Nonquantal transmission at the vestibular hair cell-calyx synapse: KLV currents modulate fast electrical and slow K+ potentials.

Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal called a calyx. The vestibular hair cell-calyx synapse supports a mysterious form of electrical transmission that does not involve gap junctions, termed nonquantal transmission (NQT). The NQT mechanism is thought to involve the flow of ions from the presynaptic hair cell to the postsynaptic calyx through low-voltage-activated channels driven by changes in cleft [K+] as K+ exits the hair cell. However, this hypothesis has not been tested with a quantitative model and the possible role of an electrical potential in the cleft has remained speculative. Here, we present a computational model that captures experimental observations of NQT and identifies features that support the existence of an electrical potential (ϕ) in the synaptic cleft. We show that changes in cleft ϕ reduce transmission latency and illustrate the relative contributions of both cleft [K+] and ϕ to the gain and phase of NQT. We further demonstrate that the magnitude and speed of NQT depend on calyx morphology and that increasing calyx height reduces action potential latency in the calyx afferent. These predictions are consistent with the idea that the calyx evolved to enhance NQT and speed up vestibular signals that drive neural circuits controlling gaze, balance, and orientation.

The Remarkable Outer Hair Cell: Proceedings of a Symposium in Honour of W. E. Brownell.

In 1985, Bill Brownell and colleagues published the remarkable observation that cochlear outer hair cells (OHCs) express voltage-driven mechanical motion: electromotility. They proposed OHC electromotility as the mechanism for the elusive "cochlear amplifier" required to explain the sensitivity of mammalian hearing. The finding and hypothesis stimulated an explosion of experiments that have transformed our understanding of cochlear mechanics and physiology, the evolution of hair cell structure and function, and audiology. Here, we bring together examples of current research that illustrate the continuing impact of the discovery of OHC electromotility.

Caregiver Experiences With Oral Bilingualism in Children Who Are Deaf and Hard of Hearing in the United States: Impact on Child Language Proficiency.

PURPOSE: Best practices recommend promoting the use of the home language and allowing caregivers to choose the language(s) that they want to use with their child who is deaf or hard of hearing (DHH). We examined whether Spanish-speaking caregivers of children who are DHH receive professional recommendations on oral bilingualism that follow best practices. We also assessed whether professional recommendations, caregiver beliefs, and language practices had an impact on child language(s) proficiency. METHOD: Sixty caregivers completed a questionnaire on demographic questions, language(s) use and recommendations, beliefs on bilingualism, and child language proficiency measures in English, Spanish, and American Sign Language (ASL). Professional recommendations on oral bilingualism were reported descriptively, and linear regression was used to identify the predictors of child language(s) proficiency. RESULTS: We found that only 23.3% of the caregivers were actively encouraged to raise their child orally bilingual. Language practices predicted child proficiency in each language (English, Spanish, and ASL), but professional recommendations and caregiver beliefs did not. CONCLUSIONS: Our results revealed that most caregivers received recommendations that do not follow current best practices. Professional training is still needed to promote bilingualism and increase cultural competence when providing services to caregivers who speak languages different from English. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.21644846.

Structural and functional diversity of mitochondria in vestibular/cochlear hair cells and vestibular calyx afferents.

Mitochondria supply energy in the form of ATP to drive a plethora of cellular processes. In heart and liver cells, mitochondria occupy over 20% of the cellular volume and the major need for ATP is easily identifiable - i.e., to drive cross-bridge recycling in cardiac cells or biosynthetic machinery in liver cells. In vestibular and cochlear hair cells the overall cellular mitochondrial volume is much less, and mitochondria structure varies dramatically in different regions of the cell. The regional demands for ATP and cellular forces that govern mitochondrial structure and localization are not well understood. Below we review our current understanding of the heterogeneity of form and function in hair cell mitochondria. A particular focus of this review will be on regional specialization in vestibular hair cells, where large mitochondria are found beneath the cuticular plate in close association with the striated organelle. Recent findings on the role of mitochondria in hair cell death and aging are covered along with potential therapeutic approaches. Potential avenues for future research are discussed, including the need for integrated computational modeling of mitochondrial function in hair cells and the vestibular afferent calyx.

Outer hair cell electromechanics as a problem in soft matter physics: Prestin, the membrane and the cytoskeleton.

The electromechanical coupling exhibited by cochlear outer hair cells is a remarkable biophysical phenomenon. These specialized cells generate forces at acoustic frequencies and enable high-frequency hearing in mammals. While there has been significant progress since the discovery of electromotility - including the discovery of the motor protein prestin - we still do not have a clear picture of how electromotility works. A particularly vexing problem is how forces, generated by a membrane-based motor, are rapidly transmitted to the underlying cytoskeleton to enable force generation on the microsecond time scales required for amplification of acoustic signals. Here we approach the problem of electromotility from the perspective of soft matter physics in light of recent ultrastructural findings from 3D electron tomography studies on outer hair cells immobilized by high-pressure freezing. We then survey our understanding of prestin-membrane and prestin-cytoskeletal interactions in the context recently published cryoelectron microscopy (cryo-EM) structures of prestin. This will lead to the proposal of a new conceptual model of electromotility consistent with conformational states observed in the pillar proteins and actin filaments. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.

Utilization of Thiopurine Metabolites and Allopurinol in Pediatric Acute Lymphoblastic Leukemia: Consideration for an Algorithmic Approach.

Persistently elevated absolute neutrophil counts during maintenance for acute lymphoblastic leukemia is a risk factor for relapse and may be related to wild-type thiopurine methyltransferase activity and overly efficient shunting of 6-mercaptopurine to hepatotoxic metabolites (6-methylmercaptopurine nucleotides), leading to low 6-thioguanine nucleotides. 6-mercaptopurine is also metabolized by xanthine oxidase, and therefore allopurinol, an inhibitor of xanthine oxidase, allows for increased 6-thioguanine nucleotides and decreased 6-methylmercaptopurine nucleotide. Here, we report our experience with allopurinol for persistently elevated absolute neutrophil count or hepatotoxicity and suggest an algorithmic approach for checking thiopurine metabolites and initiating allopurinol in acute lymphoblastic leukemia maintenance.

Supporting Equity and Inclusion of Deaf and Hard-of-Hearing Individuals in Professional Organizations.

Disability is an important and often overlooked component of diversity. Individuals with disabilities bring a rare perspective to science, technology, engineering, mathematics, and medicine (STEMM) because of their unique experiences approaching complex issues related to health and disability, navigating the healthcare system, creatively solving problems unfamiliar to many individuals without disabilities, managing time and resources that are limited by physical or mental constraints, and advocating for themselves and others in the disabled community. Yet, individuals with disabilities are underrepresented in STEMM. Professional organizations can address this underrepresentation by recruiting individuals with disabilities for leadership opportunities, easing financial burdens, providing equal access, fostering peer-mentor groups, and establishing a culture of equity and inclusion spanning all facets of diversity. We are a group of deaf and hard-of-hearing (D/HH) engineers, scientists, and clinicians, most of whom are active in clinical practice and/or auditory research. We have worked within our professional societies to improve access and inclusion for D/HH individuals and others with disabilities. We describe how different models of disability inform our understanding of disability as a form of diversity. We address heterogeneity within disabled communities, including intersectionality between disability and other forms of diversity. We highlight how the Association for Research in Otolaryngology has supported our efforts to reduce ableism and promote access and inclusion for D/HH individuals. We also discuss future directions and challenges. The tools and approaches discussed here can be applied by other professional organizations to include individuals with all forms of diversity in STEMM.

3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.

Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features.

Diflunisal inhibits prestin by chloride-dependent mechanism.

The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.

A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging.

An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5'-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (-control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z' = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

SDF-1α stiffens myeloma bone marrow mesenchymal stromal cells through the activation of RhoA-ROCK-Myosin II.

Multiple myeloma (MM) is a B lymphocyte malignancy that remains incurable despite extensive research efforts. This is due, in part, to frequent disease recurrences associated with the persistence of myeloma cancer stem cells (mCSCs). Bone marrow mesenchymal stromal cells (BMSCs) play critical roles in supporting mCSCs through genetic or biochemical alterations. Previously, we identified mechanical distinctions between BMSCs isolated from MM patients (mBMSCs) and those present in the BM of healthy individuals (nBMSCs). These properties of mBMSC contributed to their ability to preferentially support mCSCs. To further illustrate mechanisms underlying the differences between mBMSCs and nBMSCs, here we report that (i) mBMSCs express an abnormal, constitutively high level of phosphorylated Myosin II, which leads to stiffer membrane mechanics, (ii) mBMSCs are more sensitive to SDF-1α-induced activation of MYL2 through the G(i./o)-PI3K-RhoA-ROCK-Myosin II signaling pathway, affecting Young's modulus in BMSCs and (iii) activated Myosin II confers increased cell contractile potential, leading to enhanced collagen matrix remodeling and promoting the cell-cell interaction between mCSCs and mBMSCs. Together, our findings suggest that interfering with SDF-1α signaling may serve as a new therapeutic approach for eliminating mCSCs by disrupting their interaction with mBMSCs.

A three-dimensional co-culture model of the aortic valve using magnetic levitation.

The aortic valve consists of valvular interstitial cells (VICs) and endothelial cells (VECs). While these cells are understood to work synergistically to maintain leaflet structure and valvular function, few co-culture models of these cell types exist. In this study, aortic valve co-cultures (AVCCs) were assembled using magnetic levitation and cultured for 3 days. Immunohistochemistry and quantitative reverse-transcriptase polymerase chain reaction were used to assess the maintenance of cellular phenotype and function, and the formation of extracellular matrix. AVCCs stained positive for CD31 and α-smooth muscle actin (αSMA), demonstrating that the phenotype was maintained. Functional markers endothelial nitric oxide synthase (eNOS), von Willebrand factor (VWF) and prolyl-4-hydroxylase were present. Extracellular matrix components collagen type I, laminin and fibronectin also stained positive, with reduced gene expression of these proteins in three dimensions compared to two dimensions. Genes for collagen type I, lysyl oxidase and αSMA were expressed less in AVCCs than in 2-D cultures, indicating that VICs are quiescent. Co-localization of CD31 and αSMA in the AVCCs suggests that endothelial-mesenchymal transdifferentiation might be occurring. Differences in VWF and eNOS in VECs cultured in two and three dimensions also suggests that the AVCCs possibly have anti-thrombotic potential. Overall, a co-culture model of the aortic valve was designed, and serves as a basis for future experiments to understand heart valve biology.

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis.

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

Assembly of a three-dimensional multitype bronchiole coculture model using magnetic levitation.

A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle α-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.

Selective cell-surface labeling of the molecular motor protein prestin.

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

Thermoresponsive, in situ cross-linkable hydrogels based on N-isopropylacrylamide: fabrication, characterization and mesenchymal stem cell encapsulation.

Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently cross-linkable moieties in the macromers. The effects of the macromer end group, acrylate or methacrylate, and the fabrication conditions on the degradative and swelling properties of the hydrogels were investigated. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture medium at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over 8 weeks, with the methacrylated hydrogels showing greater swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture medium under the same conditions showed lower swelling compared with phosphate-buffered saline. The interplay between chemical cross-linking and thermally induced phase separation affected the swelling characteristics of the hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over 3 weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and ß-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering.

A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells.

It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2 x 10(8) primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical microdevice, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.

Three-dimensional tissue culture based on magnetic cell levitation.

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.