Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.

Background/ObjectivesFalse vocal fold (FVF) hyperfunction during phonation is thought to be a diagnostic sign of primary muscle tension dysphonia (pMTD). However, hyperfunctional patterns with phonation are also observed in typical speakers. This study tested the hypothesis that FVF posturing during quiet breathing, as measured by the curvature of FVF, could differentiate patients with pMTD from typical speakers.MethodsLaryngoscopic images were collected prospectively in 30 subjects with pMTD and 33 typical speakers. Images were acquired at the end of expiration and maximal inspiration during quiet breathing, during sustained /i/, and during loud phonation before and after a 30‐min vocal loading task. The FVF curvature (degree of concavity/convexity) was quantified using a novel curvature index (CI, >0 for hyperfunctional/convex, <0 for “relaxed”/concave) and compared between the two groups.ResultsAt end‐expiration, the pMTD group adopted a convex FVF contour, whereas the control group adopted a concave FVF contour (mean CI 0.123 [SEM 0.046] vs. −0.093 [SEM 0.030], p = 0.0002) before vocal loading. At maximal inspiration, the pMTD group had a neutral/straight FVF contour, whereas the control group had a concave FVF contour (mean CI 0.012 [SEM 0.038] vs. −0.155 [SEM 0.018], p = 0.0002). There were no statistically significant differences in FVF curvature between groups in either the sustained voiced or loud conditions. Vocal loading did not change any of these relationships.ConclusionsA hyperfunctional posture of the FVFs during quiet breathing especially at end‐expiration may be more indicative of a hyperfunctional voice disorder than supraglottic constriction during voicing.Level of Evidence3 Laryngoscope, 2023

A dominant narrative in the treatment of larynx cancer has been balancing effective oncologic therapy with the preservation of major laryngeal functions such as breathing, swallowing, and speech. The first total laryngectomy, described by Billroth in 1873, was able to maintain the airway, and, separated the pharynx from the trachea, allowing swallowing without aspiration. 1 With modern tracheoesophageal prostheses for speech, total laryngectomy provides an oncologically comprehensive operation that facilitates safe swallowing without aspiration. For this reason, total laryngectomy remains a standard of care for advanced larynx cancer and the metric by which other therapies are measured.Despite this, the permanent tracheostoma and loss of vocal cord function has motivated interest in organ preservation since the earliest days of laryngeal surgery. Partial laryngeal surgery via a 'laryngo-fissure' or external approach was attempted as early as 1834. The first report of this technique being employed for a malignancy was described by Buck in 1853. 2 In the 20th century, improved anesthetic techniques, postoperative care, and, above all, improved selection of cases suitable for the operation, improved cure rates, with Chevalier Jackson reporting a local control rate of 82% in 1927. 3 Further refinements such as the hemilaryngectomy reported by Gluck and colleagues, 4 and the supraglottic laryngectomy described by Wilfred Trotter in 1913, modified by Justo Alonso, 5 and popularized by Som, Bocca, and Ogura, followed. The most significant problem solved by these trailblazing

The amount of bone tissue present in an organism does not remain constant throughout a lifetime. Aquatic mammals generally have more dense bones than their terrestrial counterparts and there is little to no information on aquatic mammal bone development. The Florida manatee is a fully aquatic mammal which represents a good example of dense, amedullary bones. The abundance of prominent bone tissue histological features such as osteocytes, mesenchyme, and trabeculae provide a mechanism to determine how bone tissue of the same species differs due to age. The goal of this study was to evaluate key histological differences between bone samples, from manatees of various ages, in order to approximate the age using characteristics of the bone tissue. Nasal tissue including bone from manatees in the perinatal, calf, juvenile, and adult stages of development was embedded in paraffin, sectioned and stained using trichrome. The number of osteocytes was counted at 40X and 100X total magnifications. Analysis of the osteocyte quantity using a chi square test for goodness of fit shows a significant trend (p&lt;2.2*10-16) directly corresponding to the age of manatee. As the age of manatee increased, greater amounts of osteocytes were counted. Further observations indicate trabeculae are more abundant and prominent in early developmental stages (perinatal and calf) compared to the latter stages (juvenile and adult) in which mature or secondary bone is predominant. Using visual approximation, adult bone contained the largest bone volume, but showed minor signs of degradation and degeneration by osteoclast activity. These results are in agreement with the available information regarding intramembranous bone development, and successfully show a proportional relationship between age and osteocyte number. More specific data, such as the exact age of manatee and computer calculated bone volume/osteocyte lacunae count, would allow us to further model this determined correlation between bone volume and age.

Objectives: The clinical determination of vocal fold (VF) hypomobility based on laryngoscopy is subjective. Previous studies point to VF motion anomaly as the most commonly reported factor in the diagnosis of hypomobility. This study tested the hypotheses that VF angular velocities and angular range of motion (ROM) differ between the two VFs in cases of unilateral VF hypomobility.Study Design: Retrospective. Methods: Semi-automated analysis of laryngoscopic videos of 18 subjects diagnosed with unilateral VF hypomobility and 13 subjects with normal VF mobility was performed to quantify/compare the VF angular velocity and ROM between the two VFs during /i/Àsniff and laugh.Results: In the hypomobile VF group, 7 out of 15 (47%) videos with /i/Àsniff and 5 out of 8 (63%) with laugh had a statistically significant difference in the angular velocities between the VFs in either abduction or adduction. For VF ROM, 8 out of 15 (53%) /i/Àsniff videos and 4 out of 8 (50%) with laughter had a statistically significant difference between VFs. In the group without the diagnosis of VF hypomobility, 9 out of 13 subjects (69%) had no difference in VF angular velocity and ROM during either /i/Àsniff or laugh.Conclusions: Differences in VF angular velocity or ROM are measurable in a substantial subset of subjects diagnosed with unilateral VF hypomobility. Clinicians' ability to gauge VF motion goes beyond what can be extracted from frame-by-frame analysis. Other visual cues, in addition to VF angular velocity and ROM, likely contribute to the perception of unilateral VF hypomobility.