infelicitous use of beat gesture and contrastive accenting to convey contrast on both the local and global levels.

First identification of dopamine receptors in pikeperch, Sander lucioperca, during the pre-ovulatory period.

Dopamine (DA) is a ubiquitous neurotransmitter exerting a range of pleiotropic actions through two DA receptor families, the D1 and the D2. To date in vertebrates, a maximum of four receptor subtypes have been identified within the D1 family, D1 (former D1A), D5 (former D1B), D6 (former D1C and D1D) and D7 (former D1E), while the D2 family encloses five subtypes, D2, D3, D4, D8 (former D2like or D2l) and D9 (former D4-related sequence or D4-rs). In teleosts, no study has investigated in parallel all the DA receptors to identify and localize the whole receptor repertoire from both families. In pikeperch, Sander lucioperca, a species of interest for aquaculture development, the existence, number and location of the DA receptors are totally unknown. To address these questions, RNA-seq with de novo transcriptome reconstruction, functional annotation and phylogenetic analysis were performed to characterize the transcript repertoire of DA receptors in the brain of female pikeperch at the pre-ovulatory period. Ten different cDNA were identified and showed to belong to the D1 family: two D1, one D5a, one D6a and one D6b and to the D2 family: two spliced variants of D2, one D3, one D8 and one D9. Unlike zebrafish, the subtypes D4 and D7 have not yet been isolated in pikeperch. As expected D1, D3, D8 and D9 are mostly expressed in brain parts except for the cerebellum (D1 and D3). The inter-species differences in the number of DA receptors and the inter-organ differences in the gene expression of all receptors support the complexity of the dopaminergic actions in vertebrate.

Social Judgments of Digitally Manipulated Stuttered Speech: Cognitive Heuristics Drive Implicit and Explicit Bias.

Purpose People who stutter are susceptible to discrimination, stemming from negative stereotypes and social misattributions. There has been a recent push to evaluate the underlying explicit and implicit cognitive mechanisms associated with social judgments, moving away from only evaluating explicit social bias about people who stutter. The purpose of the current study was to evaluate how listeners change their implicit and explicit social (mis)attributions after hearing a people who stutter produce disfluent speech. Method The current project was an adaptation of the Byrd et al. (2017) study to evaluate listener implicit/explicit social judgments of stuttered speech across five categories (i.e., confidence, friendliness, intelligence, distractibility, and extroversion) before and after a stuttering self-disclosure. This was done by implementing a modified version of the Ferguson et al. (2019) computer mouse-tracking paradigm. Results Consistent with previous findings, participants made more explicit positive social judgments of confidence, friendliness, extroversion, and intelligence after a stuttering self-disclosure, but the distractedness category was resistant to change. Also consistent with previous findings, participants experienced a higher degree of cognitive competition (i.e., higher area under the curve) shortly after self-disclosure, which lessened over time. Conclusions Explicit and implicit biases exist, but self-disclosure significantly impacts the cognitive system of listeners. Specifically, self-disclosure may reduce explicit bias through experience and explicit belief updating, but when cognitive heuristics are strong, implicit bias may be slower to change.

Social Judgments of Digitally Manipulated Stuttered Speech: An Evaluation of Self-Disclosure on Cognition.

Purpose Persons who stutter (PWS) may be susceptible to discrimination because of negative judgments made by listeners. The current study sought to determine how the cognitive system's explicit (i.e., conscious) and implicit (i.e., nonconscious) biases about PWS are impacted by self-disclosure. Method A computer mouse-tracking paradigm was used to evaluate categorical social judgments about PWS. Computer mouse trajectories, which have been shown to reveal underlying cognitive pull or competition between opposing concepts, were used to measure implicit bias (i.e., nonconscious stereotypes). Participants were asked to explicitly categorize the speaker as either intelligent or unintelligent before and after listening to a speaker self-disclose. Mouse cursor trajectories during the explicit response categorization were used to evaluate implicit bias associated with the decision-making process. Results Results indicated that participants chose "intelligent" for a higher proportion of the trials in the disclosure condition compared to baseline, showing that listeners' explicit biases changed after listening to a self-disclosure that the speaker stutters. Results also indicated listeners exhibited a more negative implicit bias, based on computer mouse trajectories, when rating the PWS relative to the "persons who do not stutter" talker, but this negative implicit bias did seem to reduce over time after the disclosure was made. Conclusions These findings indicate that, even though explicit and implicit biases were evident when listeners heard stuttering, both explicit and implicit biases seemed to extinguish over time after a self-disclosure. Although the bias was not completely extinguished, these results provide promising evidence toward developing methods to reduce negative beliefs and reactions toward PWS. Supplemental Material http://osf.io/mwrp7/.

Structural Insights into AQP2 Targeting to Multivesicular Bodies.

Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.

The archaeal LDH-like malate dehydrogenase from Ignicoccus islandicus displays dual substrate recognition, hidden allostery and a non-canonical tetrameric oligomeric organization.

The NAD(P)-dependent malate dehydrogenases (MalDHs) and NAD-dependent lactate dehydrogenases (LDHs) are homologous enzymes involved in central metabolism. They display a common protein fold and the same catalytic mechanism, yet have a stringent capacity to discriminate between their respective substrates. The MalDH/LDH superfamily is divided into several phylogenetically related groups. It has been shown that the canonical LDHs and LDH-like group of MalDHs are primarily tetrameric enzymes that diverged from a common ancestor. In order to gain understanding of the evolutionary history of the LDHs and MalDHs, the biochemical properties and crystallographic structure of the LDH-like MalDH from the hyperthermophilic archaeon Ignicoccus islandicus (I. isl) were determined. I. isl MalDH recognizes oxaloacetate as main substrate, but it is also able to use pyruvate. Surprisingly, with pyruvate, the enzymatic activity profile looks like that of allosteric LDHs, suggesting a hidden allosteric capacity in a MalDH. The I. isl MalDH tetrameric structure in the apo state is considerably different from those of canonical LDH-like MalDHs and LDHs, representing an alternative oligomeric organization. A comparison with MalDH and LDH counterparts provides strong evidence that the divergence between allosteric and non-allosteric members of the superfamily involves homologs with intermediate, atypical properties.

Acoustic correlates of female confidence: A production and comprehension study.

In the current study, an interactive approach is used to explore possible contributors to the misattributions listeners make about female talker expression of confidence. To do this, the expression and identification of confidence was evaluated through the evaluation of talker- (e.g., talker knowledge and affective acoustic modulation) and listener-specific factors (e.g., interaction between talker acoustic cues and listener knowledge). Talker and listener contexts were manipulated by implementing a social constraint for talkers and withholding information from listeners. Results indicated that listeners were sensitive to acoustic information produced by the female talkers in this study. However, when world knowledge and acoustics competed, judgments of talker confidence by listeners were less accurate. In fact, acoustic cues to female talker confidence were more accurately used by listeners as a cue to perceived confidence when relevant world knowledge was missing. By targeting speech dynamics between female talkers and both female and male listeners, the current study provides a better understanding of how confidence is realized acoustically and, perhaps more importantly, how those cues may be interpreted/misinterpreted by listeners.

A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography.

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.

Protein-protein interactions in AQP regulation - biophysical characterization of AQP0-CaM and AQP2-LIP5 complex formation.

Protein-protein interactions play important roles in regulating human aquaporins (AQP) by gating as well as trafficking. While structural and functional studies have provided detailed knowledge of AQP transport mechanisms, selectivity as well as gating by conformational changes of loops or termini, the mechanism behind how protein-protein interactions control AQP-mediated water transport through cellular membranes remains poorly characterized. Here we explore the interaction between two human AQPs and regulatory proteins: the interaction between AQP0 and calmodulin, which mediates AQP0 gating, as well as the interaction between AQP2 and LIP5, which is involved in trafficking. Using microscale thermophoresis (MST) and fluorescence anisotropy, two methods that have the advantage of low sample consumption and detergent compatibility, we show that the interactions can be studied using both full-length AQPs and AQP peptides corresponding to the regulatory protein binding sites. However, full-length AQPs gave better reproducibility between methods and for the first time revealed that AQP0 binds CaM in a cooperative manner, which was not seen in experiments using peptides. Our study highlights that, while peptides are great tools for locating binding sites and pinpointing interacting residues, full-length proteins may give additional insights, such as binding mechanism, allostery and cooperativity, important parameters for understanding protein-protein mediated regulation in the cellular context. Our work provides a platform for further studies of AQP regulation that may be of interest for designing drugs that target AQP complexes as well as the development of artificial bio-mimetic water channels for water-purification purposes.

The Effects of Emotion Suppression During Language Planning and Production.

Purpose: Emotion regulation and language planning occur in parallel during interactive communication, but their processes are often studied separately. It has been suggested that emotion suppression and more complex language production both recruit cognitive resources. However, it is currently less clear how the language planning and production system is impacted when required to emotionally suppress outward displays of affect (i.e., expressive suppression). The purpose of the current study was to evaluate the interactive effects of emotion regulation and language production processes. Method: Through discourse analysis of a corpus of interactive dialogue, we evaluated the production of interjections (i.e., also termed "filled pauses," a type of speech disfluency) when participants regulated outward displays of emotion and when language was lexically complex (i.e., via lexical diversity). One participant (the sender) was assigned to either express or suppress affective displays during the interaction. The other person (the receiver) was given no special instructions before the interaction. The interactions were transcribed, and their linguistic content (i.e., lexical diversity, lexical alignment, and interjections) was analyzed. Results: Results indicated that participants actively suppressing outward displays of affect produced more interjections and that participants asked to emotionally regulate, both expressors and suppressors, were more disfluent when producing lexically diverse statements (2 cognitively demanding tasks). Conclusions: The current research provides support that, when suppressing emotion, one might be more disfluent when speaking. However, also when engaged in 2 simultaneous, demanding tasks of having to either upregulate or downregulate emotions and utter lexically diverse statements, the combined cognitive load may impede fluency in language production. More specifically, in the context of language planning and production, emotion suppression may pilfer resources away from the language planning and production system, leading to higher rates of disfluent speech. This finding is of particular importance because understanding the interactive effects of emotion and language production may be impactful to interventions for communication disorders.

Type IX secretion system PorM and gliding machinery GldM form arches spanning the periplasmic space.

Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N (P. gingivalis) or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-Å length that span most of the periplasmic space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior.