*4.12. Determination of Protein*

Proteins were estimated by the bicinchoninic acid (BCA) protein assay method [61], using bicinchoninic acid reagent.

## *4.13. Statistical Analysis*

Results were expressed as means ± SEM of *n* = 18 experiments (MD alone) or *n* = 22 experiments (MD plus *Coriolus*), each of which were performed, unless otherwise specified, in triplicate. Data were analyzed by one-way Analysis of Variance (ANOVA), followed by inspection of all differences by Duncan's new multiple-range test. Differences were considered significant at *p* < 0.05.

#### **5. Conclusions**

Brain cells, such as spiral ganglion neurons, possessing relatively weak endogenous antioxidant potential, show a particular need for activation of antioxidant pathways, which becomes a central prerequisite under conditions of oxidant insults, such those underlying not only the pathogenesis of MD but also acting in a broad range of age-associated diseases. Aging, in fact, is based on complex mechanisms and systemic processes, whose major gap remains insufficient knowledge about the proactive pathway shift from normal "healthy" aging to disease-associated pathological aging [75]. As a major complication of normal "healthy" aging, the increased risk of age-related diseases, such as cancer, diabetes mellitus, cardiovascular and neurodegenerative diseases, including cochleovestibular dysfunctions that can adversely affect the quality of life in general, with enhanced incidence of co-morbidities and mortality, should be considered.

We evaluated systemic oxidative stress and cellular stress response in MD patients in the absence and in the presence of treatment with a biomass preparation from *Coriolus versicolor*. It was concluded that systemic oxidative stress was reduced in MD patients treated with *Coriolus versicolor*, which was paralleled by a significant induction of vitagenes and by an increased plasma GSH vs. GSSG ratio. Vitagene up-regulation after *Coriolus versicolor* supplementation indicates a maintained response to counteract intracellular pro-oxidant status. In a contextualized global "omics" approach, the combination of redoxomics and lipidomics, being more stable than the metabolome and closer to the phenotype than the transcriptome, represents the most promising "omics" field enabling dissection and perhaps full comprehension at molecular and cellular level, of aging mechanisms and age-related processes.

Approaching the redox biology of the aging inner-ear system, as exploited in the present study, together with broadening of the potential of lipidomic analysis represents an innovative tool for monitoring at the omic level to the extent of oxidative insult and related modifications, allowing the identification of targeted antioxidative cytoprotective vitagene system proteins. The present study also highlights the importance of investigating MD as a convenient model of cochlear neurodegenerative disease.

**Author Contributions:** M.S. and P.D.M. equally contributed to this work. They were involved in experiment design, work execution and preparation of figures; M.L.O., C.A., D.C., A.T.S., and S.M. were involved in the drafting of this article; L.M. and V.C. designed, executed, and supervised the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** V.C. acknowledges support from Piano Ricerca Triennale-linea Intervento 2, University of Catania.

**Conflicts of Interest:** The authors declare no conflicts of interest.
