The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure

The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.

Effects of Sources or Formulations of Vitamin K3 on Its Stability during Extrusion or Pelleting in Swine Feed

Two studies were conducted to determine the stability of vitamin K3 (VK3) in swine diets during extrusion or pelleting. The two sources were menadione sodium bisulfite (MSB) and menadione nicotinamide bisulfite (MNB), and the three formulations were crystal micro-capsule formulation and micro-sphere formulation. The recovery of six types of VK3 in swine diets was investigated after extrusion at 100 °C or 135 °C in Experiment 1. The recovery of six types of VK3 was investigated when the diets were pelleted at 60 °C (low temperature; LT) or 80 °C (high temperature; HT) and the length to diameter ratios were 5.2:1 (low length to diameter ratio; LR) or 7.2:1 (high length to diameter ratio; HR) in Experiment 2. In Experiment 1, MNB recovery (72.74%) was higher than MSB recovery (64.67%) after extrusion, while recovery of VK3 of crystal (74.16%) was higher than the recovery of micro-capsule (65.25%) and micro-sphere (66.72%). The recovery of VK3 (70.88%) was higher when extruded at 100 °C than that at 135 °C (66.54%). In Experiment 2, MNB recovery (86.21%) was higher than MSB recovery (75.49%) after pelleting, while the recovery of VK3 of micro-capsule (85.06%) was higher than the recovery of crystal (81.40%) and micro-sphere (76.09%). The recovery of VK3 (75.50%) was lower after HTHR pelleting than LTLR (83.62%), LTHR (81.52%) or HTLR (82.76%) treatment. Our results show that MNB has greater stability than MSB. VK3 of crystal or VK3 of micro-capsule were recommended for extrusion or pelleting, respectively.

Oxidative Stress Response of Aspergillus oryzae Induced by Hydrogen Peroxide and Menadione Sodium Bisulfite

Oxidative stress response protects organisms from deleterious effects of reactive oxygen species (ROS), which can damage cellular components and cause disturbance of the cellular homeostasis. Although the defensive biochemical mechanisms have been extensively studied in yeast and other filamentous fungi, little information is available about Aspergillus oryzae. We investigated the effect of two oxidant agents (menadione sodium bisulfite, MSB, and hydrogen peroxide, H2O2) on cellular growth and antioxidant enzyme induction in A. oryzae. Results indicated severe inhibition of biomass and conidia production when high concentration of oxidants was used. Transcriptomic analysis showed an up-regulated expression of genes involved in oxidoreduction, such as catalase, glutathione peroxidase, and superoxide dismutase. In addition, it was observed that oxidative stress stimuli enhanced the expression of Yap1 and Skn7 transcription factors. Further, metabolomic analysis showed that glutathione content was increased in the oxidative treatments when compared with the control. Moreover, the content of unsaturated fatty acid decreased with oxidative treatment accompanying with the down-regulated expression of genes involved in linoleic acid biosynthesis. This study provided a global transcriptome characterization of oxidative stress response in A. oryzae, and can offer multiple target genes for oxidative tolerance improvement via genetic engineering.

Combined Effect of Bortezomib and Menadione Sodium Bisulfite on Proteasomes of Tumor Cells: The Dramatic Decrease of Bortezomib Toxicity in a Preclinical Trial

Tumor growth is associated with elevated proteasome expression and activity. This makes proteasomes a promising target for antitumor drugs. Current antitumor drugs such as bortezomib that inhibit proteasome activity have significant side effects. The purpose of the present study was to develop effective low-toxic antitumor compositions with combined effects on proteasomes. For compositions, we used bortezomib in amounts four and ten times lower than its clinical dose, and chose menadione sodium bisulfite (MSB) as the second component. MSB is known to promote oxidation of NADH, generate superoxide radicals, and as a result damage proteasome function in cells that ensure the relevance of MSB use for the composition development. The proteasome pool was investigated by the original native gel electrophoresis method, proteasome chymotrypsin-like activity—by Suc-LLVY-AMC-hydrolysis. For the compositions, we detected 10 and 20 μM MSB doses showing stronger proteasome-suppressing and cytotoxic in cellulo effects on malignant cells than on normal ones. MSB indirectly suppressed 26S-proteasome activity in cellulo, but not in vitro. At the same time, MSB together with bortezomib displayed synergetic action on the activity of all proteasome forms in vitro as well as synergetic antitumor effects in cellulo. These findings determine the properties of the developed compositions in vivo: antitumor efficiency, higher (against hepatocellular carcinoma and mammary adenocarcinoma) or comparable to bortezomib (against Lewis lung carcinoma), and drastically reduced toxicity (LD50) relative to bortezomib. Thus, the developed compositions represent a novel generation of bortezomib-based anticancer drugs combining high efficiency, low general toxicity, and a potentially expanded range of target tumors.