Single-cell multi-scale footprinting reveals the modular organization of DNA regulatory elements.

Cis-regulatory elements control gene expression and are dynamic in their structure, reflecting changes to the composition of diverse effector proteins over time1-3. Here we sought to connect the structural changes at cis-regulatory elements to alterations in cellular fate and function. To do this we developed PRINT, a computational method that uses deep learning to correct sequence bias in chromatin accessibility data and identifies multi-scale footprints of DNA-protein interactions. We find that multi-scale footprints enable more accurate inference of TF and nucleosome binding. Using PRINT with single-cell multi-omics, we discover wide-spread changes to the structure and function of candidate cis-regulatory elements (cCREs) across hematopoiesis, wherein nucleosomes slide, expose DNA for TF binding, and promote gene expression. Activity segmentation using the co-variance across cell states identifies "sub-cCREs" as modular cCRE subunits of regulatory DNA. We apply this single-cell and PRINT approach to characterize the age-associated alterations to cCREs within hematopoietic stem cells (HSCs). Remarkably, we find a spectrum of aging alterations among HSCs corresponding to a global gain of sub-cCRE activity while preserving cCRE accessibility. Collectively, we reveal the functional importance of cCRE structure across cell states, highlighting changes to gene regulation at single-cell and single-base-pair resolution.

Thyroarytenoid Muscle Gene Expression in a Rat Model of Early-Onset Parkinson's Disease.

OBJECTIVES/HYPOTHESIS: Voice disorders in Parkinson's disease (PD) are early-onset, manifest in the preclinical stages of the disease, and negatively impact quality of life. The complete loss of function in the PTEN-induced kinase 1 gene (Pink1) causes a genetic form of early-onset, autosomal recessive PD. Modeled after the human inherited mutation, the Pink1-/- rat demonstrates significant cranial sensorimotor dysfunction including declines in ultrasonic vocalizations. However, the underlying genetics of the vocal fold thyroarytenoid (TA) muscle that may contribute to vocal deficits has not been studied. The aim of this study was to identify differentially expressed genes in the TA muscle of 8-month-old male Pink1-/- rats compared to wildtype controls. STUDY DESIGN: Animal experiment with control. METHODS: High throughput RNA sequencing was used to examine TA muscle gene expression in adult male Pink1-/- rats and wildtype controls. Weighted Gene Co-expression Network Analysis was used to construct co-expression modules to identify biological networks, including where Pink1 was a central node. The ENRICHR tool was used to compare this gene set to existing human gene databases. RESULTS: We identified 134 annotated differentially expressed genes (P < .05 cutoff) and observed enrichment in the following biological pathways: Parkinson's disease (Casp7, Pink1); Parkin-Ubiquitin proteasome degradation (Psmd12, Psmd7); MAPK signaling (Casp7, Ppm1b, Ppp3r1); and inflammatory TNF-α, Nf-κB Signaling (Casp7, Psmd12, Psmd7, Cdc34, Bcl7a, Peg3). CONCLUSIONS: Genes and pathways identified here may be useful for evaluating the specific mechanisms of peripheral dysfunction including within the laryngeal muscle and have potential to be used as experimental biomarkers for treatment development. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2874-E2879, 2021.

Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

Myogenic marker expression as a function of age and exercise-based therapy in the tongue.

Degeneration of tongue muscles with aging may contribute to swallowing deficits observed in elderly people. However, the capacity for tongue muscle stem cells (SCs) to regenerate and repair the aged tongue and improve tongue strength following tongue exercise (a current clinical treatment) has never been examined. We found that the expression of regenerative, myogenic markers were impaired with age and may be related to increased expression of senescent marker p16INK4a. Tongue strength increased in young adult and old rats following exercise and was related to the expression of Pax7, MyoD, myogenin, and p16INK4a. Our study also suggests that strengthening of tongue muscles via clinical rehabilitation strategies also increased the expression of SC regenerative markers in the tongue throughout the exercise duration.

Age-related alterations in swallowing biomechanics.

BACKGROUND: Aging rodent models allow for the discovery of underlying mechanisms of cranial muscle dysfunction. Methods are needed to allow quantification of complex, multivariate biomechanical movements during swallowing. Videofluoroscopic swallow studies (VSS) are the standard of care in assessment of swallowing disorders in patients and validated quantitative, kinematic, and morphometric analysis methods have been developed. Our purpose was to adapt validated morphometric techniques to the rodent to computationally analyze swallowing dysfunction in the aging rodent. METHODS: VSS, quantitative analyses (bolus area, bolus velocity, mastication rate) and a rodent specific multivariate, morphometric computational analysis of swallowing biomechanics were performed on 20 swallows from 5 young adult and 5 old Fischer 344/Brown Norway rats. Eight anatomical landmarks were used to track the relative change in position of skeletal levers (cranial base, vertebral column, mandible) and soft tissue landmarks (upper esophageal sphincter, base of tongue). RESULTS: Bolus area significantly increased and mastication rate significantly decreased with age. Aging accounted for 77.1% of the variance in swallow biomechanics, and 18.7% of the variance was associated with swallow phase (oral vs pharyngeal). Post hoc analyses identified age-related alterations in tongue base retraction, mastication, and head posture during the swallow. CONCLUSION: Geometric morphometric analysis of rodent swallows suggests that swallow biomechanics are altered with age. When used in combination with biological assays of age-related adaptations in neuromuscular systems, this multivariate analysis may increase our understanding of underlying musculoskeletal dysfunction that contributes to swallowing disorders with aging.

Comparison Between Patient-Perceived Voice Changes and Quantitative Voice Measures in the First Postoperative Year After Thyroidectomy: A Secondary Analysis of a Randomized Clinical Trial.

Importance: Voice changes after thyroidectomy are typically attributed to recurrent laryngeal nerve injury. However, most postoperative voice changes occur in the absence of clinically evident vocal fold paralysis. To date, no study has compared the prevalence, duration, and consequences of voice-related disability from the patient perspective with use of quantitative vocal measures. Objectives: To assess the quality-of-life consequences of postthyroidectomy voice change from the perspective of patients with thyroid cancer and to compare patient-perceived voice changes with changes in quantitative vocal variables at 5 time points in the first postoperative year. Design, Setting, and Participants: This prospective mixed methods observational study within a randomized clinical trial occurred at the University of Wisconsin Hospital and Clinics. Participants were 42 patients with clinically node-negative papillary thyroid cancer without a preexisting vocal cord paralysis who were recruited and enrolled from outpatient clinics between June 6, 2014, and March 6, 2017, as part of the ongoing randomized clinical trial. Intervention: Total thyroidectomy. Main Outcomes and Measures: Semistructured interviews, symptom prevalence, and instrumental voice evaluations (laryngoscopy, phonation threshold pressure, Dysphonia Severity Index, and Voice Handicap Index) occurred at baseline (n = 42) and 2-week (n = 42), 6-week (n = 39), 6-month (n = 35), and 1-year (n = 30) postoperative time points. Results: Participants had a mean age of 48 years (interquartile range, 38-58 years; age range, 22-70 years) and were mostly female (74% [31 of 42]) and of white race/ethnicity (98% [41 of 42]). Impaired communication was the primary theme derived from patient interviews from before thyroidectomy to after thyroidectomy. Voice changes were perceived by 24 participants at 2 weeks after thyroidectomy. After surgery, voice symptoms were prevalent and persisted for 50% (21 of 42) of participants out to at least 1 year of follow-up. Quantitative vocal perturbations were detected in the Dysphonia Severity Index and Voice Handicap Index at the 2-week follow-up but returned to baseline levels by the 6-week follow-up visit. Conclusions and Relevance: Voice changes are common after surgery for papillary thyroid cancer and affect quality of life for many patients out to 1 year of follow-up. Directly querying patients about postoperative voice changes and questioning whether commonly used aerodynamic and acoustic variables detect meaningful voice changes are important in identifying patients whose quality of life has been affected by postthyroidectomy dysphonia. Trial Registration: ClinicalTrials.gov Identifier: NCT02138214.

Effect of neuromuscular electrical stimulation frequency on muscles of the tongue.

INTRODUCTION: Neuromuscular electrical stimulation (NMES) for the treatment of swallowing disorders is delivered at a variety of stimulation frequencies. We examined the effects of stimulation frequency on tongue muscle plasticity in an aging rat model. METHODS: Eighty-six young, middle-aged, and old rats were assigned to either bilateral hypoglossal nerve stimulation at 10 or 100 Hz (5 days/week, 8 weeks), sham, or no-implantation conditions. Muscle contractile properties and myosin heavy chain (MyHC) composition were determined for hyoglossus (HG) and styloglossus (SG) muscles. RESULTS: Eight weeks of 100-Hz stimulation resulted in the greatest changes in muscle contractile function with significantly longer contraction and half-decay times, the greatest reduction in fatigue, and a transition toward slowly contracting, fatigue-resistant MyHC isoforms. DISCUSSION: NMES at 100-Hz induced considerable changes in contractile and phenotypic profiles of HG and SG muscles, suggesting higher frequency NMES may yield a greater therapeutic effect. Muscle Nerve, 2018.

Age-related effect of cell death on fiber morphology and number in tongue muscle.

INTRODUCTION: Multiple pathways may exist for age-related tongue muscle degeneration. Cell death is one mechanism contributing to muscle atrophy and decreased function. We hypothesized with aging, apoptosis, and apoptotic regulators would be increased, and muscle fiber size and number would be reduced in extrinsic tongue muscles. METHODS: Cell death indices, expression of caspase-3 and Bcl-2, and measures of muscle morphology and number were determined in extrinsic tongue muscles of young and old rats. RESULTS: Significant increases in cell death, caspase-3, and Bcl-2 were observed in all extrinsic tongue muscles along with reductions in muscle fiber number in old rats. DISCUSSION: We demonstrated that apoptosis indices increase with age in lingual muscles and that alterations in apoptotic regulators may be associated with age-related degeneration in muscle fiber size and number. These observed apoptotic processes may be detrimental to muscle function, and may contribute to degradation of cranial functions with age. Muscle Nerve 57: E29-E37, 2018.

Simulation of KTP Laser-Based Zenker Diverticulotomy with a Porcine Model and Laryngeal Dissection Station.

Zenker's diverticulum is a rare cause of progressive dysphagia that is treated surgically. KTP laser-based diverticulotomy is one effective treatment. Developing a simulation model is helpful for rare conditions. Pigs have a natural hypopharyngeal pouch similar to a diverticulum. We present a model for performing rigid endoscopic KTP laser diverticulotomy in a porcine model using a laryngeal dissection station. Eleven pigs were examined to confirm presence of the hypopharyngeal pouch. A specimen was mounted on the modified laryngeal dissection station and a KTP laser-based diverticulotomy was performed. Novel aspects include use of the laryngeal dissection station and application of the model for simulating rigid endoscopic KTP laser diverticulotomy. This model allows trainees to practice equipment setup, positioning of the laryngoscope to isolate the cricopharyngeal bar, tissue handling, laser safety techniques, and use of the KTP laser through the laryngoscope under microscopic visualization.

The effects of treadmill running on aging laryngeal muscle structure.

OBJECTIVES/HYPOTHESIS: Age-related changes in laryngeal muscle structure and function may contribute to deficits in voice and swallowing observed in elderly people. We hypothesized that treadmill running, an exercise that increases respiratory drive to upper airway muscles, would induce changes in thyroarytenoid muscle myosin heavy chain (MHC) isoforms that are consistent with a fast-to-slow transformation in muscle fiber type. STUDY DESIGN: Randomized parallel group controlled trial. METHODS: Fifteen young adult and 14 old Fischer 344/Brown Norway rats received either treadmill running or no exercise (5 days/week/8 weeks). Myosin heavy chain isoform composition in the thyroarytenoid muscle was examined at the end of 8 weeks. RESULTS: Significant age and treatment effects were found. The young adult group had the greatest proportion of superfast-contracting MHCIIL isoform. The treadmill running group had the lowest proportion of MHCIIL and the greatest proportion of MHCIIx isoforms. CONCLUSION: Thyroarytenoid muscle structure was affected both by age and treadmill running in a fast-to-slow transition that is characteristic of exercise manipulations in other skeletal muscles. LEVEL OF EVIDENCE: NA. Laryngoscope, 126:672-677, 2016.

Tongue muscle plasticity following hypoglossal nerve stimulation in aged rats.

INTRODUCTION: Age-related decreases in tongue muscle mass and strength have been reported. It may be possible to prevent age-related tongue muscle changes using neuromuscular electrical stimulation (NMES). Our hypothesis was that alterations in muscle contractile properties and myosin heavy chain composition would be found after NMES. METHODS: Fifty-four young, middle-aged, and old 344/Brown Norway rats were included in this study. Twenty-four rats underwent bilateral electrical stimulation of the hypoglossal nerves for 8 weeks and were compared with control or sham rats. Muscle contractile properties and myosin heavy chain (MHC) in the genioglossus (GG), styloglossus (SG), and hyoglossus (HG) muscles were examined. RESULTS: Compared with unstimulated control rats, we found reduced muscle fatigue, increased contraction and half-decay times, and increased twitch and tetanic tension. Increased type I MHC was found, except for in GG in old and middle-aged rats. CONCLUSION: Transitions in tongue muscle contractile properties and phenotype were found after NMES.

Differential effects of targeted tongue exercise and treadmill running on aging tongue muscle structure and contractile properties.

Age-associated changes in tongue muscle structure and strength may contribute to dysphagia in elderly people. Tongue exercise is a current treatment option. We hypothesized that targeted tongue exercise and nontargeted exercise that activates tongue muscles as a consequence of increased respiratory drive, such as treadmill running, are associated with different patterns of tongue muscle contraction and genioglossus (GG) muscle biochemistry. Thirty-one young adult, 34 middle-aged, and 37 old Fischer 344/Brown Norway rats received either targeted tongue exercise, treadmill running, or no exercise (5 days/wk for 8 wk). Protrusive tongue muscle contractile properties and myosin heavy chain (MHC) composition in the GG were examined at the end of 8 wk across groups. Significant age effects were found for maximal twitch and tetanic tension (greatest in young adult rats), MHCIIb (highest proportion in young adult rats), MHCIIx (highest proportion in middle-aged and old rats), and MHCI (highest proportion in old rats). The targeted tongue exercise group had the greatest maximal twitch tension and the highest proportion of MHCI. The treadmill running group had the shortest half-decay time, the lowest proportion of MHCIIa, and the highest proportion of MHCIIb. Fatigue was significantly less in the young adult treadmill running group and the old targeted tongue exercise group than in other groups. Thus, tongue muscle structure and contractile properties were affected by both targeted tongue exercise and treadmill running, but in different ways. Studies geared toward optimizing dose and manner of providing targeted and generalized tongue exercise may lead to alternative tongue exercise delivery strategies.

Effects of aging on thyroarytenoid muscle regeneration.

OBJECTIVES/HYPOTHESIS: Regenerative properties of age-associated changes in the intrinsic laryngeal muscles following injury are unclear. The purpose of this study was to investigate the regenerative properties of the thyroarytenoid (TA) muscle in an aging rat model. The hypothesis was that following myotoxic injury, old animals would exhibit a decrease in mitotic activities of muscle satellite cells when compared with younger rats, suggesting reduced regenerative potential in the aging rat TA muscle. STUDY DESIGN: Animal group comparison. METHODS: Regeneration responses following injury to the TA muscle were examined in 18 young adult, middle-aged, and old Fischer 344/Brown Norway rats. TA muscle fiber cross-sectional area (CSA), satellite cell mitosis (number/fiber), and regeneration index (CSA injured side/CSA noninjured side) were measured and compared across age groups. RESULTS: Young adult animals had a significantly higher regeneration index than the middle-aged and old groups. Within the lateral region of the TA muscle (LTA), the regeneration index was significantly higher in the young adult animals than in the middle-aged and old animals. The regeneration index of the medial TA was significantly higher than the LTA across all age groups. CONCLUSIONS: The regenerative capacity of the TA muscle is impaired with increasing age.