Vesicular glutamatergic transmission in noise-induced loss and repair of cochlear ribbon synapses

Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3

Sensorineural correlates of failed functional recovery after natural regeneration of vestibular hair cells in adult mice

Vestibular hair cells (HCs) are mechanoreceptors that sense head motions by modulating the firing rate of vestibular ganglion neurons (VGNs), whose central processes project to vestibular nucleus neurons (VNNs) and cerebellar neurons. We explored vestibular function after HC destruction in adult Pou4f3+/DTR (DTR) mice, in which injections of high-dose (50 ng/g) diphtheria toxin (DT) destroyed most vestibular HCs within 2 weeks. At that time, DTR mice had lost the horizontal vestibulo-ocular reflex (aVORH), and their VNNs failed to upregulate nuclear cFos expression in response to a vestibular stimulus (centrifugation). Five months later, 21 and 14% of HCs were regenerated in utricles and horizontal ampullae, respectively. The vast majority of HCs present were type II. This degree of HC regeneration did not restore the aVORH or centrifugation-evoked cFos expression in VNNs. The failure to regain vestibular pathway function was not due to degeneration of VGNs or VNNs because normal neuron numbers were maintained after HC destruction. Furthermore, sinusoidal galvanic stimulation at the mastoid process evoked cFos protein expression in VNNs, indicating that VGNs were able to regulate VNN activity after HC loss. aVORH and cFos responses in VNNs were robust after low-dose (25 ng/g) DT, which compared to high-dose DT resulted in a similar degree of type II HC death and regeneration but spared more type I HCs in both organs. These findings demonstrate that having more type I HCs is correlated with stronger responses to vestibular stimulation and suggest that regenerating type I HCs may improve vestibular function after HC loss

Neptune Odyssey: A Flagship Concept for the Exploration of the Neptune–Triton System

The Neptune Odyssey mission concept is a Flagship-class orbiter and atmospheric probe to the Neptune-Triton system. This bold mission of exploration would orbit an ice-giant planet to study the planet, its rings, small satellites, space environment, and the planet-sized moon Triton. Triton is a captured dwarf planet from the Kuiper Belt, twin of Pluto, and likely ocean world. Odyssey addresses Neptune system-level science, with equal priorities placed on Neptune, its rings, moons, space environment, and Triton. Between Uranus and Neptune, the latter is unique in providing simultaneous access to both an ice giant and a Kuiper Belt dwarf planet. The spacecraft - in a class equivalent to the NASA/ESA/ASI Cassini spacecraft - would launch by 2031 on a Space Launch System or equivalent launch vehicle and utilize a Jupiter gravity assist for a 12 yr cruise to Neptune and a 4 yr prime orbital mission; alternatively a launch after 2031 would have a 16 yr direct-to-Neptune cruise phase. Our solution provides annual launch opportunities and allows for an easy upgrade to the shorter (12 yr) cruise. Odyssey would orbit Neptune retrograde (prograde with respect to Triton), using the moon's gravity to shape the orbital tour and allow coverage of Triton, Neptune, and the space environment. The atmospheric entry probe would descend in ~37 minutes to the 10 bar pressure level in Neptune's atmosphere just before Odyssey's orbit-insertion engine burn. Odyssey's mission would end by conducting a Cassini-like "Grand Finale,"passing inside the rings and ultimately taking a final great plunge into Neptune's atmosphere

Tailoring hydrogel degradation and drug release via neighboring amino acid-controlled ester hydrolysis

We present a versatile scheme to rationally modulate the hydrolysis rate of ester bonds in hydrophilic polymer networks via adjacent charged amino acids. As soluble model systems, two cysteine-bearing oligopeptides containing either positively charged arginine (GRCRGGRCRG, termed R-linker) or negatively charged aspartic acid (GDCDGGDCDG, termed D-linker) were linked to monomethoxy PEG-acrylate via Michael-type addition, and the hydrolysis rate of the conjugates was monitored using HPLC. A ca. 6-fold difference in hydrolysis kinetics of the conjugates was determined, positively charged arginine leading to an increased hydrolysis rate (t1/2 of 6.56 days vs. 36.1 days for the R- and D-linker containing conjugates, respectively). As a first step towards utilizing this concept to create tunable matrices for drug delivery and tissue engineering, the above peptides were crosslinked into hybrid hydrogels (R-gels and D-gels) by mixing with 4-arm PEG-acrylate at variable stoichiometric ratios. The physicochemical gel properties were characterized and gel degradation kinetics were quantified by monitoring the gel weight change over time at pH 7.4 and 37 °C. Differences in ester hydrolysis rates of individual chains translated into a ca. 12-fold difference in hydrogel degradation rate (R-gels: t1/2 = 7.53 days, D-gels: t1/2 = 86.6 days). Finally, the gel release kinetics of covalently linked bovine serum albumin (BSA) was also shown to be highly dependent on the charge of adjacent amino acids (R-gels: t1/2 = 3.32 days, D-gels: t1/2 = 32.1 days). With the availability of 20 natural amino acids as building blocks to modulate the chemical environment in close proximity of labile esters, we expect this work will provide a generalizable framework for the engineering of hybrid polymer-co-peptide gels with tunable and predictive degradation and drug release properties

Targeted genomic integration of a selectable floxed dual fluorescence reporter in human embryonic stem cells.

The differentiation of pluripotent stem cells involves transition through a series of specific cell states. To understand these cell fate decisions, the field needs improved genetic tools for the labeling, lineage tracing and selection of specific cell types from heterogeneous differentiating populations, particularly in the human embryonic stem cell (hESC) system. We used zinc finger nuclease technology to stably insert a unique, selectable, floxed dual-fluorescence reporter transgene into the AAVS1 locus of RUES2 hESCs. This "stoplight" transgene, mTmG-2a-Puro, strongly expresses membrane-localized tdTomato red fluorescent protein until Cre-dependent recombination causes a switch to expression of membrane-localized enhanced green fluorescent protein (eGFP) and puromycin resistance. First, to validate this system in undifferentiated cells, we transduced transgenic hESCs with a lentiviral vector driving constitutive expression of Cre and observed the expected phenotypic switch. Next, to demonstrate its utility in lineage-specific selection, we transduced differentiated cultures with a lentiviral vector in which the striated muscle-specific CK7 promoter drives Cre expression. This yielded near-homogenous populations of eGFP(+) hESC-derived cardiomyocytes. The mTmg-2a-Puro hESC line described here represents a useful new tool for both in vitro fate mapping studies and the selection of useful differentiated cell types

RAFT homo- and copolymerization of N-acryloyl-morpholine, piperidine, and azocane and their self-assembled structures

We present new polymeric amphiphiles derived from N-acryloyl derivatives of the cyclic secondary amines: morpholine, piperidine, and azocane polymerized by reversible addition -fragmentation transfer (RAFT) polymerization. Both homopolymerization and block copolymerization of N-acryloylmorpholine (AM), N-acryloylpiperidine (AP), and N-acryloylazocane (AH) were carried out. The block copolymeric amphiphiles, poly[(N-acryloylmorpholine)-block-(N-acryloylpiperidine)] (PAM-PAP) and poly[(N-acryloylmorpholine)-block-(N-acryloylazocane)] (PAM-PAH) were investigated, PAM being a hydrophile, and PAP and PAH being hydrophobes. Moreover, to compare PAM as a hydrophilic block with poly(ethylene glycol) (PEG), poly[(ethylene glycol)block-(N-acryloylpiperidine)] (PEG-PAP) was also formed. In all cases, the degree of polymerization was well-controlled and polymers were obtained in monomodal distributions. The macroamphiphile aggregates in water were reproducibly well-formed by dialysis with a size range between 10 and 70 nm as characterized by dynamic light scattering (DLS). The morphology of the aggregates was examined by transmission electron microscopy (TEM). All aggregates formed from PAM-PAP and PAM-PAH series, up to 0.76 and 0.85 hydrophobic weight fraction, respectively, revealed spherical micelles, whereas coexistence of spherical micelles and/or polymersomes was observed from PEG-PAP at a hydrophobic weight fraction of 0.91. From study of copolymer segregation behavior, PEG-PAP and PAM-PAH span the weak segregation region (WSR) as well as the strong segregation region (SSR), whereas PAM-PAP is positioned in the WSR, owing to the greater hydrophobicity of PAH than PAP. PAM yielded similar aggregation results to PEG when copolymerized with hydrophobic blocks. As a model drug, everolimus was loaded in PAM(0.15)-PAH(0.85) micelles. After loading the drug, the micelle hydrodynamic diameter was slightly increased from 43 +/- 0.1 to 52 +/- 1.8 nm. Everolimus was encapsulated with 60 +/- 7.8% of efficiency and was released over 3 wk in PBS (pH 7.4, 10 mM) at 37 degrees C

Soundfield hearing for patients with cochlear implants and hearing aids

There are several important issues that need to be discussed in order to fully understand and question the potential advantages associated with bilateral cochlear implants. Our approach in this chapter is to propose what we believe are the key questions involved in considering bilateral cochlear implantation

Cre expression mediates a fluorescence switch in undifferentiated mTmG-2a-Puro floxed reporter hESCs.

<p>(<b>A</b>) Flow cytometry showing changing numbers of tdTomato⁺ and eGFP⁺ RUES2 mTmG-2a-Puro cells at various timepoints after transduction with an EF1α-Cre lentivirus. (<b>B</b>) Quantitation of the fluorescence switch shown in panel A (n = 3 biological replicates). (<b>C</b>) Scatter plot of undifferentiated mTmG-2a-Puro cells treated with EF1α-Cre lentivirus and then selected with puromycin for 48 hours. Note that the resultant cultures were >99% eGFP⁺ by flow cytometry (i). Comparison of untreated (red) and EF1α-Cre treated, puromycin selected (blue) undifferentiated mTmG-2a-Puro cells (ii). Quantitation of flow cytometry data from three independent experiments in which cells were transduced with EF1α-Cre and puromycin selected (iii). (<b>D</b>) Phase contrast and fluorescent photomicrographs of undifferentiated mTmG-2a-Puro cells (i), mTmG-2a-Puro cells treated with EF1α-Cre lentivirus (ii), and mTmG-2a-Puro cells treated with EF1α-Cre and selected with puromycin (iii). Scale bars are 50 µm. Error bars represent +/− one standard error of the mean. (<b>E</b>) mTmG-2a-Puro cells transduced with EF1α-Cre lentivirus were immunostained with an anti-Cre recombinase antibody. Note that Cre (magenta) co-localizes only with eGFP⁺ cell nuclei. Scale bar is 10 µm.</p