The process of gentamicin-induced hair cell damage includes the activation of oxidative stress processes. Sestrins, as stress-responsive proteins, protect cells against oxidative stress. Sestrins, particularly Sestrin-2, suppress excessive reactive oxygen species (ROS) accumulation and inhibit mammalian target of rapamycin complex 1 (mTORC1). Thus, we addressed the role of Sestrin-2 in the regulation of sensory hair cell survival after gentamicin exposure. Here, we show that Sestrins were expressed in the inner ear tissues, and Sestrin-2 immunolocalized in sensory hair cells and spiral ganglion (SG). The expression of Sestrin-2 was unchanged, and later downregulated, in gentamicin-treated explants from wild-type mice in vitro. Compared with wild-type mice, Sestrin-2 knockout mice exhibited significantly greater hair cell loss in gentamicin-treated cochlear explants. Significant downregulation of phosphorylation of AMP-activated protein kinase alpha (AMPKα) and upregulation of the 70-kDa ribosomal protein S6 kinase (p70S6K) were measured in wild-type cochlear explants exposed to gentamicin compared with their untreated controls. Such regulatory effect was not observed between explants from untreated and gentamicin-treated knockout mice. The gentamicin effect on mTOR signaling was rapamycin-sensitive. Thus, our data provide evidence that Sestrin-2 plays an important role in the protection of hair cells against gentamicin, and the mTOR signaling pathway appears to be modulated by gentamicin during hair cell death.

Exosomes are nanovesicles part of a recently described intercellular communication system. Their properties seem promising as a biomarker in cancer research, where more sensitive monitoring and therapeutic applications are desperately needed. In the case of head and neck squamous cell carcinoma (HNSCC), overall survival often remains poor, although huge technological advancements in the treatment of this disease have been made. In the following review, diagnostic and therapeutic properties are highlighted and summarised. Impressive first results have been obtained but more research is needed to implement these innovative techniques into daily clinical routines.

Extracellular vesicles (EVs) have recently come into the spotlight as potential cancer biomarkers. Isolation of pure EVs is complex, so wider use requires reliable and time-efficient isolation methods. In the present study, galectin-based magnetic glycan recognition particles, EXÖBead ® were investigated for their practicality as a novel EV isolation technique, exemplified here for squamous cell carcinoma of the head and neck. Analysis of the isolation method showed a high concentration of pure EVs with detection of specific EV markers such as CD9, CD63, CD81 and TSG101. No apolipoprotein A1 was shown in the isolates, indicating low contamination of this isolation technique compared with size exclusion chromatography. In addition, common leukocyte antigen (CD45), three HNSCC [epithelial cell adhesion molecule (EpCAM), pan-cytokeratin and programmed death-ligand 1 (PD-L1)] and PanEV markers (premixed CD9, CD63 and CD81 anti-bodies) were measured by bead-based flow cytometry (BFC). BFC revealed that CD45 Neg PanEV + , EpCAM + PanEV + and PD-L1 + PanEV + were significantly higher in tumor patients compared with healthy control plasma. CD45 Neg PanEV + and CD45 + PanEV + carrying two or three HNSCC biomarkers were also significantly higher in tumor patients compared with healthy controls (BFC). Comparison of the functional immunosuppression effect of eluted tumor patient plasma EVs from EXÖBead ® and commercial polyethylene glycol isolation showed a significant tumor-dependent increase in concentration of EVs. A peripheral blood mononuclear cell activation assay also showed that the T-cell functionality of tumor patient plasma EVs isolated with EXÖBead ® was preserved in vitro . In conclusion, isolation using galectin-based magnetic glycan recognition particles is a novel method for isolating plasma EVs with low lipoprotein contamination. Bead-based flow cytometry provided an easy way to understand EV subpopulations. EXÖBead ® therefore showed great potential as a new isolation tool with high throughput capacity that could potentially be used in a clinical setting.