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 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.

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.

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.

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.

Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5.

The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser256, Ser261, Ser264, and Thr269), of which Ser256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications.

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.

Aquaporin Protein-Protein Interactions.

Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein-protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein-protein interactions; dividing the interactions into three types: (1) interactions between aquaporin tetramers; (2) interactions between aquaporin monomers within a tetramer (hetero-tetramerization); and (3) transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

Insights into the Hendra virus NTAIL-XD complex: Evidence for a parallel organization of the helical MoRE at the XD surface stabilized by a combination of hydrophobic and polar interactions.

The Hendra virus is a member of the Henipavirus genus within the Paramyxoviridae family. The nucleoprotein, which consists of a structured core and of a C-terminal intrinsically disordered domain (N(TAIL)), encapsidates the viral genome within a helical nucleocapsid. N(TAIL) partly protrudes from the surface of the nucleocapsid being thus capable of interacting with the C-terminal X domain (XD) of the viral phosphoprotein. Interaction with XD implies a molecular recognition element (MoRE) that is located within N(TAIL) residues 470-490, and that undergoes α-helical folding. The MoRE has been proposed to be embedded in the hydrophobic groove delimited by helices α2 and α3 of XD, although experimental data could not discriminate between a parallel and an antiparallel orientation of the MoRE. Previous studies also showed that if the binding interface is enriched in hydrophobic residues, charged residues located close to the interface might play a role in complex formation. Here, we targeted for site directed mutagenesis two acidic and two basic residues within XD and N(TAIL). ITC studies showed that electrostatics plays a crucial role in complex formation and pointed a parallel orientation of the MoRE as more likely. Further support for a parallel orientation was afforded by SAXS studies that made use of two chimeric constructs in which XD and the MoRE were covalently linked to each other. Altogether, these studies unveiled the multiparametric nature of the interactions established within this complex and contribute to shed light onto the molecular features of protein interfaces involving intrinsically disordered regions.

Apelin (APLN) and Apelin Receptor (APLNR) in Human Ovary: Expression, Signaling, and Regulation of Steroidogenesis in Primary Human Luteinized Granulosa Cells.

Apelin (APLN) is a recently discovered adipokine involved in the regulation of various metabolic functions. Its receptor, APLNR, is expressed in reproductive tissues, however, its role in human ovarian cells is unknown. In this study, we identified APLN and APLNR in human ovarian follicles and analyzed their expression in granulosa cells and follicular fluid obtained from obese and nonobese patients, with or without polycystic ovary syndrome (PCOS). We also investigated the effect of APLN on steroidogenesis in cultured human luteinized granulosa cells (hGCs) from nonobese patients without PCOS. Using RT-PCR and immunoblotting, we found that APLN and APLNR were expressed in hGCs and cumulus and theca cells. We confirmed these data immunohistochemically and observed that APLNR and APLN are present in human oocytes at different stages of follicular development. In patients with PCOS, we observed that follicular fluid APLN concentration and granulosa cell APLN and APLNR mRNA expression was higher than that observed in control patients. In cultured hGCs from nonobese patients without PCOS, insulin-like growth factor 1 (IGF1) increased APLNR expression, and recombinant human APLN (APLN-13 and APLN-17) increased both basal and IGF1-induced steroid secretion. These effects on steroid production were reversed when cultured in the presence of ML221, an APLNR antagonist, which was associated with an increased 3beta-hydrosteroid dehydrogenase (HSD3B) protein concentration. We showed that these effects were dependent on the activation of the AKT and MAPK3/1 pathways using pharmacological inhibitors. Our results show that APLN and APLNR are present in human ovarian cells and APLN increases IGF1-induced steroidogenesis in granulosa cells through an increase in HSD3B protein expression and activation of the MAPK3/1 and Akt pathways. Therefore, APLN and APLNR may play a role in human follicular development and the pathogenesis of PCOS.

Opposing roles for mammary epithelial-specific PPARγ signaling and activation during breast tumour progression.

BACKGROUND: Among women worldwide, breast cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related deaths. Improved understanding of breast tumourigenesis may facilitate the development of more effective therapies. Peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor that regulates genes involved in insulin sensitivity and adipogenesis. Previously, we showed, using 7,12-dimethylbenz [a] anthracene (DMBA)-treated haploinsufficient PPARγ mice, that PPARγ suppresses breast tumour progression; however, the PPARγ expressing cell types and mechanisms involved remain to be clarified. Here, the role of PPARγ expression and activation in mammary epithelial cells (MG) with respect to DMBA-mediated breast tumourigenesis was investigated. METHODS: PPARγ MG knockout (PPARγ-MG KO) mice and their congenic, wild-type controls (PPARγ-WT) were treated once a week for six weeks by oral gavage with 1 mg DMBA dissolved in corn oil and maintained on a normal chow diet. At week 7, mice were randomly divided into those maintained on a normal chow diet (DMBA Only; PPARγ-WT: n = 25 and PPARγ-MG KO: n = 39) or those receiving a diet supplemented with the PPARγ ligand, rosiglitazone (ROSI, 4 mg/kg/day) (DMBA + ROSI; PPARγ-WT: n = 34 and PPARγ-MG KO: n = 17) for the duration of the 25-week study. RESULTS: Compared to DMBA Only-treated PPARγ-WTs, both breast tumour susceptibility and serum levels of proinflammatory and chemotactic cytokines, namely IL-4, eotaxin, GM-CSF, IFN-γ, and MIP-1α, were decreased among PPARγ-MG KOs. Cotreatment with ROSI significantly reduced breast tumour progression among PPARγ-WTs, correlating with increased BRCA1 and decreased VEGF and COX-2 protein expression levels in breast tumours; whereas, surprisingly DMBA + ROSI-treated PPARγ-MG KOs showed increased breast tumourigenesis, correlating with activation of COX-2. CONCLUSION: These novel data suggest MG-specific PPARγ expression and signaling is critical during breast tumourigenesis, and may serve as a strong candidate predictive biomarker for response of breast cancer patients to the use of therapeutic strategies that include PPARγ ligands.

Explicit and implicit cognitive processes of the public towards people who stutter.

PURPOSE: The Public Opinion Survey of Human Attributes - Stuttering (POSHA-S, St. Louis, 2013) was developed as a standard measure of public attitudes about people who stutter. As with any survey-based methods, threats to validity may occur because of social desirability bias. Using computer mouse-tracking, we were interested in observing changes in cognition that are manifested in intentionality through action by evaluating underlying cognitive processes that drive social judgments of people who stutter. METHODS: Twenty-two women, 1 non-binary person, and 47 men reported using a computer mouse to complete an online, remote, and modified version of the POSHA-S. Responses were categorized as correct/helpful or incorrect/unhelpful relative to each component of the POSHA-S and were used as measures of explicit cognitive processes. Computer-mouse trajectory metrics, including area under the curve (AUC) and reaction time (RT), were used to measure implicit cognitive processes. RESULTS: Although participants' explicit responses were significantly more likely to be correct/helpful than incorrect/unhelpful, with endorsement of correct/helpful prompts 77 % of the time, participants also endorsed incorrect/unhelpful prompts more than half (i.e., 52 %) of the time. Familiarity with people who stutter was associated with disagreeing with incorrect/unhelpful prompts. As indicated by greater AUC, participants exhibited significantly more implicit cognitive processes indicating competition when responding "disagree" compared to "agree", regardless of whether the prompts were correct/helpful or incorrect/unhelpful. Similarly, participants took significantly longer to respond to prompts with "disagree" rather than "agree". CONCLUSION: The findings of this study offer evidence of participants reporting cognitive processes that are overall more correct/helpful than incorrect/unhelpful, in their explicit responses to the dichotomous response tasks of the POSHA-S. However, these findings are tempered by evidence of a tendency to agree with statements in the measure and suggest the need for further research to increase understanding of how to measure and improve explicit and implicit cognitive processes related to people who stutter.

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.

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.

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.

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.

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.