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

Launching the T-cell-lineage developmental programme

Multipotent blood progenitor cells enter the thymus and begin a protracted differentiation process in which they gradually acquire T-cell characteristics while shedding their legacy of developmental plasticity. Notch signalling and basic helix-loop-helix E-protein transcription factors collaborate repeatedly to trigger and sustain this process throughout the period leading up to T-cell lineage commitment. Nevertheless, the process is discontinuous with separately regulated steps that demand roles for additional collaborating factors. This Review discusses new evidence on the coordination of specification and commitment in the early T-cell pathway; effects of microenvironmental signals; the inheritance of stem-cell regulatory factors; and the ensemble of transcription factors that modulate the effects of Notch and E proteins, to distinguish individual stages and to polarize T-cell-lineage fate determination

Brief communication: landslide motion from cross correlation of UAV-derived morphological attributes

Unmanned aerial vehicles (UAVs) can provide observations of high spatio-temporal resolution to enable operational landslide monitoring. In this research, the construction of digital elevation models (DEMs) and orthomosaics from UAV imagery is achieved using structure-from-motion (SfM) photogrammetric procedures. The study examines the additional value that the morphological attribute of "openness", amongst others, can provide to surface deformation analysis. Image-cross-correlation functions and DEM subtraction techniques are applied to the SfM outputs. Through the proposed integrated analysis, the automated quantification of a landslide's motion over time is demonstrated, with implications for the wider interpretation of landslide kinematics via UAV surveys

Comparison between the Parks Medical Doppler and the Mano Médical Vet BP Doppler for recording indirect systemic blood pressure in conscious dogs

Background: The acquisition of systemic blood pressure (SBP) provides valuable information regarding cardiovascular function and tissue perfusion in human and veterinary species.Aim: To evaluate the agreement between Parks Medical Doppler (PMD) and Mano Médical Vet BP (MMVBP) Doppler for assessing SBP in conscious dogs.Methods: 40 client-owned dogs were prospectively enrolled; SBP measurements were acquired by a single operator using the PMD and then the MMVBP. The mean of five consecutive measurements for each device was classified according to target organ damage (TOD) risk scores (1: <140 mmHg; 2: 140–160 mmHg; 3: 160–180 mmHg; 4: ≥ 180 mmHg).Results: Total mean SBP for the devices was not statistically different (p = 0.77). However, the Bland–Altman analysis revealed wide limits of agreement (LoA), with MMVBP slightly underestimating SBP compared to PMD (bias = −0.6 mmHg, 95% LoA: −26.3 to 25.09). Both devices correlated well (r = 0.8269; p < 0.0001) and had identically acceptable intra-observer repeatability (coefficients of variation = 4.09% for MMVBP and 3.86% for PMD). Four dogs (10%) had a TOD score of 3 by one device but scored <3 with the other.Conclusion: A good agreement and correlation was observed between the PMD and the MMVBP, suggesting that both devices can be used interchangeably for assessment of SBP in conscious dogs. The wide LoA observed between both devices was most likely associated with intraindividual variability in SBP over time

TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines

A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-012-1217-5) contains supplementary material, which is available to authorized users

Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.

We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms

A preliminary investigation of additive manufacture to fabricate human nail plate surrogates for pharmaceutical testing

In vitro permeation studies using nail clippings or nail plates are commonly used in the development of transungual formulations. However, there are ethical, safety and cost issues associated with sourcing such tissues. Herein, we describe a preliminary approach is described for the design and manufacture of a human nail model surrogate based on 3D printing. To evaluate these 3D printed constructs, nails were mounted in conventional glass Franz cells and a commercial antifungal lacquer formulation containing ciclopirox olamine was applied daily to the surrogate printed surfaces for a period of 14 days. On days 8 and 14, the surfaces of the 3D printed nails were washed with ethanol to remove excess formulation. Confocal Raman spectroscopy (CRS) was used to profile the drug in the 3D printed nail. At the end of the Franz cell studies, no drug was observed in the receptor phase. CRS studies confirmed penetration of the active into the model nails with reproducible depth profiles. Our ongoing work is focused on synthesising commercial and non-commercial printable resins that can replicate the physical and chemical characteristics of the human nail. This will allow further evaluation of actives for ungual therapy and advance the development of the surrogate nail tissue model

Staphylococcus aureus Activates the NLRP3 Inflammasome in Human and Rat Conjunctival Goblet Cells

The conjunctiva is a moist mucosal membrane that is constantly exposed to an array of potential pathogens and triggers of inflammation. The NACHT, leucine rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) is a Nod-like receptor that can sense pathogens or other triggers, and is highly expressed in wet mucosal membranes. NLRP3 is a member of the multi-protein complex termed the NLRP3 inflammasome that activates the caspase 1 pathway, inducing the secretion of biologically active IL-1β, a major initiator and promoter of inflammation. The purpose of this study was to: (1) determine whether NLRP3 is expressed in the conjunctiva and (2) determine whether goblet cells specifically contribute to innate mediated inflammation via secretion of IL-1β. We report that the receptors known to be involved in the priming and activation of the NLRP3 inflammasome, the purinergic receptors P2X4 and P2X7 and the bacterial Toll-like receptor 2 are present and functional in conjunctival goblet cells. Toxin-containing Staphylococcus aureus (S. aureus), which activates the NLRP3 inflammasome, increased the expression of the inflammasome proteins NLRP3, ASC and pro- and mature caspase 1 in conjunctival goblet cells. The biologically active form of IL-1β was detected in goblet cell culture supernatants in response to S. aureus, which was reduced when the cells were treated with the caspase 1 inhibitor Z-YVAD. We conclude that the NLRP3 inflammasome components are present in conjunctival goblet cells. The NRLP3 inflammasome appears to be activated in conjunctival goblet cells by toxin-containing S. aureus via the caspase 1 pathway to secrete mature IL1-β. Thus goblet cells contribute to the innate immune response in the conjunctiva by activation of the NLRP3 inflammasome