Voltage-gated sodium channels (VGSCs) are transiently expressed in cochlear hair cells before hearing onset and play an indispensable role in shaping spontaneous activity. In this study, we showed that Na ? currents shaped the spontaneous action potentials in developing mouse inner hair cells (IHCs) by decreasing the time required for the membrane potential to reach the actionpotential threshold. In immature IHCs, we identified 9 known VGSC subtypes (Nav1.1a-1.9a), among which Nav1.7a was the most highly expressed subtype and the main contributor to Na ? currents in developing hair cells. Electrophysiological recordings of two cochlea-specific Nav1.7 variants (CbmNav1.7a and CbmNav1.7b) revealed a novel loss-of-function mutation (C934R) at the extracellular linker between segments 5 and 6 of domain II. In addition, post-transcriptional modification events, such as alternative splicing and RNA editing, amended the gating properties and kinetic features of CbmNav1.7a (C934). These results provide molecular and functional characteristics of VGSCs in mammalian IHCs and their contributions to spontaneous physiological activity during cochlear maturation.

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Previous studies have reported that rearing infant rat pups in continuous moderate-level noise delayed the formation of topographic representational order and the refinement of response selectivity in the primary auditory (A1) cortex. The present study further verified that exposure to long-term moderate-intensity white noise (70 dB sound pressure level) from postnatal day (P) 12 to P30 elevated the hearing thresholds of infant rats. Compared with age-matched control rats, noise exposure (NE) rats had elevated hearing thresholds ranging from low to high frequencies, accompanied by decreased amplitudes and increased latencies of the two initial auditory brainstem response waves. The power of raw local field potential oscillations and high-frequency β oscillation in the A1 cortex of NE rats were larger, whereas the power of high-frequency γ oscillation was smaller than that of control rats. In addition, the expression levels of five glutamate receptor (GluR) subunits in the A1 cortex of NE rats were decreased with laminar specificity. These results suggest that the altered neural excitability and decreased GluR expression may underlie the delay of functional maturation in the A1 cortex, and may have implications for the treatment of hearing impairment induced by environmental noise.

BackgroundSolid tumors remain a challenge for chimeric antigen receptor T (CAR-T) cell therapy due to lack of tumor-specific antigens, often-hard-to-penetrate tumor structure, and hostile tumor microenvironment (TME) for T-cell activation and survival. Interleukin (IL)-2 is an essential cytokine central to the initiation and maintenance of T-cell-mediated immune responses, which is usually downregulated in TME. IL-2 has been shown to improve the effectiveness of various T-cell-based therapies for solid tumors. However, systemic administration of IL-2 has been shown to elicit immunosuppression through regulatory T cells and cause capillary leak syndrome, both of which limit its use in T-cell immunotherapies, such as CAR-T. To improve the resistance of CAR-T to TME and to enhance its persistence, expansion and efficacy in vivo, we designed novel second generation mesothelin (MSLN)-specific CAR constructs (MSLN-CAR-T-IL-2tb) that incorporate secretory form of IL-2 variants (IL-2tb). IL-2tb produced by MSLN-CAR-T-IL-2tb improves cell viability, expansion, and potency, and reduces immunosuppression which could also potentially stimulate endogenous polyclonal tumor-infiltrating lymphocytes in solid tumors.MethodsMSLN-CAR-T-IL-2tb and MSLN-CAR-T cells were generated by lentiviral transduction. To assess in vitro proliferation, CAR-T cells were repeatedly cocultured with MSLN-expressing tumor cell lines and CAR+ T cells were enumerated. CAR-T cell apoptosis, memory phenotype and exhaustion were monitored by flow cytometry at various time points. MSLN-CAR-T-IL-2tb and MSLN-CAR-T cell resistance to TME was tested in vitro. Cytotoxicity was determined using RTCA- or luciferase-based assays. CAR-T tumoricidal activity in vivo was evaluated in human cell line-derived xenograft models using severe immunodeficient mice.ResultsMSLN-CAR-T-IL-2tb and MSLN-CAR-T demonstrated comparable efficacies in short-term tumor killing assays in vitro against multiple tumor cell lines expressing varying levels of MSLN in the presence of exogenous IL-2. However, when cultured in the absence of IL-2, MSLN-CAR-T-IL-2tb was much longer-lasting than MSLN-CAR-T in terms of CAR-T cell viability, proliferation, and persistence. MSLN-CAR-T-IL-2tb was more cytotoxic against multiple MSLN-expressing tumor cells, including MDA-MB-231 and HCC70 (triple-negative breast cancers) and OVCAR-3 (ovarian cancer). Moreover, in multiple xenograft mouse models, MSLN-CAR-T-IL-2tb showed very potent and durable anti-tumor responses.ConclusionsMSLN-CAR-T cells expressing a secretory form of IL-2 variant were able to maintain long-term proliferation and cytotoxicity which could be partly due to the reduced immunosuppression in the TME. Autocrine and paracrine loops of IL-2 can further improve CAR-T functionality in solid tumor and could be a promising strategy for clinical application.Ethics ApprovalAll animal experiments were conducted in facilities accredited by the service providers’ Institutional Review Boards.