Shibo Sun

Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor, acting via the regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterized, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here we report a focused CRISPR–Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B ₂ ) as a modulator of ferroptosis sensitivity. We demonstrate that riboflavin supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. Our findings provide a rational strategy to modulate the FSP1–antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients, as well as their contributions to a cell’s antioxidant capacity.

Covalent inhibitors are an attractive targeting strategy that has expanded the development of degraders to target poorly druggable proteins including the E3 ligase RNF4. We show that RNF4 is a potential vulnerability of AML. High RNF4 expression levels correlate with poor patient survival and depletion of RNF4 results in increased sensitivity of AML cells to antileukemic drugs. Therefore, we aimed to develop chemical degraders (PROTACs) of RNF4 using a known covalent RNF4 ligand (CCW16), containing a chloro- N -acetamide group, as well as established E3 ligands targeting CRBN or VHL. However, while CCW16 and CCW16-derived PROTACs react potently with cysteines in recombinant RNF4, in cells, CCW16 forms covalent bonds with a large number of proteins, including peroxiredoxins. Consequently, CCW16 based PROTACs do not trigger degradation of RNF4, but induce the ferroptosis marker heme oxygenase-1 and impair cell viability in a distinct, RNF4-independent, ferroptotic cell death pathway. We hypothesize that other chloro- N -acetamide-containing E3 ligase ligands would also induce ferroptosis. Indeed, the RNF114 ligand EN219 also strongly induces ferroptosis, suggesting that ligands harboring this electrophile induce undesired off-target toxicity.

Milk thistle is a traditional medicinal herb. Silybin is a medicinal component found in the seed coat of milk thistle, which has liver-protective and anti-cancer properties. Conventional studies have focused on the extraction of silybin with organic reagents, which was inapplicable to the food industry. This study aims to develop a fermented milk containing silybin and protein from the milk thistle seeds. A three step procedure was developed, comprising homogenization of milk thistle seeds, NaHCO₃ heat treatment, and microbial fermentation. The silybin was characterized by high performance liquid chromatography, and the protein was quantified and electrophorized. It was found that the homogenization step was essential for the preparation of protein, and the NaHCO₃ heat treatment was the crucial step in obtaining silybin. The optimal NaHCO₃ treatment settings were 1% NaHCO₃, 60°C, and 3 h, and the optimal strains for microbial fermentation were L131 (Rummeliibacillus stabekisii) and RS72 (Lactobacillus plantarum). The silybin yield in the fermented milk reached 11.24–12.14 mg/g seeds, accounting for 72.6–78.4% of the total silybin in the milk thistle seeds, and the protein yield reached 121.8–129.6 mg/g seeds. The fermented milk had a slightly sweet yoghurt-like flavor and could be used as a dietary supplement for silybin and protein.

The black soldier fly (BSF), Hermetia illucens, has emerged as a promising species for waste bioconversion and source of antimicrobial proteins (AMPs). However, there is a scarcity of research on the element transformation efficiency and molecular characterization of AMPs derived from waste management. Here, food waste treatment was performed using BSF larvae (BSFL) in a C/N ratio of 21:1–10:1, with a focus on the C/N-dependent element bioconversion, AMP antimicrobial activity, and transcriptome profiling. The C-larvae transformation rates were found to be similar among C/Ns (27.0–35.5%, p = 0.109), while the N-larvae rates were different (p = 0.001), with C/N 21:1–16:1 (63.5–75.0%) being higher than C/N 14:1–10:1 (35.0–45.7%). The C/N ratio did not alter the antimicrobial spectrum of AMPs, but did affect the activities, with C/N 21:1 being significantly lower than C/N 18:1–10:1. The lysozyme genes were found to be significantly more highly expressed than the cecropin, defensin, and attacin genes in the AMP gene family. Out of 51 lysozyme genes, C/N 18:1 and C/N 16:1 up-regulated (p < 0.05) 14 and 12 genes compared with C/N 21:1, respectively, corresponding to the higher activity of AMPs. Overall, the element bioconversion efficiency and AMP expression can be enhanced through C/N ratio manipulation, and the C/N-dependent transcriptome regulation is the driving force of the AMP difference.

Industrial processing of raspberry juice and wine generates considerable byproducts of raspberry pomace. Ellagic acids/ellagitannins, being characterized by their antioxidant and antiproliferation properties, constitute the majority of polyphenolics in the pomace and are valuable for recovery. In the present study, we developed a novel procedure with sodium bicarbonate assisted extraction (SBAE) to recover ellagic acid from raspberry wine pomace. Key parameters in the procedure, i.e., sodium bicarbonate concentration, temperature, time and solid/liquid (S/L) ratio, were investigated by single factor analysis and optimized subsequently by Response Surface Methodology (RSM). Optimal parameters for the SBAE method here were found to be 1.2% (w/v) NaHCO₃, 1:93 (w/v) S/L ratio, 22 min and 100 °C. Under these conditions, the ellagic acid yield was 6.30 ± 0.92 mg/g pomace with an antioxidant activity of 79.0 ± 0.96 μmol Trolox eq/g pomace (DPPH assay), which are 2.37 and 1.32 times the values obtained by extraction with methanol–acetone–water solvent, respectively. The considerable improvement in ellagic acid extraction efficiency could be highly attributed to the reactions of lipid saponification and ellagitannin hydrolysis resulted from sodium bicarbonates. The present study has established an organic solvent-free method for the extraction of ellagic acid from raspberry wine pomace, which is feasible and practical in nutraceutical applications.

Cadmium (Cd) as a ubiquitous toxic heavy metal in the environment, causes severe hazards to human health, such as cellular stress and organ injury. Selenium (Se) was reported to reduce Cd toxicity and the mechanisms have been intensively studied so far. However, it is not yet crystal clear whether the protective effect of Se against Cd-induced cytotoxicity is related to selenoproteins in nerve cells or not. In this study, we found that Cd inhibited selenoprotein thioredoxin reductase 1 (TrxR1; TXNRD1) and decreased the expression level of TrxR1, resulting in cellular oxidative stress, and Se supplements ameliorated Cd-induced cytotoxicity in SH-SY5Y cells. Mechanistically, the detoxification of Se against Cd is attributed to the increase of the cellular TrxR activity and upregulated TrxR1 protein level, culminating in strengthened antioxidant capacity. Results showed that Se supplements attenuated the ROS production and apoptosis in SH-SY5Y cells, and significantly mitigated Cd-induced SH-SY5Y cell death. This study may be a valuable reference for shedding light on the mechanism of Cd-induced cytotoxicity and the role of TrxR1 in Se-mitigated cytotoxicity of Cd in neuroblast cells, which may be helpful for understanding the therapeutic potential of Cd and Se in treating or preventing neurodegenerative diseases, like Alzheimer’s disease (AD) and Parkinson’s disease (PD). Graphical abstract

Food colorants are widely used by humans in food production and preparation; however, their potential toxicity requires an in-depth analysis. In this study, five out of 15 commercial food colorants, namely, lutein, betanin, caramel, crocin and chlorophyll, significantly inhibited wild type selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1) in vitro. The hyperactive Sec⁴⁹⁸ residue of TrxR1 was targeted by those five colorants, which was confirmed by the site-directed mutagenesis of TrxR1. Furthermore, two colorants, chlorophyll and betanin, triggered the oligomerization of TrxR1. A chlorophyll-derived compound, chlorophyllin, irreversibly inhibited the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) reducing activity of TrxR1 with K_inact = 6.96 × 10⁻³ ± 0.49 × 10⁻³ µM⁻¹ min⁻¹. Moreover, chlorophyllin reduced the cellular TrxR activity, leading to reactive oxygen species (ROS) accumulation and, subsequently, promoting cancer cell death. In conclusion, this study might contribute to understand the food safety of commercial colorants and provide chemotherapeutic compounds by targeting TrxR1.

Biowaste treatment by black soldier fly larvae (BSFL, Hermetia illucens) has received global research interest and growing industrial application. Larvae farming conditions, such as temperature, pH, and moisture, have been critically examined. However, the substrate carbon to nitrogen ratio (C/N), one of the key parameters that may affect larval survival and bioconversion efficiency, is significantly less studied. The current study aimed to compare the nitrogen supplying effects of 9 nitrogen species (i.e., NH₄Cl, NaNO₃, urea, uric acid, Gly, L-Glu, L-Glu:L-Asp (1:1, w/w), soybean flour, and fish meal) during food waste larval treatment, and further examine the C/N effects on the larval development and bioconversion process, using the C/N adjustment with urea from the initial 21:1 to 18:1, 16:1, 14:1, 12:1, and 10:1, respectively. The food wastes were supplied with the same amount of nitrogen element (1 g N/100 g dry wt) in the nitrogen source trial and different amount of urea in the C/N adjustment trial following larvae treatment. The results showed that NH₄Cl and NaNO₃ caused significant harmful impacts on the larval survival and bioconversion process, while the 7 organic nitrogen species resulted in no significant negative effect. Further adjustment of C/N with urea showed that the C/N range between 18:1 and 14:1 was optimal for a high waste reduction performance (73.5–84.8%, p < 0.001) and a high larvae yield (25.3–26.6%, p = 0.015), while the C/N range of 18:1 to 16:1 was further optimal for an efficient larval protein yield (10.1–11.1%, p = 0.003) and lipid yield (7.6–8.1%, p = 0.002). The adjustment of C/N influenced the activity of antioxidant enzymes, such as superoxide dismutase (SOD, p = 0.015), whereas exerted no obvious impact on the larval amino acid composition. Altogether, organic nitrogen is more suitable than NH₄Cl and NaNO₃ as the nitrogen amendment during larval food waste treatment, addition of small amounts of urea, targeting C/N of 18:1–14:1, would improve the waste reduction performance, and application of C/N at 18:1–16:1 would facilitate the larval protein and lipid bioconversion process.

Cytosolic thioredoxin reductase (TXNRD1) is an important selenoprotein that participates in the reduction of thioredoxin and many other redox-related substrates. The enhancement of ROS production to cause cancer cell death is an effective anticancer strategy. Herein, we found that menadione substantially increased ROS generation via interaction with TXNRD1. To elucidate the mechanism behind this, various TXNRD1 mutant proteins were used to investigate the relationship between ROS production and the reaction between enzymes and menadione. A mutation at the C-terminal active site -GCUG of TXNRD1 to -GSSG or -GC, or the N-terminal active site C59S, C64S, or the deletion of the C-terminal 16 amino acid residues caused the loss of TXNRD1 activity needed for the reduction of menadione and therefore resulted in the loss of the ROS production ability of menadione. In contrast, the mutation of -GCUG to -GCCG resulted in an increase in the TXNRD1 activity towards the reduction of menadione, thus leading to an increase in ROS production. The co-treatment of the TXNRD1 inhibitor aurothioglucose and menadione could significantly alleviate the efficiency of ROS generation in vitro and increase the viability of A549 cells. Moreover, menadione could be reduced by the glutathione system and caused ROS production with less efficiency. These results demonstrate that TXNRD1 can serve as an effective source to generate ROS, which may provide a novel anticancer method based on the use of menadione.