Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging.

Single-cell (SC) analysis provides unique insight into individual cell dynamics and cell-to-cell heterogeneity. Here, we utilize trapped ion mobility separation coupled with dual-polarity ionization mass spectrometry imaging (MSI) to enable high-throughput in situ profiling of the SC lipidome. Multimodal SC imaging, in which dual-polarity-mode MSI is used to perform serial data acquisition runs on individual cells, significantly enhanced SC lipidome coverage.

On-tissue amidation of sialic acid with aniline for sensitive imaging of sialylated N-glycans from FFPE tissue sections via MALDI mass spectrometry.

Spatial visualization of glycans within clinical tissue samples is critical for discovery of disease-relevant glycan dysregulations. Herein, we develop an on-tissue derivatization strategy for sensitive spatial visualization of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections, based on amidation of sialic acid residues with aniline. The sialylated N-glycans were stabilized and given enhanced signal intensity owing to selective capping of a phenyl group to the sialic acid residue after aniline labeling.

An atlas of protein turnover rates in mouse tissues.

Protein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins-including those of the extracellular matrix-in a set of biologically important mouse tissues.

Quantification and molecular imaging of fatty acid isomers from complex biological samples by mass spectrometry.

Elucidating the isomeric structure of free fatty acids (FAs) in biological samples is essential to comprehend their biological functions in various physiological and pathological processes. Herein, we report a novel approach of using peracetic acid (PAA) induced epoxidation coupled with mass spectrometry (MS) for localization of the C[double bond, length as m-dash]C bond in unsaturated FAs, which enables both quantification and spatial visualization of FA isomers from biological samples. Abundant diagnostic fragment ions indicative of the C[double bond, length as m-dash]C positions were produced upon fragmentation of the FA epoxides derived from either in-solution or on-tissue PAA epoxidation of free FAs.

High-resolution magnetic resonance and mass spectrometry imaging of the human larynx.

High-resolution, noninvasive and nondestructive imaging of the subepithelial structures of the larynx would enhance microanatomic tissue assessment and clinical decision making; similarly, in situ molecular profiling of laryngeal tissue would enhance biomarker discovery and pathology readout. Towards these goals, we assessed the capabilities of high-resolution magnetic resonance imaging (MRI) and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) imaging of rarely reported paediatric and adult cadaveric larynges that contained pathologies. The donors were a 13-month-old male, a 10-year-old female with an infraglottic mucus retention cyst and a 74-year-old female with advanced polypoid degeneration and a mucus retention cyst.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections.

Proteomic and Genomic Methylation Signatures of Idiopathic Subglottic Stenosis.

Objective: Idiopathic subglottic stenosis (iSGS) is a chronic inflammatory condition that causes dyspnea and affects middle-aged women of White race and non-Latino or Hispanic ethnicity. To better characterize its phenotype and pathogenesis, we assessed the proteomic and genomic methylation signatures of subglottic tissue collected from iSGS patients compared to controls.

Study Design: Molecular analysis of clinical biospecimens.

Dynamics of vitamin A uptake, storage, and utilization in vocal fold mucosa.

Objective: Extrahepatic vitamin A is housed within organ-specific stellate cells that support local tissue function. These cells have been reported in the vocal fold mucosa (VFM) of the larynx; however, it is unknown how vitamin A reaches and is disseminated among VFM target cells, how VFM storage and utilization vary as a function of total body stores, and how these parameters change in the context of pathology. Therefore, in this study, we investigated fundamental VFM vitamin A uptake and metabolism.

Reversal of Vocal Fold Mucosal Fibrosis Using siRNA against the Collagen-Specific Chaperone Serpinh1.

Vocal fold (VF) mucosal fibrosis results in substantial voice impairment and is recalcitrant to current treatments. To reverse this chronic disorder, anti-fibrotic therapies should target the molecular pathology of aberrant collagen accumulation in the extracellular matrix. We investigated the therapeutic potential of siRNA against Serpinh1, a collagen-specific chaperone that enables cotranslational folding and assembly of procollagens in the endoplasmic reticulum.

Differentiated fibrocytes assume a functional mesenchymal phenotype with regenerative potential.

Fibrocytes (FCs) are hematopoietic lineage cells that migrate to sites of injury, transition to a mesenchymal phenotype, and help to mediate wound repair. Despite their relevance to human fibrotic disorders, there are few data characterizing basic FC biology. Herein, using proteomic, bioenergetic, and bioengineering techniques, we conducted deep phenotypic characterization of differentiating and mature FCs.

Serum retinyl esters are positively correlated with analyzed total liver vitamin A reserves collected from US adults at time of death.

Background: Minimal human data exist on liver vitamin A (VA) compared with serum biomarkers. Cutoffs of 5% and 10% total serum VA as retinyl esters (REs) suggest a VA intoxication diagnosis.

Objectives: We compared total liver VA reserves (TLRs) with the percentage of total serum VA as REs to evaluate hypervitaminosis with the use of US adult autopsy samples.

Practice variations in voice treatment selection following vocal fold mucosal resection.

Objectives/hypothesis: To characterize initial voice treatment selection following vocal fold mucosal resection in a Medicare population.

Study Design: Retrospective analysis of a large, nationally representative Medicare claims database.

Methods: Patients with > 12 months of continuous Medicare coverage who underwent a leukoplakia- or cancer-related vocal fold mucosal resection (index) procedure during calendar years 2004 to 2009 were studied.

Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds.

Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared with those obtained from experimentally naïve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and α-smooth muscle actin synthesis, and enhanced contractile function.

Protein turnover during in vitro tissue engineering.

Repopulating acellular biological scaffolds with phenotypically appropriate cells is a promising approach for regenerating functional tissues and organs. Under this tissue engineering paradigm, reseeded cells are expected to remodel the scaffold by active protein synthesis and degradation; however, the rate and extent of this remodeling remain largely unknown. Here, we present a technique to measure dynamic proteome changes during in vitro remodeling of decellularized tissue by reseeded cells, using vocal fold mucosa as the model system.

Bioengineered vocal fold mucosa for voice restoration.

Patients with voice impairment caused by advanced vocal fold (VF) fibrosis or tissue loss have few treatment options. A transplantable, bioengineered VF mucosa would address the individual and societal costs of voice-related communication loss. Such a tissue must be biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration and physiologically capable of maintaining a barrier against the airway lumen.

Proteomic analysis of naturally-sourced biological scaffolds.

A key challenge to the clinical implementation of decellularized scaffold-based tissue engineering lies in understanding the process of removing cells and immunogenic material from a donor tissue/organ while maintaining the biochemical and biophysical properties of the scaffold that will promote growth of newly seeded cells. Current criteria for evaluating whole organ decellularization are primarily based on nucleic acids, as they are easy to quantify and have been directly correlated to adverse host responses. However, numerous proteins cause immunogenic responses and thus should be measured directly to further understand and quantify the efficacy of decellularization.

Microarray-based characterization of differential gene expression during vocal fold wound healing in rats.

The vocal fold (VF) mucosa confers elegant biomechanical function for voice production but is susceptible to scar formation following injury. Current understanding of VF wound healing is hindered by a paucity of data and is therefore often generalized from research conducted in skin and other mucosal systems. Here, using a previously validated rat injury model, expression microarray technology and an empirical Bayes analysis approach, we generated a VF-specific transcriptome dataset to better capture the system-level complexity of wound healing in this specialized tissue.

Clinical features and surgical outcomes following closed reduction of arytenoid dislocation.

Importance: Arytenoid dislocation is a rare condition characterized by vocal fold immobility and is easily mistaken as recurrent laryngeal nerve paralysis.

Objective: To describe the presenting features, multimodal diagnostic evaluation, and surgical outcomes following closed reduction (CR) of arytenoid dislocation.

Design, Setting, And Participants: Prospective case series at a single academic medical center.

Interspecies comparison of stellate cell-containing macula flavae and vitamin A storage in vocal fold mucosa.

The macula flavae (MF), populated by vitamin A-storing stellate cells (SCs), are believed to play a fundamental role in development, maintenance and repair of the vocal fold (VF) mucosa; however, to date, they have mostly been examined in observational human cadaver studies. Here, we conducted an interspecies comparison of MF and SC phenotype, as well as vitamin A quantification and localization, in human, pig, dog, rabbit and rat VF mucosae. MF containing vitamin A-positive SCs were only identified in human and rat specimens.

Correlation between Reflux and multichannel intraluminal impedance pH monitoring in untreated volunteers.

Objectives/hypothesis: Although probable causative agents have been identified (e.g., refluxate components, tobacco smoke), the definitive mechanism for inflammation-related laryngeal mucosal damage remains elusive.

TGF-β3 modulates the inflammatory environment and reduces scar formation following vocal fold mucosal injury in rats.

Transforming growth factor (TGF)-β1 and TGF-β3 have been reported to exert differential effects on wound healing, and possibly even account for tissue-specific differences in scar formation. Scarring is particularly detrimental in the vocal fold mucosa (VFM), where destruction of the native extracellular matrix causes irreparable biomechanical changes and voice impairment. Here, in a series of in vitro and in vivo experiments, we identified differences in TGF-β1 and TGF-β3 transcription and immunolocalization to various cell subpopulations in naïve and injured rat VFM, compared with oral mucosa (which undergoes rapid healing with minimal scar) and skin (which typically heals with scar).

Proteomic analysis of a decellularized human vocal fold mucosa scaffold using 2D electrophoresis and high-resolution mass spectrometry.

Natural biologic scaffolds for tissue engineering are commonly generated by decellularization of tissues and organs. Despite some preclinical and clinical success, in vivo scaffold remodeling and functional outcomes remain variable, presumably due to the influence of unidentified bioactive molecules on the scaffold-host interaction. Here, we used 2D electrophoresis and high-resolution mass spectrometry-based proteomic analyses to evaluate decellularization effectiveness and identify potentially bioactive protein remnants in a human vocal fold mucosa model.

Nonlinear dynamic-based analysis of severe dysphonia in patients with vocal fold scar and sulcus vocalis.

Objective: The primary goal of this study was to evaluate a nonlinear dynamic approach to the acoustic analysis of dysphonia associated with vocal fold scar and sulcus vocalis.

Study Design: Case-control study.

Methods: Acoustic voice samples from scar/sulcus patients and age-/sex-matched controls were analyzed using correlation dimension (D2) and phase plots, time-domain based perturbation indices (jitter, shimmer, signal-to-noise ratio [SNR]), and an auditory-perceptual rating scheme.