Faculty Bibliography

Motivation/UNASSIGNED:Shotgun DNA sequencing provides sensitive detection of all 182 HPV types in tissue and body fluid. However, existing computational methods either produce false positives misidentifying HPV types due to shared sequences among HPV, human, and prokaryotes, or produce false negative since they identify HPV by assembled contigs requiring large abundant of HPV reads. Results/UNASSIGNED:We designed HPViewer with two custom HPV reference databases masking simple repeats and homology sequences respectively and one homology distance matrix to hybridize these two databases. It directly identified HPV from short DNA reads rather than assembled contigs. Using 100,100 simulated samples, we revealed that HPViewer was robust for samples containing either high or low number of HPV reads. Using 12 shotgun sequencing samples from respiratory papillomatosis, HPViewer was equal to VirusTAP, and Vipie and better than HPVDetector with the respect to specificity and was the most sensitive method in the detection of HPV types 6 and 11. We demonstrated that contigs-based approaches had disadvantages of detection of HPV. In 1,573 sets of metagenomic data from 18 human body sites, HPViewer identified 104 types of HPV in a body-site associated pattern and 89 types of HPV co-occurring in one sample with other types of HPV. We demonstrated HPViewer was sensitive and specific for HPV detection in metagenomic data. Availability/UNASSIGNED:HPViewer can be accessed at https://github.com/yuhanH/HPViewer/. Contact/UNASSIGNED:Zhiheng.pei@nyumc.org. Supplementary information/UNASSIGNED:Supplementary data are available at Bioinformatics online.

OBJECTIVES/HYPOTHESIS/OBJECTIVE:Our laboratory and others hypothesized that Smad3 is a principle mediator of the fibrotic phenotype in the vocal folds (VFs), and we further posited that alteration of Smad3 expression through short interfering (si)RNA holds therapeutic promise, yet delivery remains challenging. To address this issue, we employed a novel synthetic oligomer, lipitoid, complexed with siRNA to improve stability and cellular uptake with the goal of increased efficiency of RNA-based therapeutics. STUDY DESIGN/METHODS:In vitro study and in vivo animal model. METHODS:In vitro, lipitoid cytotoxicity was quantified via colorimetric and LIVE/DEAD assays in immortalized human VF fibroblasts and primary rabbit VF fibroblasts. In addition, optimal incubation interval and solution for binding siRNA to lipitoid for intracellular delivery were determined. In vivo, a rabbit model of VF injury was employed to evaluate Smad3 knockdown following locally injected lipitoid-complexed siRNA. RESULTS:In vitro, lipitoid did not confer additional toxicity compared to commercially available reagents. In addition, 20-minute incubation in 1× phosphate-buffered saline resulted in maximal Smad3 knockdown. In vivo, Smad3 expression increased following VF injury. This response was significantly reduced in injured VFs at 4 and 24 hours following injection (P = .035 and .034, respectively). CONCLUSIONS:The current study is the first to demonstrate targeted gene manipulation in the VFs as well as the potential utility of lipitoid for localized delivery of genetic material in vivo. Ideally, these data will serve as a platform for future investigation regarding the functional implications of therapeutic gene manipulation in the VFs. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2017.

OBJECTIVES/HYPOTHESIS: Although the primary goal of medialization laryngoplasty is to improve glottic closure, implant placement is also likely to alter the biomechanical properties of the vocal fold (VF). We sought to employ novel, nanoscale technology to quantify these properties following medialization based on the hypothesis that different medialization materials will likely yield differential biomechanical effects. STUDY DESIGN: Ex vivo. METHODS: Nine pig larynges were divided into three groups: control, Silastic (Dow Corning, Midland, Michigan, U.S.A.) block medialization, or Gore-Tex (W.L. Gore & Associates, Newark, Delaware) medialization. Laryngoplasty was performed on excised, intact larynges. The larynges were then bisected in the sagittal plane and each subjected to dynamic nanomechanical analysis (nano-DMA) at nine locations using a 250-mum flat-tip punch and frequency sweep-load profile across the free edge of the VF and inferiorly along the conus elasticus. RESULTS: Silastic block and Gore-Tex implant introduced increased storage and loss moduli. Overall, storage moduli mean (maximum) increased from 38 kilopascals (kPa) (119) to 72 kPa (422) and 129 kPa (978) in control, Gore-Tex, and Silastic implants, respectively. Similarly, loss moduli increased from 13 kPa (43) to 22 kPa (201) and 31 kPa (165), respectively. Moduli values varied widely by location in the Silastic block and Gore-Tex groups. At the free VF edge, mean (maximum) storage moduli were lowest in the Gore-Tex group, 20 kPa (44); compared to control, 34.5 kPa (86); and Silastic, 157.9 kPa (978), with similar loss and complex moduli trends. CONCLUSION: Medialization laryngoplasty altered VF structure biomechanical properties; Silastic and Gore-Tex implants differentially impact these properties. LEVEL OF EVIDENCE: NA. Laryngoscope, 2017.

OBJECTIVES/HYPOTHESIS: A secure airway is critical to study obstructive disorders of the larynx and trachea in preclinical models. Tracheostomy has been described in rabbits, swine, canines, and other mammals using tracheostomy tubes or permanent stomas. No studies specifically evaluated morbidity and mortality associated with these models, and existing studies using tracheostomy make little mention of tracheostomy-related complications. We assessed the management, complications, and mortality associated with tracheostomy in a rabbit model that has recently gained significant attention. STUDY DESIGN: In vivo. METHODS: Twenty-two female rabbits underwent tubeless tracheotomy. Rabbits were monitored hourly for the first 8 hours, with progressively increasing intervals between evaluations up to 7 days. A suctioning and tracheal moisture protocol was employed, and animals with signs of crusting or impending airway compromise underwent therapeutic bronchoscopy. RESULTS: Nine of 22 (41%) rabbits succumbed to tracheostomy-related complications, ranging from 1 to 7 days after tracheotomy. The experiment consisted of two study groups. The preliminary group of 10 rabbits studied over a 4-day period had 40% mortality. After implementing modified preventive therapy guidelines and a new humidification system, the second group of 12 rabbits studied over a 7-day period had 42% mortality. Average time to unrecoverable complication was 2.2 days (median = 2 days). Cause of death was airway obstruction in four animals and respiratory depression in three animals, and two animals were found unresponsive. CONCLUSION: Tracheostomy in preclinical rabbit models should be temporally limited, and investigators should anticipate tracheostomy-related complications during study design. LEVEL OF EVIDENCE: NA. Laryngoscope, 2017.

OBJECTIVES/HYPOTHESIS: Given that the vocal folds are active organs of respiration, reports of dyspnea in the context of glottic insufficiency are not uncommon. We hypothesize that improved glottal closure via framework surgery or vocal fold augmentation improves dyspnea symptoms. STUDY DESIGN: Retrospective review. METHODS: Charts of patients undergoing procedures to correct glottal insufficiency, either via vocal fold augmentation (VFA) or medialization laryngoplasty (ML) between December 2012 and September 2015 were reviewed (n = 189). Modified Borg Dyspnea Scale (MBDS) and Modified Medical Research Council Dyspnea Scale (MMRCDS) data were collected before and after intervention. Age, body mass index (BMI), and sex, as well as pulmonary and cardiac comorbidities were considered. Subgroup analysis was performed on individuals with subjective dyspnea prior to intervention. RESULTS: For the entire cohort, differences in the MMRCDS and MBDS were not statistically different pre- and postintervention (P = .20 and P = .12, respectively). Patients with BMI <30 experienced more improvement on the MBDS (P = .03). Both the MMRCDS and MMBDS improved post-procedure (P = .001 and P = .001, respectively) in patients reporting dyspnea prior to intervention. CONCLUSIONS: Patients with glottic insufficiency and dyspnea prior to intervention to improve glottic closure had a significant reduction in dyspnea following treatment. Conversely, subjects without complaints of dyspnea prior to intervention had variable outcomes with regard to dyspnea symptoms. Additionally, based on data from the entire cohort, VFA or ML did not worsen dyspnea symptoms. These data may assist in counseling and/or selection of patients considered for procedures to improve glottic closure. LEVEL OF EVIDENCE: 4 Laryngoscope, 2017.

OBJECTIVES/HYPOTHESIS: To describe the distribution of recurrent respiratory papillomatosis (RRP) lesions across 21 laryngeal anatomic regions in previously untreated patients at initial presentation to provide insight regarding the natural history of RRP. STUDY DESIGN: Multi-institutional, retrospective case series. METHODS: Initial laryngoscopic examination videos of 83 previously untreated patients with adult-onset RRP were reviewed. Papilloma locations were recorded using a 21-region laryngeal schematic. Multivariate analyses by anatomic subsite were conducted for the entire population and for subgroups stratified by sex, age, and proton pump inhibitor (PPI) usage. Heat maps were generated, hierarchically color coding the anatomic distribution of disease. RESULTS: In this cohort, RRP was most likely to occur on the true vocal folds (TVFs) and anterior commissure (P < .0001, odds ratio [OR]: 7.02); within the TVFs, the membranous vocal folds (MVFs) were most likely to be affected (P < .0001, OR: 3.56). The cohort was predominantly male (80.7%); males had a higher average number of affected sites (P = .005) and were more likely to have lesions in any laryngeal subsite (P < .0001, OR: 2.88,) compared to females. PPI users were more likely than nonusers to have disease in any laryngeal subsite (P = .0037, OR: 1.62), particularly in the posterior and subglottic regions (P = .0061, OR: 2.53). Age was not correlated with lesion prevalence or distribution. CONCLUSIONS: In untreated patients presenting to three laryngology clinics, the MVFs were most likely to be affected by RRP. Males had more anatomic sites affected by papilloma than females. The influence of PPI use on RRP distribution warrants further investigation. LEVEL OF EVIDENCE: 4. Laryngoscope, 2017.

Due to their anatomical location, vocal folds are highly susceptible to injury from external and internal stressors that can lead to irreversible damage and changes in function. As the structure and composition of the vocal folds are heavily linked to their unique function, we hypothesize that a vocal fold-derived extracellular matrix (ECM) would be the ideal scaffold in a regenerative approach to vocal fold repair. Our group has previously described a porcine-derived vocal fold lamina propria ECM (VFLP-ECM)¹. In order to optimize the delivery modality of the VFLP-ECM, we have developed an injectable hydrogel form of the ECM scaffold and have studied the effects of tissue specificity using human vocal fold fibroblasts (hVFF) and human peripheral blood-derived macrophages (hPB-Macrophages). Both cell types play unique roles during the inflammatory and wound healing response at the site of vocal fold injury. In the present study, we compare VFLP-ECM with other ECM hydrogels (such as collagen, heart, bladder) in their ability to activate and modify gene expression of hVFFs and hPB-macrophages. This information will help us tailor the VFLP-ECM hydrogel to mod-ulate the environment present during vocal fold injury

Importance/UNASSIGNED:Compromised cough effectiveness is correlated with dysphagia and aspiration. Glottic insufficiency likely yields decreased cough strength and effectiveness. Although vocal fold augmentation favorably affects voice and likely improves cough strength, few data exist to support this hypothesis. Objective/UNASSIGNED:To assess whether vocal fold augmentation improves peak airflow measurements during maximal-effort cough following augmentation. Design, Setting, and Participants/UNASSIGNED:This case series study was conducted in a tertiary, academic laryngology clinic. Participants included 14 consecutive individuals with glottic insufficiency due to vocal fold paralysis, which was diagnosed via videostrobolaryngoscopy as a component of routine clinical examination. All participants who chose to proceed with augmentation were considered for the study whether office-based or operative augmentation was planned. Postaugmentation data were collected only at the first follow-up visit, which was targeted for 14 days after augmentation but varied on the basis of participant availability. Data were collected from June 5, 2014, to October 1, 2015. Data analysis took place between October 2, 2015, and March 3, 2017. Main Outcomes and Measures/UNASSIGNED:Peak airflow during maximal volitional cough was quantified before and after vocal fold augmentation. Participants performed maximal coughs, and peak expiratory flow during the maximal cough was captured according to American Thoracic Society guidelines. Results/UNASSIGNED:Among the 14 participants (7 men and 7 women), the mean (SD) age was 62 (18) years. Three types of injectable material were used for vocal fold augmentation: carboxymethylcellulose in 5 patients, hyaluronic acid in 5, and calcium hydroxylapatite in 4. Following augmentation, cough strength increased in 11 participants and decreased cough strength was observed in 3. Peak airflow measurements during maximal cough varied from a decrease of 40 L/min to an increase of 150 L/min following augmentation. When preaugmentation and postaugmentation peak airflow measurements were compared, the median improvement was 50 L/min (95% CI, 10-75 L/min; P = .01). Immediate peak airflow measurements during cough collected within 30 minutes of augmentation varied when compared with measurements collected at follow-up (103-380 vs 160-390 L/min). Conclusions and Relevance/UNASSIGNED:Peak airflow during maximal cough may improve with vocal fold augmentation. Additional assessment and measurements are needed to further delineate which patients will benefit most regarding their cough from vocal fold augmentation.

OBJECTIVE: Recent reports highlight the efficacy of small interfering RNA (siRNA) targeting SMAD3 to regulate transforming growth factor beta (TGF-beta)-mediated fibroplasia in vocal fold fibroblasts. The current study sought to investigate SMAD3 expression during wound healing in vivo and quantify the downstream transcriptional events associated with SMAD3 knockdown in vitro. STUDY DESIGN: In vivo and in vitro. METHODS: Unilateral vocal fold injury was created in a rabbit model. SMAD3 and SMAD7 mRNA expression was quantified at 1 hour and 1, 3, 7, 14, 30, 60, and 90 days following injury. In vitro, multi-gene analysis technology was employed in our immortalized human vocal-fold fibroblast cell line following TGF-beta1 stimulation +/- SMAD3 knockdown across time points. RESULTS: SMAD3 mRNA expression increased following injury; upregulation was significant at 3 and 7 days compared to control (both P < 0.001). SMAD7 mRNA was also upregulated at 3, 7, and 14 days (P = 0.02, P < 0.001, and P < 0.001, respectively). In vitro, SMAD3 knockdown reduced the expression of multiple profibrotic, TGF-beta signaling, and extracellular matrix metabolism genes at 6 and 24 hours following TGF-beta1 stimulation. CONCLUSION: Cumulatively, these data support SMAD3 as a potential master regulator of TGF-beta-mediated fibrosis. SMAD3 transcription peaked 7 days following injury. Multi-gene analysis indicated that the therapeutic effectiveness of SMAD3 knockdown may be related to regulation of downstream mediators of fibroplasia and altered TGF-beta signaling. LEVEL OF EVIDENCE: NA. Laryngoscope, 2017.

OBJECTIVES/HYPOTHESIS: NR4A1 was recently identified as an endogenous inhibitor of transforming growth factor (TGF)-beta-induced fibrosis, and the role of this nuclear receptor has not been elucidated in tissue health or the response to injury in the vocal folds. Given the clinical implications of vocal fold fibrosis, we investigated NR4A1 expression during vocal fold wound healing in vivo and the regulatory roles of NR4A1 on vocal fold fibroblasts (VFFs) in vitro with the ultimate goal of developing targeted therapies for this challenging patient population. STUDY DESIGN: In vivo and in vitro. METHODS: In vivo, the temporal pattern of NR4A1 mRNA expression was quantified following rat vocal fold injury. In vitro, the role of NR4A1 on TGF-beta1-mediated transcription of genes underlying fibrosis as well as myofibroblast differentiation and collagen gel contraction was quantified in our human VFF line. Small interfering RNA was employed to alter NR4A1 expression to further elucidate this complex system. RESULTS: Nr4a1 mRNA increased 1 day after injury and peaked at 7 days. Knockdown of NR4A1 resulted in upregulation of COL1A1 and TGF-beta1, with TGF-beta1 stimulation (both P < .001) in VFFs. NR4A1 knockdown also resulted in increased alpha-smooth muscle actin-positive cells (P = .013) and contraction (P = .002) in response to TGF-beta1. CONCLUSIONS: NR4A1 has not been described in vocal fold health or disease. Upregulation of TGF-beta following vocal fold injury was concurrent with increased NR4A1 expression. These data provide a foundation for the development of therapeutic strategies given persistent TGF-beta signaling in vocal fold fibrosis. LEVEL OF EVIDENCE: N/A Laryngoscope, 2017.