The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs.

Innate immune signaling by Toll-like receptors (TLRs) involves receptor phosphorylation, which helps to shape and drive key inflammatory outputs, yet our understanding of the kinases and mechanisms that mediate TLR phosphorylation is incomplete. Spleen tyrosine kinase (Syk) is a nonreceptor protein tyrosine kinase, which is known to relay adaptive and innate immune signaling, including from TLRs. However, TLRs do not contain the conserved dual immunoreceptor tyrosine-based activation motifs that typically recruit Syk to many other receptors. One possibility is that the Syk-TLR association is indirect, relying on an intermediary scaffolding protein. We previously identified a role for the palmitoylated transmembrane adapter protein SCIMP in scaffolding the Src tyrosine kinase Lyn, for TLR phosphorylation, but the role of SCIMP in mediating the interaction between Syk and TLRs has not yet been investigated. Here, we show that SCIMP recruits Syk in response to lipopolysaccharide-mediated TLR4 activation. We also show that Syk contributes to the phosphorylation of SCIMP and TLR4 to enhance their binding. Further evidence pinpoints two specific phosphorylation sites in SCIMP critical for its interaction with Syk-SH2 domains in the absence of immunoreceptor tyrosine-based activation motifs. Finally, using inhibitors and primary macrophages from SCIMP-/- mice, we confirm a functional role for SCIMP-mediated Syk interaction in modulating TLR4 phosphorylation, signaling, and cytokine outputs. In conclusion, we identify SCIMP as a novel, immune-specific Syk scaffold, which can contribute to inflammation through selective TLR-driven inflammatory responses.

Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS.

Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-D-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.

Generalizable Protein Biosensors Based on Synthetic Switch Modules.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

Voicing contrast of stop consonant production in the speech of individuals with Parkinson disease ON and OFF dopaminergic medication.

The aims of this study were to: 1) compare voicing contrast in speakers with Parkinson disease (PD) and healthy controls by comparing the separation of voice onset time (VOT) distributions of voiced and voiceless stop consonants and 2) to determine whether the administration of dopaminergic medication affected VOT separation in speakers with PD. Data from a previous study by Fisher and Goberman (2010) were used to compare the VOT measures obtained from a group of speakers with PD with both ON and OFF medication, and a group of healthy controls. Supplementing the previous findings, the current analysis revealed that individuals with PD exhibited significantly less contrast between voiced and voiceless VOT than that observed in healthy speakers. Medication administration did not affect VOT contrast as no differences in VOT separation were observed between the ON and OFF medication states.

An Inexpensive, Open-Source USB Arduino Data Acquisition Device for Chemical Instrumentation.

Many research and teaching labs rely on USB data acquisition devices to collect voltage signals from instrumentation. However, these devices can be cost-prohibitive (especially when large numbers are needed for teaching labs) and require software to be developed for operation. In this article, we describe the development and use of an open-source USB data acquisition device (with 16-bit acquisition resolution) built using simple electronic components and an Arduino Uno that costs under $50. Additionally, open-source software written in Python is included so that data can be acquired using nearly any PC or Mac computer with a simple USB connection. Use of the device was demonstrated for a sophomore-level analytical experiment using GC and a CE-UV separation on an instrument used for research purposes.

Gender Perception of Speech: Dependence on Fundamental Frequency, Implied Vocal Tract Length, and Source Spectral Tilt.

OBJECTIVE: To investigate how listeners use fundamental frequency, implied vocal tract length, and source spectral tilt to infer speaker gender. METHODS: Sound files each containing the vowels /i, æ, ɑ, u/ interspersed by brief silences were synthesized. Each of the 210 stimuli was a combination of 10 values for fundamental frequency and 7 values for implied vocal tract length (and the associated formant frequencies) ranging from male-typical to female-typical, and 3 values for source spectral tilt approximating the voice qualities of breathy, normal, and pressed. Twenty-three listeners judged each synthesized "speaker" as "female" or "male." Generalized linear mixed model analysis was used to determine the extent to which fundamental frequency, implied vocal track length, and spectral tilt influenced listener judgment. RESULTS: Increasing fundamental frequency and decreasing implied vocal tract length resulted in increased probability of female judgment. Two interactions were identified: An increase in fundamental frequency and also a decrease in source spectral tilt (more negative) resulted in a greater increase in the probability of female judgment when the vocal tract length was relatively short. CONCLUSIONS: The relationships among fundamental frequency, implied vocal tract length, source spectral tilt, and probability of female judgment changed across the range of normal values, suggesting that the relative contributions of fundamental frequency and implied vocal tract length to gender perception varied over the ranges studied. There was no threshold of fundamental frequency or implied vocal tract length that dramatically shifted the perception between male and female.

Cross-Cultural Adaptation, Validation, and Diagnostic Accuracy of the Voice Fatigue Handicap Questionnaire-Persian Version.

OBJECTIVE: The current study aimed to determine the psychometric characteristics of a translated version of the Voice Fatigue Handicap Questionnaire in Persian by examining the validity, reliability, and diagnostic accuracy using data collected from people with and without dysphonia. STUDY DESIGN: Cross-sectional study. METHOD: The original questionnaire was translated from Italian to Persian using the International Quality of Life Assessment criteria. The translated version was completed by 122 people with dysphonia and 49 people without dysphonia. The validity, reliability, and diagnostic accuracy of the Voice Fatigue Handicap Questionnaire Persian-version were evaluated. A panel of five voice-specializing Speech and Language Pathologists and 20 native Persian speakers (with dysphonia) in a pilot study confirmed the translated version for content and face validity, respectively. The internal consistency was evaluated by Cronbach's alpha and McDonald omega coefficients. The intraclass correlation coefficient was used to examine the test-retest reliability. For discriminant validity, the mean total and subscale scores of respondents with and without dysphonia were compared through independent t tests. Finally, sensitivity, specificity, and ideal cut-off value were determined using the receiver-operating characteristic curve. RESULTS: Cronbach's alpha coefficient was 0.965 for the total score (functional: α = 0.883, emotional: α = 0.944, physical: α = 0.917). Also, the McDonald omega coefficient was 0.966 for the total score (functional: ω = 0.889, emotional: ω = 0.933, physical: ω = 0.920). The intraclass correlation coefficient for measuring the test-retest reliability was 0.971 for the total score (functional: ICC=0.937, emotional: ICC=0.954, physical ICC=0.976). The results of the independent samples t tests indicated that the dysphonia group obtained significantly higher mean scores (total and subscale scores) than the group without dysphonia. The cutoff value of 13.5 was determined based on the optimal balance of sensitivity (0.992) and specificity (0.918). CONCLUSION: The Voice Fatigue Handicap Questionnaire-Persian version is a valid and reliable questionnaire to identify individuals at high risk of vocal fatigue and subsequent dysphonia.

Systemic Racism in Communication Sciences and Disorders Academic Programs: A Commentary on Trends in Racial Representation.

PURPOSE: As a discipline, communication sciences and disorders (CSD) has struggled to address equity and inclusion for students, professionals, and scholars from historically excluded racial groups. Recent publications in this periodical that have begun to confront systemic racism in the discipline have been met with some expected resistance. In this commentary, I attempt to support and expand an argument made by Ellis and Kendall (2021), namely, that systemic racism has been and continues to be a normal and persistent feature of our academic programs. A comparison to U.S. Census data suggests that Asian, Black, Indigenous, Native, Latino/a/e, and multiracial CSD professionals are represented to a drastically lower rate compared with their representation in the population at large. Furthermore, publicly available data summaries indicate that there is a reduction in the level of racial diversity that is associated with an increase in White representation across the entire progression of the professional training and certification process, with the greatest level of diversity at the undergraduate level followed by the graduate and professional levels. CONCLUSIONS: A general knowledge of social and legal history in the United States would suggest that the relative reduction in representation across the academic and professional levels of the CSD disciplines results from policy and practice patterns that serve to preserve a White-dominant culture in our profession and exclude People of Color. Continued efforts broadly based in critical race studies may prove as a useful tool to identify, confront, and transform current policy and practice patterns in our national organization, academic programs, and accrediting bodies that have produced and sustained levels of inequality and White dominance in our programs and disciplines.

Effect of clear speech on acoustic measures of dysprosody in Parkinson disease for different reading tasks.

PURPOSE: The purpose of the study was to determine the effect of clear speech instruction on acoustic measures of dysprosody between reading passages of differing linguistic content for speakers with and without Parkinson Disease (PD). METHOD: Ten speakers with PD and 10 controls served as participants and read five simple and three standard reading stimuli twice. First, speakers read habitually and then following clear speech instruction. Acoustic measures of fundamental frequency variation (semitone standard deviation, STSD), articulation rate, and between-complex pause durations were calculated. RESULT: Results indicated speakers with PD exhibited less fundamental frequency variation than controls across reading stimuli and instructions. All speakers exhibited lower STSD and longer between-complex pause durations for the standard compared to simple reading stimuli. For clear speech, all speakers reduced articulation rate and increased between-complex pause durations in both simple and standard reading stimuli. However, speakers with PD exhibited a significantly less robust reduction in articulation rate for clear speech than control speakers for all reading stimuli. CONCLUSION: Linguistic content of reading stimuli contributes to differences in fundamental frequency variation and pause duration for all speakers. All speakers reduced articulation rate for clear speech compared to habitual instruction, but speakers with PD did so to a lesser extent than controls. Linguistic content of reading stimuli to examine dysprosody in PD should be considered for clinical application.

Order Affects Clear and Loud Speech Response.

PURPOSE: The purpose of this investigation was to examine the impact of instruction order on the speech production response when adopting higher effort speaking styles, specifically loud and clear speech. METHOD: Speech intensity, lip aperture range, and speech rate data were collected from 24 talkers who repeated the utterance "Buy Bobby a puppy" using habitual, clear, and loud speech. Participants were assigned in quasi-random fashion to one of two groups: a Clear-Loud Group (11 participants; order: habitual-clear-loud) or a Loud-Clear Group (13 participants; order: habitual-loud-clear). RESULTS: Talkers in the Clear-Loud Group exhibited higher speech intensity during the loud style compared with those who performed the Loud-Clear Group. Furthermore, talkers in the Clear-Loud Group retained the increases lip aperture range and reductions in speech rate associated with the clear style when producing the loud style. Conversely, talkers in the Loud-Clear Group exhibited significant increases in lip aperture range between the habitual and loud styles and between the loud and clear styles. Additionally, the Loud-Clear Group exhibited a reduction in speech rate only during the clear style, as no differences in speech rate were observed between the habitual and loud styles. CONCLUSIONS: These findings may suggest that producing a higher effort style leads to carry-over effects in subsequent styles. Future research should investigate factors that moderate the degree of order effects for both research and clinical purposes. For instance, if generalizable, the compounding carry-over effects may prove advantageous for certain clinical populations.

Quantifying Spatial and Temporal Variability of Clear and Loud Speech Produced While Performing a Concurrent Visuomotor Task.

PURPOSE: The purpose of this research note was to quantify the impact of concurrent performance of an attention-demanding secondary task on utterance-to-utterance movement variability associated with higher effort speaking styles, namely, clear and loud speech. METHOD: Lip and jaw kinematics collected as part of a prior study were analyzed. Participants repeated "Buy Bobby a puppy" using habitual, loud, and clear speech styles in isolation and while performing a secondary tracking task. The lip aperture (LA) signal was segmented based on opening and closing gestures associated with the utterance. The standard deviation of each segment duration was calculated to quantify temporal variability. To quantify spatial variability, each segment was first time normalized. The mean standard deviation of the overlapping time-normalized LA amplitudes was computed for each segment from the repetitions produced in each speech style and condition (speaking in isolation vs. speaking while tracking). A relative measure of spatial variation was also computed to account for the potential impact of articulatory scaling. RESULTS: Clear speech was associated with greater temporal and spatial variability than the habitual and loud styles. In the habitual style, talkers also exhibited a slight reduction in absolute spatial variability when speaking while tracking compared to speaking in isolation. The reduction in absolute spatial variability was likely associated with the concomitant reduction in LA range of motion, as there was no change in the relative spatial variability between conditions. CONCLUSION: The current investigation expands prior work by quantifying spatial and temporal characteristics of different speaking styles performed in isolation and while concurrently performing an attention-demanding visuomotor task.

Prolonged Processing Time for Manual and Vocal Responses in Parkinson Disease: A Psychological Refractory Period Paradigm Study.

PURPOSE: The purpose of this study was to examine a potential increased cognitive processing bottleneck within Parkinson disease (PD) by extending a previous overlapping task methodology. Additionally, this study extends previous overlapping task methodology in PD to examine the influence of modality (vocal vs. manual) on response delays in overlapping tasks in PD. METHOD: This study used the psychological refractory period (PRP) paradigm (overlapping-task paradigm) to study processing limitations as participants complete two tasks that increasingly overlap in time. Three levels of temporal overlap of tasks were utilized to vary cognitive demands on manual and vocal response time tasks. Ten participants with PD (PwPD) and 12 participants without PD were included in this study. RESULTS: Participants with PD demonstrated response time delays across temporal overlap conditions (likely indicating motor deficits) along with a larger increase in response delays in the most overlapped, cognitively taxing condition (likely indicating longer central processing bottleneck). Additionally, modality did not influence response times differently in overlapping task conditions or within participant groups. CONCLUSION: An extension of previous overlapping task methodologies within a complex task was successful in demonstrating an increased central processing deficit across manual and vocal response delays in PD, regardless of modality of response.

Reliability of a Linguistic Segmentation Procedure Specified by Systemic Functional Linguistics to Examine Extemporaneous Speech.

PURPOSE: Extemporaneous speech tasks provide an ecologically valid sample to examine speech acoustics, but differing methodologies exist in the literature for segmentation. Therefore, the purpose of this study was to examine the utility and reliability of a segmentation approach for extemporaneous speech specified by systemic functional linguistics (SFL) and its potential research and clinical applications. METHOD: Ten speakers without communication disorders served as participants in this study, and they responded to self-selected extemporaneous speaking prompts. Two expert analysts and one clinician analyst utilized a segmentation procedure specified by SFL to segment the extemporaneous speech samples into clauses and clause complexes. Intra- and interrater reliability were calculated for each analyst and pair of analysts. Acoustic measures of duration, speech rate, and intercomplex pause durations were calculated for each clause complex. RESULTS: Analyses for both intra- and interrater reliability revealed high percent agreement that was significantly greater than chance for expert and clinician analysts and between each pair of analysts (p < .001). Acoustic analyses revealed expected variation in number and duration of spoken syllables of clause complexes between and within speakers. CONCLUSIONS: The segmentation approach for extemporaneous speech specified by SFL is a reliable method for trained analysts that is informed by lexico-grammar and allows for acoustic measurement of speech production. It is also a reliable method for clinician analysts for speakers without communication disorders, and future work will investigate its utility for speakers with motor speech disorders. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.22357138.

Development of epistatic YES and AND protein logic gates and their assembly into signalling cascades.

The construction and assembly of artificial allosteric protein switches into information and energy processing networks connected to both biological and non-biological systems is a central goal of synthetic biology and bionanotechnology. However, designing protein switches with the desired input, output and performance parameters is challenging. Here we use a range of reporter proteins to demonstrate that their chimeras with duplicated receptor domains produce YES gate protein switches with large (up to 9,000-fold) dynamic ranges and fast (minutes) response rates. In such switches, the epistatic interactions between largely independent synthetic allosteric sites result in an OFF state with minimal background noise. We used YES gate protein switches based on ß-lactamase to develop quantitative biosensors of therapeutic drugs and protein biomarkers. Furthermore, we demonstrated the reconfiguration of YES gate switches into AND gate switches controlled by two different inputs, and their assembly into signalling networks regulated at multiple nodes.

Over the rainbow: structural characterization of the chromoproteins gfasPurple, amilCP, spisPink and eforRed.

Anthozoan chromoproteins are highly pigmented, diversely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp2-hybridized N-acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp3-hybridized trans-peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.

Practice Mediates Bidirectional Dual-Task Interference When Performing a Novel Sequential Nonword Repetition Task.

Introduction The current study examined the extent to which practice amount mediates dual-task interference patterns associated with concurrent performance of a novel speech task and attention-demanding visuomotor task. Method A Sequential Nonword Repetition Task was used to examine the effect of practice on interference associated with concurrent performance of a Visuomotor Pursuit Task. Twenty-five young adult participants were assigned to either an Extended Practice Group or a Limited Practice Group and performed a novel Sequential Nonword Repetition Task in isolation and while performing a concurrent visuomotor pursuit rotor task. Results Participants in the Limited Practice Group who were afforded a limited amount of practice exhibited dual-task interference (i.e., dual-task performance reductions) for both the speech and visuomotor tasks (i.e., bidirectional dual-task interference). Conversely, participants in the Extended Practice Group who were afforded extended practice exhibited little-to-no observable dual-task interference on the nonword repetition task. Conclusion Data from the current investigation suggest that the amount of initial practice mediates the degree of dual-task interference observed when a novel speech production task is performed with an attention-demanding Visuomotor Pursuit Task. Supplemental Material https://doi.org/10.23641/asha.14608071.

Tracking the Costs of Clear and Loud Speech: Interactions Between Speech Motor Control and Concurrent Visuomotor Tracking.

Purpose Prior work has demonstrated that competing tasks impact habitual speech production. The purpose of this investigation was to quantify the extent to which clear and loud speech are affected by concurrent performance of an attention-demanding task. Method Speech kinematics and acoustics were collected while participants spoke using habitual, loud, and clear speech styles. The styles were performed in isolation and while performing a secondary tracking task. Results Compared to the habitual style, speakers exhibited expected increases in lip aperture range of motion and speech intensity for the clear and loud styles. During concurrent visuomotor tracking, there was a decrease in lip aperture range of motion and speech intensity for the habitual style. Tracking performance during habitual speech did not differ from single-task tracking. For loud and clear speech, speakers retained the gains in speech intensity and range of motion, respectively, while concurrently tracking. A reduction in tracking performance was observed during concurrent loud and clear speech, compared to tracking alone. Conclusions These data suggest that loud and clear speech may help to mitigate motor interference associated with concurrent performance of an attention-demanding task. Additionally, reductions in tracking accuracy observed during concurrent loud and clear speech may suggest that these higher effort speaking styles require greater attentional resources than habitual speech.

Connecting Artificial Proteolytic and Electrochemical Signaling Systems with Caged Messenger Peptides.

Enzymatic polypeptide proteolysis is a widespread and powerful biological control mechanism. Over the last few years, substantial progress has been made in creating artificial proteolytic systems where an input of choice modulates the protease activity and thereby the activity of its substrates. However, all proteolytic systems developed so far have relied on the direct proteolytic cleavage of their effectors. Here, we propose a new concept where protease biosensors with a tunable input uncage a signaling peptide, which can then transmit a signal to an allosteric protein reporter. We demonstrate that both the cage and the regulatory domain of the reporter can be constructed from the same peptide-binding domain, such as calmodulin. To demonstrate this concept, we constructed a proteolytic rapamycin biosensor and demonstrated its quantitative actuation on fluorescent, luminescent, and electrochemical reporters. Using the latter, we constructed sensitive bioelectrodes that detect the messenger peptide release and quantitatively convert the recognition event into electric current. We discuss the application of such systems for the construction of in vitro sensory arrays and in vivo signaling circuits.

A Rationally and Computationally Designed Fluorescent Biosensor for d-Serine.

Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability, and conformational change to accomplish diverse functions in small molecule transport, sensing, and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here, we have engineered a d-alanine-specific SBP into a fluorescence biosensor with specificity for the signaling molecule d-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (KD = 6.7 ± 0.5 µM), specificity (40-fold increase vs glycine), thermostability (Tm = 79 °C), and dynamic range (∼14%). This sensor allowed measurement of physiologically relevant changes in d-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippocampal slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity, and thermostability and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.

Caged Activators of Artificial Allosteric Protein Biosensors.

The ability of proteins to interconvert unrelated biochemical inputs and outputs underlays most energy and information processing in biology. A common conversion mechanism involves a conformational change of a protein receptor in response to a ligand binding or a covalent modification, leading to allosteric activity modulation of the effector domain. Designing such systems rationally is a central goal of synthetic biology and protein engineering. A two-component sensory system based on the scaffolding of modules in the presence of an analyte is one of the most generalizable biosensor architectures. An inherent problem of such systems is dependence of the response on the absolute and relative concentrations of the components. Here we use the example of two-component sensory systems based on calmodulin-operated synthetic switches to analyze and address this issue. We constructed "caged" versions of the activating domain thereby creating a thermodynamic barrier for spontaneous activation of the system. We demonstrate that the caged biosensor architectures could operate at concentrations spanning 3 orders of magnitude and are applicable to electrochemical, luminescent, and fluorescent two-component biosensors. We analyzed the activation kinetics of the caged biosensors and determined that the core allosteric switch is likely to be the rate limiting component of the system. These findings provide guidance for predictable engineering of robust sensory systems with inputs and outputs of choice.