Age-related hearing loss (ARHL) is an increasing and gradual sensorineural hearing dysfunction. Oxidative stress is an essential factor in developing ARHL; additionally, premature senescence of auditory cells induced by oxidative stress can produce hearing loss. Hydrogen peroxide (H2O2) represents a method commonly used to generate cellular senescence in vitro. The objective of the present paper is to study H2O2-induced senescence patterns in three auditory cell lines (House Ear Institute-Organ of Corti 1, HEI-OC1; organ of Corti, OC-k3, and stria vascularis, SV-k1 cells) to elucidate the intrinsic mechanisms responsible for ARHL. The auditory cells were exposed to H2O2 at different concentrations and times. The results obtained show different responses of the hearing cells concerning cell growth, β-galactosidase activity, morphological changes, mitochondrial activation, levels of oxidative stress, and other markers of cell damage (Forkhead box O3a, FoxO3a, and 8-oxoguanine, 8-oxoG). Comparison between the responses of these auditory cells to H2O2 is a helpful method to evaluate the molecular mechanisms responsible for these auditory cells’ senescence. Furthermore, this in vitro model could help develop anti-senescent therapeutic strategies for the treatment of AHRL.
Labeling of food allergens in accordance with legal regulations is important to protect the health of allergic consumers. The requirements for detecting allergens in foods involve adequate specificity and sensitivity to identify very small amounts of the target allergens in complex food matrices and processed foods. In this work, one hundred commercial samples were analyzed for walnut detection using three different methods: a sandwich enzyme-linked immunosorbent assay (ELISA) kit based on polyclonal antibodies, a direct ELISA using a recombinant multimeric scFv, and a real time PCR. The most sensitive method was real time PCR followed by sandwich ELISA kit and multimeric scFv ELISA. There was agreement between the three methods for walnut detection in commercial products, except for some heat-treated samples or those that contained pecan. The walnut ELISA kit was less affected by sample processing than was the multimeric scFv ELISA, but there was cross-reactivity with pecan, producing some false positives that must be confirmed by real time PCR. According to the results obtained, 7.0 to 12.6% of samples (depending on the analytical method) contained walnut but did not declare it, confirming there is a risk for allergic consumers. Moreover, there was one sample (3.7%) labelled as containing walnut but that tested negative for this tree nut. Genetic and immunoenzymatic techniques offer complementary approaches to develop a reliable verification for walnut allergen labeling.
Real-time PCR (polymerase chain reaction) has shown to be a very effective technology for the detection of food allergens. The protocol described herein consists on a real-time PCR assay targeting the plant ITS (Internal Transcribed Spacer) region, using species-specific primers and hydrolysis probes (Taqman) dual labeled with a reporter fluorophore at the 5' end (6-carboxyfluorescein, FAM) and a quencher fluorophore at the 3' end (Blackberry, BBQ). The species-specific real-time PCR systems (primers/probe) described in this work allowed the detection of different nuts (peanut, hazelnut, pistachio, almond, cashew, macadamia, walnut and pecan), common allergens present in commercial food products, with a detection limit of 0.1 mg/kg.
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