Closed-loop network of skin-interfaced wireless devices for quantifying vocal fatigue and providing user feedback.

Vocal fatigue is a measurable form of performance fatigue resulting from overuse of the voice and is characterized by negative vocal adaptation. Vocal dose refers to cumulative exposure of the vocal fold tissue to vibration. Professionals with high vocal demands, such as singers and teachers, are especially prone to vocal fatigue. Failure to adjust habits can lead to compensatory lapses in vocal technique and an increased risk of vocal fold injury. Quantifying and recording vocal dose to inform individuals about potential overuse is an important step toward mitigating vocal fatigue. Previous work establishes vocal dosimetry methods, that is, processes to quantify vocal fold vibration dose but with bulky, wired devices that are not amenable to continuous use during natural daily activities; these previously reported systems also provide limited mechanisms for real-time user feedback. This study introduces a soft, wireless, skin-conformal technology that gently mounts on the upper chest to capture vibratory responses associated with vocalization in a manner that is immune to ambient noises. Pairing with a separate, wirelessly linked device supports haptic feedback to the user based on quantitative thresholds in vocal usage. A machine learning-based approach enables precise vocal dosimetry from the recorded data, to support personalized, real-time quantitation and feedback. These systems have strong potential to guide healthy behaviors in vocal use.

A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.

TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

Path to autonomous soil sampling and analysis by ground-based robots.

Good site characterization is essential for the selection of remediation alternatives for impacted soils. The value of site characterization is critically dependent on the quality and quantity of the data collected. Current methods for characterizing impacted soils rely on expensive manual sample collection and off-site analysis. However, recent advances in terrestrial robotics and artificial intelligence offer a potentially revolutionary set of tools and methods that will help to autonomously explore natural environments, select sample locations with the highest value of information, extract samples, and analyze the data in real-time without exposing humans to potentially hazardous conditions. A fundamental challenge to realizing this potential is determining how to design an autonomous system for a given investigation with many, and often conflicting design criteria. This work presents a novel design methodology to navigate these criteria. Specifically, this methodology breaks the system into four components - sensing, sampling, mobility, and autonomy - and connects design variables to the investigation objectives and constraints. These connections are established for each component through a survey of existing technology, discussion of key technical challenges, and highlighting conditions where generality can promote multi-application deployment. An illustrative example of this design process is presented for the development and deployment of a robotic platform characterizing salt-impacted oil & gas reserve pits. After calibration, the relationship between the in situ robot chloride measurements and laboratory-based chloride measurements had a good linear relationship (R2-value = 0.861) and statistical significance (p-value = 0.003).

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline.

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m6A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single-nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

RNA matchmaking in chromatin regulation.

Beyond being the product of gene expression, RNA can also influence the regulation of chromatin. The majority of the human genome has the capacity to be transcribed and the majority of the non-protein-coding transcripts made by RNA Polymerase II are enriched in the nucleus. Many chromatin regulators can bind to these ncRNAs in the nucleus; in some cases, there are clear examples of direct RNA-mediated chromatin regulation mechanisms stemming from these interactions, while others have yet to be determined. Recent studies have highlighted examples of chromatin regulation via RNA matchmaking, a term we use broadly here to describe intermolecular base-pairing interactions between one RNA molecule and an RNA or DNA match. This review provides examples of RNA matchmaking that regulates chromatin processes and summarizes the technical approaches used to capture these events.

Clipping of arginine-methylated histone tails by JMJD5 and JMJD7.

Two of the unsolved, important questions about epigenetics are: do histone arginine demethylases exist, and is the removal of histone tails by proteolysis a major epigenetic modification process? Here, we report that two orphan Jumonji C domain (JmjC)-containing proteins, JMJD5 and JMJD7, have divalent cation-dependent protease activities that preferentially cleave the tails of histones 2, 3, or 4 containing methylated arginines. After the initial specific cleavage, JMJD5 and JMJD7, acting as aminopeptidases, progressively digest the C-terminal products. JMJD5-deficient fibroblasts exhibit dramatically increased levels of methylated arginines and histones. Furthermore, depletion of JMJD7 in breast cancer cells greatly decreases cell proliferation. The protease activities of JMJD5 and JMJD7 represent a mechanism for removal of histone tails bearing methylated arginine residues and define a potential mechanism of transcription regulation.

Recruitment and allosteric stimulation of a histone-deubiquitinating enzyme during heterochromatin assembly.

Heterochromatin formation in budding yeast is regulated by the silent information regulator (SIR) complex. The SIR complex comprises the NAD-dependent deacetylase Sir2, the scaffolding protein Sir4, and the nucleosome-binding protein Sir3. Transcriptionally active regions present a challenge to SIR complex-mediated de novo heterochromatic silencing due to the presence of antagonistic histone post-translational modifications, including acetylation and methylation. Methylation of histone H3K4 and H3K79 is dependent on monoubiquitination of histone H2B (H2B-Ub). The SIR complex cannot erase H2B-Ub or histone methylation on its own. The deubiquitinase (DUB) Ubp10 is thought to promote heterochromatic silencing by maintaining low H2B-Ub at sub-telomeres. Here, we biochemically characterized the interactions between Ubp10 and the SIR complex machinery. We demonstrate that a direct interaction between Ubp10 and the Sir2/4 sub-complex facilitates Ubp10 recruitment to chromatin via a co-assembly mechanism. Using hydrolyzable H2B-Ub analogs, we show that Ubp10 activity is lower on nucleosomes compared with H2B-Ub in solution. We find that Sir2/4 stimulates Ubp10 DUB activity on nucleosomes, likely through a combination of targeting and allosteric regulation. This coupling mechanism between the silencing machinery and its DUB partner allows erasure of active PTMs and the de novo transition of a transcriptionally active DNA region to a silent chromatin state.

Proteomic profiling of yeast heterochromatin connects direct physical and genetic interactions.

Heterochromatin domains are stably repressed chromatin structures composed of a core assembly of silencing proteins that condense adjacent nucleosomes. The minimal heterochromatin structure can serve as a platform for recruitment of complementary regulatory factors. We find that a reconstituted budding yeast heterochromatin domain can act as a platform to recruit multiple factors that play a role in regulating heterochromatin function. We uncover the direct interaction between the SIR heterochromatin complex and a chromosomal boundary protein that restricts the spread of heterochromatin. We find that the SIR complex relieves a mechanism of auto-inhibition within the boundary protein Yta7, allowing the Yta7 bromodomain to engage chromatin. Our results suggest that budding yeast shares with other eukaryotes the ability to establish complex heterochromatin domains that coordinate multiple mechanisms of silencing regulation through physical interactions.

SILAC-MS Profiling of Reconstituted Human Chromatin Platforms for the Study of Transcription and RNA Regulation.

DNA packaged into chromatin is the core structure of the human genome. Nearly all eukaryotic genome regulation must interface with this genomic structure, and modification of the chromatin can influence molecular mechanisms that regulate the underlying DNA. Many processes are governed by regulated stepwise assembly mechanisms that build complex machinery on chromatin to license a specific activity such as transcription. Transcriptional activators drive the initial steps of gene expression, regulated in part by chromatin. Here we describe tools to study the stepwise assembly of protein complexes on chromatin in a highly controlled manner using reconstituted human chromatin platforms and quantitative proteomic profiling. We profile the early steps in transcriptional activation and highlight the potential for understanding the multiple ways chromatin can influence transcriptional regulation. We also describe modifications of this approach to study the activity of a long noncoding RNA to act as a dynamic scaffold for proteins to be recruited to chromatin. This approach has the potential to provide a more comprehensive understanding of important macromolecular complex assembly that occurs on the human genome. The reconstituted nature of the chromatin substrate offers a tunable system that can be trapped at specific substeps to understand how chromatin interfaces with genome regulation machinery.

The interplay of histone H2B ubiquitination with budding and fission yeast heterochromatin.

Mono-ubiquitinated histone H2B (H2B-Ub) is important for chromatin regulation of transcription, chromatin assembly, and also influences heterochromatin. In this review, we discuss the effects of H2B-Ub from nucleosome to higher-order chromatin structure. We then assess what is currently known of the role of H2B-Ub in heterochromatic silencing in budding and fission yeasts (S. cerevisiae and S. pombe), which have distinct silencing mechanisms. In budding yeast, the SIR complex initiates heterochromatin assembly with the aid of a H2B-Ub deubiquitinase, Ubp10. In fission yeast, the RNAi-dependent pathway initiates heterochromatin in the context of low H2B-Ub. We examine how the different silencing machineries overcome the challenge of H2B-Ub chromatin and highlight the importance of using these microorganisms to further our understanding of H2B-Ub in heterochromatic silencing pathways.

An RNA matchmaker protein regulates the activity of the long noncoding RNA HOTAIR.

The human long noncoding RNA (lncRNA) HOTAIR acts in trans to recruit the Polycomb repressive complex 2 (PRC2) to the HOXD gene cluster and to promote gene silencing during development. In breast cancers, overexpression of HOTAIR increases metastatic potential via the repression of many additional genes. It has remained unclear what factors determine HOTAIR-dependent PRC2 activity at specific genomic loci, particularly when high levels of HOTAIR result in aberrant gene silencing. To identify additional proteins that contribute to the specific action of HOTAIR, we performed a quantitative proteomic analysis of the HOTAIR interactome. We found that the most specific interaction was between HOTAIR and the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a member of a family of proteins involved in nascent mRNA processing and RNA matchmaking. Our data suggest that A2/B1 are key contributors to HOTAIR-mediated chromatin regulation in breast cancer cells: A2/B1 knockdown reduces HOTAIR-dependent breast cancer cell invasion and decreases PRC2 activity at the majority of HOTAIR-dependent loci. We found that the B1 isoform, which differs from A2 by 12 additional amino acids, binds with highest specificity to HOTAIR. B1 also binds chromatin and associates preferentially with RNA transcripts of HOTAIR gene targets. We furthermore demonstrate a direct RNA-RNA interaction between HOTAIR and a target transcript that is enhanced by B1 binding. Together, these results suggest a model in which B1 matches HOTAIR with transcripts of target genes on chromatin, leading to repression by PRC2.

Global profiling of hnRNP A2/B1-RNA binding on chromatin highlights LncRNA interactions.

Long noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Automated acoustic analysis of 50-kHz ultrasonic vocalizations using template matching and contour analysis.

Ultrasonic vocalizations are a useful tool for inferring affective states in the rat and have been incorporated in research paradigms modeling important human conditions. While the majority of studies report the quantity or rate of observed ultrasonic vocalizations, growing evidence suggests that critical data may be contained in the acoustic features of individual vocalizations. Thus, the goal of the present study was to develop and validate a method for measuring acoustic parameters of ultrasonic vocalizations that were collected using automatic template detection. Acoustic parameters derived using this method were found to be comparable to those collected using commercially available software.

Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

Vocal Health Assessment of Professional Performers Returning to the Stage After the COVID-19 Pandemic Shutdown.

OBJECTIVES/HYPOTHESIS: This study assessed the vocal health of performers returning to full-time performance after the COVID-19 pandemic shutdown and investigated how differences in voice usage, exposure to voice care professionals, and vocal pathology before and during the pandemic contributed to variability in self-perceived and instrumental vocal outcome measures. STUDY DESIGN: This was a prospective, case-control observational study conducted at a single outpatient site. METHODS: Twenty-two patients, 11 cases and 11 controls, were enrolled for the study. All participants were full-time singing professionals prior to the COVID-19 pandemic. Cases were recruited from patients presenting to a tertiary care voice center for vocal or pharyngeal complaints. Controls were healthy volunteers recruited from the general population of professional singers in the surrounding metropolitan area. All participants provided responses to the Voice Handicap Index-10, Evaluation of Ability to Sing Easily, and Laryngopharyngeal Measure of Perceived Sensation validated questionnaires as well as a study survey with questions regarding vocal use and history prior to and during the pandemic. All participants underwent instrumental acoustic and videostroboscopic voice evaluations. RESULTS: Cases had poorer outcome measures overall and were more likely to report their voices were worse at study enrollment when compared to their prepandemic perception (P = 0.027). Cases tended to be older and less likely to have pursued alternative employment during the pandemic that involved increased speaking voice use (27% vs 55%), but these differences were not statistically significant. CONCLUSIONS: There was a variable response among performers to the prolonged hiatus from performing during the COVID-19 pandemic. Those with poorer outcomes tended to be older and may have used their voice less during the pandemic. These findings are consistent with detraining periods in the exercise physiology literature and support the construct of treating vocal performers as vocal athletes.

Rapid Implementation of Teletherapy for Voice Disorders: Challenges and Opportunities for Speech-Language Pathologists.

INTRODUCTION: The COVID-19 pandemic necessitated a rapid restructuring of the clinical management of voice and upper airway disorders by speech-language pathologists (SLPs). As in-person therapy sessions were suspended, voice-specialized SLPs across healthcare settings shifted to online teletherapy. In this survey study, we queried voice therapists on their experiences with and opinions regarding the adoption of teletherapy into routine clinical practice. METHODS: Voice-specialized SLPs were recruited nationwide to complete an online survey which included questions about the usability of software and hardware, patient management, the effectiveness of therapy, overall satisfaction, and suggestions for improvement. RESULTS: 48 participants completed the survey. The majority of respondents reported frequent technical difficulties and poor access to or understanding of appropriate equipment. Overall, participants endorsed better patient access, attendance, and compliance, as well as increased scheduling flexibility. While 95% of the respondents stated they would recommend teletherapy to another SLP, only 20% supported a shift to exclusively virtual sessions. Forty percent of respondents endorsed a hybrid model consisting of initial in-person sessions followed by virtual ones. DISCUSSION: Incorporating teletherapy into clinical voice practice has, for the most part, followed Carl May's normalization process theory framework, in that clinicians have invested understanding, training, time and effort, and appraisal into its implementation. However, the unusually rapid pace of change necessitated by the pandemic has presented its own set of challenges. Given the inherent conveniences of virtual therapy, the online modality is likely here to stay. It is critical that we understand the facilitators and barriers to its successful adoption.

Effects of Participation in a Structured Choral Program on the Voice of Older Adults.

OBJECTIVES: To examine the effects of short-term and long-term engagement with structured choral singing on vocal function and quality of life outcomes in older adults. METHODS: Two groups of older adult singers over 55 years, one with fewer than 4 semesters and one with 4 or more semesters singing in a chorale, were assessed at 3 time points: baseline, after 1 semester of singing, and either after 1 semester of rest or after 1 semester of rest and 1 semester more of singing. Acoustic and aerodynamic measures, voice-related quality of life ratings, and measures of singing accuracy were obtained. Percent change between time points were calculated to determine three outcomes: improvement, lack of change, or worsening of measures across time. RESULTS: Long-term average spectrum (LTAS), difference in first and second harmonics and estimated subglottic pressure were significantly more likely to improve after a semester of singing with less experience singers, and LTAS continued to improve after a semester of rest. Flow was significantly more likely to improve with more singing experience after a semester of singing. Aerodynamic variables consistently changed in more experienced singers and improvement was maintained over the three visits. No significant changes occurred over time for singing accuracy for any singer type. Self-perception of singing voice continued to improve with more singing experience. CONCLUSIONS: This study demonstrated that for older adults in good health, regular singing provided a mechanism for maintaining speaking voice over time.

Characterization of Primary Muscle Tension Dysphonia Using Acoustic and Aerodynamic Voice Metrics.

OBJECTIVES/HYPOTHESIS: The objectives of this study were to (1) identify optimal clusters of 15 standard acoustic and aerodynamic voice metrics recommended by the American Speech-Language-Hearing Association (ASHA) to improve characterization of patients with primary muscle tension dysphonia (pMTD) and (2) identify combinations of these 15 metrics that could differentiate pMTD from other types of voice disorders. STUDY DESIGN: Retrospective multiparametric METHODS: Random forest modeling, independent t-tests, logistic regression, and affinity propagation clustering were implemented on a retrospective dataset of 15 acoustic and aerodynamic metrics. RESULTS: Ten percent of patients seen at the New York University (NYU) Voice Center over two years met the study criteria for pMTD (92 out of 983 patients), with 65 patients with pMTD and 701 of non-pMTD patients with complete data across all 15 acoustic and aerodynamic voice metrics. PCA plots and affinity propagation clustering demonstrated substantial overlap between the two groups on these parameters. The highest ranked parameters by level of importance with random forest models-(1) mean airflow during voicing (L/sec), (2) mean SPL during voicing (dB), (3) mean peak air pressure (cmH2O), (4) highest F0 (Hz), and (5) CPP mean vowel (dB)-accounted for only 65% of variance. T-tests showed three of these parameters-(1) CPP mean vowel (dB), (2) highest F0 (Hz), and (3) mean peak air pressure (cmH2O)-were statistically significant; however, the log2-fold change for each parameter was minimal. CONCLUSION: Computational models and multivariate statistical testing on 15 acoustic and aerodynamic voice metrics were unable to adequately characterize pMTD and determine differences between the two groups (pMTD and non-pMTD). Further validation of these metrics is needed with voice elicitation tasks that target physiological challenges to the vocal system from baseline vocal acoustic and aerodynamic ouput. Future work should also place greater focus on validating metrics of physiological correlates (eg, neuromuscular processes, laryngeal-respiratory kinematics) across the vocal subsystems over traditional vocal output measures (eg, acoustics, aerodynamics) for patients with pMTD. LEVEL OF EVIDENCE: II.

Longitudinal Effects of Base of Tongue Concurrent Chemoradiation Therapy in a Pre-Clinical Model.

BACKGROUND/OBJECTIVES: Base of tongue (BOT) dysfunction is common following oropharyngeal concurrent chemoradiation therapy (CCRT). We present a clinically relevant animal model quantifying the effects of CCRT on tongue strength and elasticity over time. METHODS: Fifty-three male and 53 female Sprague-Dawley rats were randomized to control or experimental groups. Experimental animals received cisplatin, 5-fluorouracil, and 5 fractions of 7 Gy directed to the BOT. Controls received no intervention. At 2 weeks, 5 months, or 10 months after CCRT, animals underwent non-survival surgery to measure twitch and tetanic tongue strength, which were analyzed using multivariate linear mixed effects models. Tongue displacement, a surrogate for tongue elasticity, was also determined via stress-strain testing and analyzed via a multivariate linear mixed effects model. RESULTS: Reporting the combined results of both sexes, the estimated experimental group mean peak twitch forces became more divergent over time compared to controls, being 8.3% lower than controls at 2 weeks post-CCRT, 15.7% lower at 5 months, and 31.6% lower at 10 months. Estimated experimental group mean peak tetanic forces followed a similar course and were 2.9% lower than controls at 2 weeks post CCRT, 20.7% lower at 5 months, and 27.0% lower at 10 months. Stress-strain testing did not find CCRT to have a significant effect on tongue displacement across experimental timepoints. CONCLUSIONS: This study demonstrates an increasing difference in tongue strength over time between controls and animals exposed to CCRT. Tongue elasticity was not significantly affected by CCRT, suggesting that changes in strength may not be caused by fibrosis. LEVEL OF EVIDENCE: NA Laryngoscope, 133:1455-1461, 2023.