Protecting mammalian hair cells from aminoglycoside-toxicity: assessing phenoxybenzamine’s potential

Aminoglycosides (AGs) are widely used antibiotics because of their low cost and high efficacy against gram-negative bacterial infection. However, AGs are ototoxic,causing the death of sensory hair cells in the inner ear. Strategies aimed at developing or discovering agents that protect against aminoglycoside ototoxicity have focused on inhibiting apoptosis or more recently, on preventing antibiotic uptake by the hair cells. Recent screens for ototoprotective compounds using the larval zebrafish lateral line identified phenoxybenzamine as a potential protectant for aminoglycoside induced hair cell death. Here we used live imaging of FM1-43 uptake as a proxy for aminoglycoside entry, combined with hair-cell death assays to evaluate whether phenoxybenzamine can protect mammalian cochlear hair cells from the deleterious effects of the aminoglycoside antibiotic neomycin. We show that phenoxybenzamine can block FM1-43 entry into mammalian hair cells in a reversible and dose-dependent manner, but pre-incubation is required for maximal inhibition of entry. We observed differential effects of phenoxybenzamine on FM1-43 uptake in the two different types of cochlear hair cell in mammals, the outer hair cells (OHCs) and inner hair cells (IHCs).The requirement for pre-incubation and reversibility suggests an intracellular rather than an extracellular site of action for phenoxybenzamine. We also tested the efficacy of phenoxybenzamine as an otoprotective agent. In mouse cochlear explants the hair cell death resulting from 24 h exposure to neomycin was steeply dose-dependent, with 50% cell death occurring at ~230 uM for both IHC and OHC. We used 250 uM neomycin in subsequent hair-cell death assays. At 100 uM with 1 h pre-incubation, phenoxybenzamine conferred significant protection to both IHCs and OHCs, however at higher concentrations phenoxybenzamine itself showed clear signs of ototoxicity and an additive toxic effect when combined with neomycin. These data do not support the use of phenoxybenzamine as a therapeutic agent in mammalian inner ear. Our findings do share parallels with the observations from the zebrafish lateral line model but they also highlight the necessity for validation in the mammalian system and the potential for differential effects on sensory hair cells from different species, in different systems and even between cells in the same organ

A comparative investigation of the wear characteristics of a high power diode laser generated single-stage tile grout and commercial epoxy tile grout

A comparative study of a single-stage ceramic tile grout, generated using a 60 W high power diode laser (HPDL), and a commercially available tile grout has determined the wear characteristics of the two materials. Within both normal and corrosive environmental conditions, the single-stage ceramic tile grout proved to have a superior wear rate over the epoxy tile grout, 0.9 mg/cm2/h compared with 125 mg/cm2/h when in an HNO3 environment respectively. Likewise, life assessment testing revealed that the single-stage ceramic tile grout gave an increase in wear life of 4 to 42 times over the commercially available epoxy tile grout, depending upon the corrosive environment. It is believed that the economic and material benefits to be gained from the deployment of such an effective and efficient means of sealing ceramic tiles could be significant

Aminoglycoside-Induced Phosphatidylserine Externalization in Sensory Hair Cells Is Regionally Restricted, Rapid, and Reversible

The aminophospholipid phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane. During certain cellular processes, including apoptosis, PS translocates to the outer leaflet and can be labeled with externally applied annexin V, a calcium-dependent PS-binding protein. In mouse cochlear cultures, annexin V labeling reveals that the aminoglycoside antibiotic neomycin induces rapid PS externalization, specifically on the apical surface of hair cells. PS externalization is observed within ~75 s of neomycin perfusion, first on the hair bundle and then on membrane blebs forming around the apical surface. Whole-cell capacitance also increases significantly within minutes of neomycin application, indicating that blebbing is accompanied by membrane addition to the hair cell surface. PS externalization and membrane blebbing can, nonetheless, occur independently. Pretreating hair cells with calcium chelators, a procedure that blocks mechanotransduction, or overexpressing a phosphatidylinositol 4,5-biphosphate (PIP2)-binding pleckstrin homology domain, can reduce neomycin-induced PS externalization, suggesting that neomycin enters hair cells via transduction channels, clusters PIP2, and thereby activates lipid scrambling. The effects of short-term neomycin treatment are reversible. After neomycin washout, PS is no longer detected on the apical surface, apical membrane blebs disappear, and surface-bound annexin V is internalized, distributing throughout the supranuclear cytoplasm of the hair cell. Hair cells can therefore repair, and recover from, neomycin-induced surface damage. Hair cells lacking myosin VI, a minus-end directed actin-based motor implicated in endocytosis, can also recover from brief neomycin treatment. Internalized annexin V, however, remains below the apical surface, thereby pinpointing a critical role for myosin VI in the transport of endocytosed material away from the periphery of the hair cell

An emerging role for stress granules in neurodegenerative disease and hearing loss

Stress granules (SGs) are membrane-less cytosolic assemblies that form in response to stress (e.g., heat, oxidative stress, hypoxia, viral infection and UV). Composed of mRNA, RNA binding proteins and signalling proteins, SGs minimise stress-related damage and promote cell survival. Recent research has shown that the stress granule response is vital to the cochlea's response to stress. However, emerging evidence suggests stress granule dysfunction plays a key role in the pathophysiology of multiple neurodegenerative diseases, several of which present with hearing loss as a symptom. Hearing loss has been identified as the largest potentially modifiable risk factor for dementia. The underlying reason for the link between hearing loss and dementia remains to be established. However, several possible mechanisms have been proposed including a common pathological mechanism. Here we will review the role of SGs in the pathophysiology of neurodegenerative diseases and explore possible links and emerging evidence that they may play an important role in maintenance of hearing and may be a common mechanism underlying age-related hearing loss and dementia

siRNA Delivery to the Glomerular Mesangium Using Polycationic Cyclodextrin Nanoparticles Containing siRNA

There is an urgent need for new therapies that can halt or reverse the course of chronic kidney disease with minimal side-effect burden on the patient. Small interfering RNA (siRNA) nanoparticles are new therapeutic entities in clinical development that could be useful for chronic kidney disease treatment because they combine the tissue-specific targeting properties of nanoparticles with the gene-specific silencing effects of siRNA. Recent reports have emerged demonstrating that the kidney, specifically the glomerulus, is a readily accessible site for nanoparticle targeting. Here, we explore the hypothesis that intravenously administered polycationic cyclodextrin nanoparticles containing siRNA (siRNA/CDP-NPs) can be used for delivery of siRNA to the glomerular mesangium. We demonstrate that siRNA/CDP-NPs localize to the glomerular mesangium with limited deposition in other areas of the kidney after intravenous injection. Additionally, we report that both mouse and human mesangial cells rapidly internalize siRNA/CDP-NPs in vitro and that nanoparticle uptake can be enhanced by attaching the targeting ligands mannose or transferrin to the nanoparticle surface. Lastly, we show knockdown of mesangial enhanced green fluorescent protein expression in a reporter mouse strain following iv treatment with siRNA/CDP-NPs. Altogether, these data demonstrate the feasibility of mesangial targeting using intravenously administered siRNA/CDP-NPs

Drug-induced stress granule formation protects sensory hair cells in mouse cochlear explants during ototoxicity

Stress granules regulate RNA translation during cellular stress, a mechanism that is generally presumed to be protective, since stress granule dysregulation caused by mutation or ageing is associated with neurodegenerative disease. Here, we investigate whether pharmacological manipulation of the stress granule pathway in the auditory organ, the cochlea, affects the survival of sensory hair cells during aminoglycoside ototoxicity, a common cause of acquired hearing loss. We show that hydroxamate (-)-9, a silvestrol analogue that inhibits eIF4A, induces stress granule formation in both an auditory cell line and ex-vivo cochlear cultures and that it prevents ototoxin-induced hair-cell death. In contrast, preventing stress granule formation using the small molecule inhibitor ISRIB increases hair-cell death. Furthermore, we provide the first evidence of stress granule formation in mammalian hair cells in-vivo triggered by aminoglycoside treatment. Our results demonstrate that pharmacological induction of stress granules enhances cell survival in native-tissue, in a clinically-relevant context. This establishes stress granules as a viable therapeutic target not only for hearing loss but also other neurodegenerative diseases.EI:595 - Action on Hearing Loss; 091092/Z/09/Z - Wellcome Trust (Wellcome); MR/N004329/1 - RCUK | Medical Research Council (MRC)Published versio

Kentucky Labor Supply and Demand Surveys

Excerpt from the executive summary: The Center for Business and Economic Research at the University of Kentucky (CBER), along with its partners, the Survey Research Center at the University of Kentucky (UK-SRC), the Survey Research Center in the Urban Studies Institute at the University of Louisville (UL-SRC), and the Department of Economics at the University of Louisville, is pleased to present this final report on the findings of the Kentucky labor supply and demand surveys sponsored by the Kentucky Cabinet for Workforce Development. The two universities have put together a consortium including some of the best scholars in the region in the areas of labor economics, local economic development, and survey design and administration

Calixpyrroles, calixpyridinopyrroles and calixpyridines

The present invention provides calixpyrrole, calixpyridinopyrrole, and calixpyridine macrocycles, having 4, 5, 6, 7, or 8 heterocyclic rings, as well as syntheses, derivatives, conjugates, multimers, and solid supports thereof. Such macrocycles have proved to be effective and selective ion- and neutral molecule-binding agents forming supramolecular ensembles, and ion- and neutral molecule-separation agents. The macrocycles are fully meso-non-hydrogen-substituted porphyrinogens, a few molecules of which were previously known but not recognized as possessing anion- or molecule-binding properties. The binding mode is noncovalent, primarily that of hydrogen-bonding, thereby providing a new mode for liquid chromatography, that of Hydrogen Bonding Liquid Chromatography. Further useful applications of the macrocycles provided herein include environmental remediation by removal of undesired ions or neutral molecules, and removal of phosphate for kidney dialysis.Board of Regents, University of Texas Syste