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SummaryOxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5‐diaminofluorescein diacetate and 2′,7′‐dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2‐hydroxyestradiol, menadione, 4‐hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol‐peroxidase and, to a lesser extent, 2′,7′‐dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

Our previous studies have shown that p53 isoform expression is altered in breast cancer and related to prognosis. In particular, a high ∆40p53:p53α ratio is associated with worse disease-free survival. In this manuscript, the influence of altered Δ40p53 and p53α levels on the response to standard of care DNA-damaging agents used in breast cancer treatment was investigated in vitro. Our results revealed that a high Δ40p53:p53α ratio causes cells to respond differently to doxorubicin and cisplatin treatments. Δ40p53 overexpression significantly impairs the cells’ sensitivity to doxorubicin through reducing apoptosis and DNA damage, whereas Δ40p53 knockdown has the opposite effect. Further, a high Δ40p53:p53α ratio inhibited the differential expression of several genes following doxorubicin and promoted DNA repair, impairing the cells’ canonical response. Overall, our results suggest that the response of breast cancer cells to standard of care DNA-damaging therapies is dependent on the expression of p53 isoforms, which may contribute to outcomes in breast cancer.

Background Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan; however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound—ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc. Methods Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017–2020) was analyzed. Results GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months (P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not (P = .012). Conclusions This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug; however, GsONC201 was never used as a monotherapy.

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