Antioxidants

Nitric oxide metabolism-based protein post-translational modifications, such as reversible S- nitrosylation, have been at the pinnacle of plant redox research owing to their significant correlation with seed dormancy, interaction with other signaling molecules, plant development and metabolism, biotic and abiotic plant stress responses, immune defense responses against plant pathogens, and senescence. The rapid interconversion of reactive nitrogen species, the abrogation of nitric oxide homeostasis by exogenous supplementation of NO donors and scavengers, the lack of spatio-temporal specificity of NO signaling, and the limited bioavailability or assay sensitivity for detection often limit the effectiveness of identifying and characterizing S-nitrosothiols in plants. Hitherto unknown, we report the first experimental evidence of the total in vivo S-nitrosoproteome in Oryza sativa L. subsp. indica, comprising 134 PSNOs, enriched with 169 putative sites susceptible to S-nitrosylation, without any exogenous supplementation of NO donors. In the present study, mercuric salt- driven facile decomposition of S-nitrosoproteins in the presence of nitrone spin trap 5,5- dimethyl-1-pyrroline N-oxide, resulting in the synthesis of DMPO-nitrone adducts with PSNO-derived protein thiyl radicals in O. sativa, has been demonstrated as an efficient and novel strategy for characterizing the PSNOs using mass spectrometry analysis. The evidence of physiological levels of PSNOs was further re-examined in a bi-directional qualitative and quantitative approach involving the 2,3-diaminonaphthalene assay in tandem with fluorescence-based visualization and fluorometric quantification. In silico analyses, involving both functional enrichment and pathway prediction analyses, have furthermore revealed unique protein-protein interaction networks and signaling pathways among the S- nitrosoproteome candidates and their predictable physiological roles in O. sativa indica, awaiting further in vitro validation for their functional correlation in response to S- nitrosylation. In conclusion, the present study provides novel evidence of nitric oxide signaling in rice cultivars under physiological conditions, bringing new insights into the potential in vivo transnitrosylation of regulatory or active-site cysteine thiols.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in older adults. It is characterised by early retinal pigment epithelium (RPE) dysfunction followed by progressive photoreceptor degeneration. Cigarette smoking is a major environmental risk factor for AMD, and hydroquinone (HQ), a redox-active cigarette smoke component, induces oxidative stress and apoptosis in RPE cells. To analyse how RPE stress contributes to photoreceptor degeneration, we employed a retinal co-culture model composed of human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells in conjunction with porcine neuroretina explants. Exposure to HQ induced oxidative stress in iPSC-RPE cells as well as retinal photoreceptors (RPR), resulting in apoptosis, executed at least in part by caspase activation. Concomitantly, HQ caused endoplasmic reticulum (ER) stress (ERAD) in RPR followed by their degeneration, evidenced by reduced outer nuclear layer (ONL) rows and shortened RPR outer segments (OS). Based on earlier results, which suggest a perturbation of proteostasis due to HQ, we tested whether ML240, a bona fide inhibitor of valosin-containing protein (VCP), would influence the degree of degenerative activities. ML240 did not prevent HQ-induced apoptosis in iPSC-RPE cells. However, it significantly preserved photoreceptor integrity, retaining OS length and cone density in HQ-stressed co-cultures. Proteomic analysis suggested that ML240 reshapes stress response patterns of the HQ-exposed neuroretina, as evidenced by a reduction in ERAD-associated markers, increased levels of antioxidant response proteins, and the preservation of cytochrome c enrichment in photoreceptor inner segments, which indicates improved mitochondrial integrity consistent with the observed preservation of photoreceptor structure. Together, these findings establish the iPSC-RPE/neuroretina co-culture as a platform to analyse pathophysiological features of AMD, dissect cell type-specific retinal responses to environmental stress and test neuroprotective pharmacological approaches to protect photoreceptors in oxidative stress-associated retinal degeneration.

Mitochondrial dysfunction and oxidative stress represent two interconnected, primary causes for Diabetic Neuropathy (DN); however, the majority of currently available anti-diabetic therapies have focused on glucose control as opposed to neurodegenerative downstream effects. Corilagin, is an ellagitannin having high anti-oxidant properties; however, it has not been evaluated against hyperglycemia induced neuronal injury. The present study demonstrates the ability of Corilagin to protect against mitochondrial dysfunction via models of diabetic nephropathy and cerebral ischemia. High glucose (50 mM, 24 hr) was utilized to induce diabetes like conditions in the SH-SY5Y human neuroblastoma Cell Line. High glucose induced significant decreases in cell viability, increases in intracellular and mitochondrial reactive oxygen species, depletion of reduced glutathione reserves, induces apoptosis, and causes mitochondrial depolarization and fragmentation. Corilagin pre-treatment attenuated each of these high-glucose induced effects by protecting against mitochondrial membrane potential loss and maintaining mitochondrial network morphology while reducing apoptotic cell fraction relative to glucose alone. Additionally, these protective effects were accompanied by restoration of AMPK phosphorylation and up-regulation of SIRT1, PGC1 and TFAM, components that are part of the principal signaling pathway that regulates mitochondrial biogenesis; therefore, therefore, this pathway may contribute mechanistically to the cyto-protective effect of Corilagin. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/740444v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1be4a92org.highwire.dtl.DTLVardef@11d6e9org.highwire.dtl.DTLVardef@1346757org.highwire.dtl.DTLVardef@16c9f1e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Proposed mechanism underlying the neuroprotective effects of Corilagin against high glucose-induced mitochondrial dysfunction.High glucose suppresses AMPK phosphorylation, leading to downregulation of the SIRT1-PGC-1-TFAM signaling axis, increased intracellular and mitochondrial reactive oxygen species (ROS), glutathione depletion, apoptosis, mitochondrial depolarization, and mitochondrial fragmentation. Corilagin pretreatment restores AMPK activation and the downstream SIRT1-PGC-1-TFAM pathway, thereby reducing oxidative stress, preserving intracellular glutathione, preventing apoptosis, maintaining mitochondrial membrane potential, and protecting mitochondrial network integrity. C_FIG

Dehydroascorbic acid (DHA), the oxidized form of vitamin C, exhibits specific toxicity against KRAS-mutant cancer cells; however, the early metabolic reprogramming mechanisms leading to cell death remain incompletely understood. In this study, we reveal the non-linear, dose-dependent response of the intracellular metabolome during the ultra-early phase of 1-hour DHA exposure. Under 1 mM DHA exposure, an early significant accumulation of the primary catabolite erythrulose (p = 0.030) and moderate consumption of glutamate (p = 0.0086) were observed, while core TCA cycle homeostasis and cell viability were preserved. In contrast, under 10 mM DHA exposure, an acute and profound alteration of the metabolic profile was observed. Along with the significant accumulation of glycerate, indicative of a glycolytic blockade, and the terminal peroxidation product oxalate, core TCA cycle intermediates such as -ketoglutarate and malate showed rapid depletion (Log2FC < -2.0, p < 10-7). Furthermore, coupled with this acute energy depletion, a statistically significant accumulation of free methionine (Log2FC = 1.14, p = 0.011), which was below the limit of detection in the control, was identified alongside profound depletions of glutamate (p < 0.001) and cysteine (p = 0.0027). These results strongly suggest that the initial response to high-dose DHA is not merely a linear accumulation of oxidative stress, but rather an "abrupt metabolic switch" characterized by rapid TCA cycle dysfunction followed by the early signature of a methionine trap.

All-trans-retinal (atRAL) is a photoreactive aldehyde generated during visual pigment regeneration, and impaired atRAL clearance contributes to oxidative stress and retinal degeneration. Retinol dehydrogenase 12 (RDH12) reduces atRAL to all-trans-retinol, yet the temporal mechanisms linking its upstream enzymatic activity to downstream cellular stress adaptations remain poorly defined. Here, we utilized stable RDH12-expressing HEK293T cells to match proteomic and lipidomic profiles and performed cell viability assays to map the transition from acute atRAL stress to cellular recovery. RDH12 expression increased cellular tolerance to retinaldehyde-induced cytotoxicity. Acute atRAL exposure engaged the supply arm of the KEAP1-NRF2 program with increased HMOX1, SLC7A11, GCLC, MGST2, and MGST3, while the terminal glutathione peroxidase arm declined. Acute exposure was accompanied by membrane remodeling, indicated by increased glycerophospholipid enzyme abundance and increased stress-signaling ceramides and lysophosphatidylcholines. Concurrently, acute stress induced upregulation of the transferrin receptor (TFRC) and SLC11A2, which influence intracellular iron flux and enable Fenton chemistry that can lead to oxidative stress. During recovery, RDH12-expressing cells exhibited a distinct post-atRAL molecular state characterized by increased abundance of phosphatidylcholines (PCs) and ether-linked PCs, and proteome changes associated with cytoskeletal organization and antioxidant processes. Furthermore, we performed a pharmacological rescue with Ferrostatin-1, which indicated that atRAL-induced cytotoxicity in RDH12-expressing cells is dependent, in part, on lipid radical propagation and lipid peroxidation, supporting ferroptosis-regulated cell death. Collectively, the multiomics and biological findings establish a novel temporal sequence for retinaldehyde toxicity, showing that RDH12 is a critical component of upstream metabolic defense for acute aldehyde clearance, late-stage lipid detoxification, and adaptive membrane lipid homeostasis.

Background: Selenium is a vital trace element involved in antioxidant defence and cardiovascular health. Although selenium deficiency is implicated in cardiomyopathies, its early cardiac effects and underlying mechanisms remain poorly defined. Methods: C57BL6/Njr mice were fed either a selenium deficient or control diet for 12 weeks. Systemic selenium status, cardiac function by echocardiography, left ventricular (LV) transcriptomic profiles, redox balance, and circadian pathway markers were assessed, including sex-specific analyses. Results: Selenium deficiency reduced plasma selenium levels without inducing overt cardiac hypertrophy or fibrosis. Echocardiography showed preserved ejection fraction and fractional shortening but reduced global longitudinal strain, indicating early systolic dysfunction. Cardiac stress markers were increased predominantly in male mice. Left ventricular RNA sequencing revealed enrichment of pathways related to cardiac remodelling, redox regulation, mitochondrial function, and circadian rhythm. Additional protein and metabolic analyses supported sex-specific redox circadian alterations, with males showing a more pronounced stress response profile. Conclusions: Dietary selenium deficiency induces early myocardial dysfunction and molecular remodelling before overt cardiac failure. These changes are associated with redox and circadian pathway disruption and show sex specific features, suggesting that selenium contributes to cardiac homeostasis through sex dependent redox circadian regulation.

Animals experience stressors in a variety of contexts that result in activation of neuroendocrine and cellular stress responses. Release of stress hormones can disrupt or restore redox homeostasis, and the resulting changes in oxidative states, physiology and behavior vary by an individuals stress coping style. However, oxidative stress can also directly modulate neuroendocrine stress signaling. To what extent individual differences in brain antioxidant levels alter behavioral stress levels is not well understood. The present study investigated how N-acetylcysteine amide (NACA), an antioxidant and glutamate-modulating compound, regulates stress behavior across zebrafish (Danio rerio) with different stress coping styles (proactive, reactive). Following 24-hour exposure to NACA or control conditions, we quantified individual and composite stress behaviors using a Light-Dark Test (LDT). As expected, both proactive fish and NACA-treated fish showed significantly lower stress behaviors compared to reactive and control animals, respectively. Notably, stress-reducing effects of NACA were only seen in those with a reactive stress coping style. Overall, our data suggest that antioxidant mechanisms (e.g., glutathione system) may be key in facilitating the distinct behavioral and physiological responses to stressors that characterize alternative stress coping styles. The results underscore how individual differences in stress coping style and redox state can influence behavioral responses to stress.

Cloudberry (Rubus chamaemorus L.)-derived nanovesicles (NVs) represent a promising but still poorly characterized class of plant-derived vesicles with potential relevance for skin-related applications. Here, we isolated cloudberry fruit-derived NVs and investigated their physicochemical and molecular properties, cellular uptake, cytocompatibility, and functional effects in human dermal fibroblasts (HDF) and HaCaT keratinocytes. Nanoparticle tracking analysis and transmission electron microscopy confirmed a nanosized vesicle preparation with characteristic round morphology, while protein quantification supported reproducible isolation of NV-associated material. In vitro, cloudberry NVs showed concentration-dependent effects on cell viability and proliferation, with lower doses being better tolerated. Labelled NVs were internalized by both HDF and HaCaT cells in a time-dependent manner. Under oxidative stress conditions, cloudberry NVs reduced H2O2-induced senescence-associated {beta}-galactosidase staining in HDFs and exerted cytoprotective effects in both cell lines, alongside measurable cell-free antioxidant activity in the DPPH assay. In scratch wound-healing assays, cloudberry NVs modulated wound closure in a dose-dependent manner, with the lowest tested concentration showing the most favorable response. UHPLC-MS/MS-based proteomics and metabolomics further indicated the presence of diverse secondary metabolites and stress-related protein cargo. Together, these results support the view that cloudberry-derived NVs are biologically active plant nanovesicles with potential utility in skin-related regenerative applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/741293v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@c9094dorg.highwire.dtl.DTLVardef@81a1b5org.highwire.dtl.DTLVardef@9fafe2org.highwire.dtl.DTLVardef@1d421d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

The participation of sulfhydryl or thiol functions in a multitude of protein posttranslational modifications, although reflects on the redox versatility of cysteine residues, but their assessment in a dynamic cellular milieu involving the facile inter-conversion of SH to SSG, S-S, SNO, and S-R has been overwhelmingly difficult despite theirimplications in protein folding, enzyme structure and function, signalling and detoxification pathways, and pathophysiological ramifications.The current methodology, in contrast to a wide variety of cumbersome and prolonged techniques,repurposes the conventional DTNB assay for a hassle-free qualitative and quantitative analysisof redox-modified single or multiple susceptible thiol residues of cysteines in pure proteins as well as in a complex mixture of proteins.In this study, we document the thiol content, bearing the susceptibility to undergo reversible, oxidative thiol modifications, utilizing reverse DTNB assay in cell-free lysates and purified proteins that might provide a possible framework for dissecting the physiological phenomena behind the concealment of the susceptible cysteines through their redox-modified forms.

Background: Macrophages play a central role in systemic iron homeostasis by recycling iron from senescent erythrocytes through the only known cellular iron exporter, ferroportin (Fpn). Previous studies have shown that Fpn localizes to membrane microdomains, or lipid rafts, which are important for its function and regulation by hepcidin. Iron strongly promotes the enrichment of Fpn in lipid rafts. However, the proteomic composition of these domains during iron overload remains largely unexplored. Objective: This study aimed to characterize iron-induced changes in the macrophage lipid raft proteome in order to identify potential functional partners of Fpn and better define the cellular mechanisms involved in the response to iron overload. Methods: Quantitative iTRAQ-based proteomic analysis was performed on detergent-resistant membranes (DRMs) isolated by iodixanol density-gradient ultracentrifugation from J774a.1 macrophages treated with iron-NTA (FeNTA). Key proteomic observations were further validated in both J774a.1 cells and bone marrow-derived macrophages (BMDMs) using western blot, cell-surface biotinylation, and confocal immunofluorescence microscopy. The regulatory role of the transcription factor Nrf2 was also assessed using Nrf2-knockout mouse models. Results: In addition of Fpn, our lipid raft proteome identified 79 proteins significatively upregulated upon iron treatment, including, antioxidant proteins like the peroxiredoxin-1 (Prdx1), the glucose-6-phosphate dehydrogenase X-linked (G6pdx), as well as heme oxygenase-1 (Hmox1). Bioinformatic and functional analyses indicated that this response is associated with nuclear translocation of Nrf2, which is required for the transcriptional induction of Fpn and Hmox1. Whereas Fpn was detected in lipid rafts under basal conditions, Hmox1 was recruited to these domains only after iron or heme treatment. Confocal microscopy and cell-surface biotinylation assays confirmed that Hmox1 and Fpn colocalize at the plasma membrane, particularly within caveolae, specialized invaginated lipid raft domains. Conclusions: Iron or heme overload induces a major remodeling of the macrophage membrane, leading to the formation of specialized lipid raft platforms enriched in antioxidant and iron handling proteins. The coordinated localization of Hmox1 and Fpn at the cell surface suggests a coupled mechanism linking heme catabolism to iron export. This organization may help limit intracellular iron accumulation and protect the plasma membrane from iron-mediated lipid peroxidation.

Anaerobic ammonium-oxidizing (anammox) bacteria are essential players in the global nitrogen cycle, responsible for converting ammonium and nitrite directly to nitrogen gas. Anammox bacteria have unique features such as a specialized cellular compartment - the anammoxosome. Candidatus (Ca.) Brocadia pituitae genome, as other anammox bacteria, encodes for a diversity of hydroxylamine oxidoreductase (HAO) paralogs, often pointed out as the enzymes responsible for most of the reactions of the anammox cycle. One of this Ca. B. pituitae HAO paralogs is an 840-amino acids protein, named here as BpMHAO, that stands out for its unprecedented domain organization, which includes a multicopper oxidase-like (MCo-like) domain followed by the HAO-like one. Sequence and structural analyses classified this MCo-like domain as homologous to the small laccase family. Spectroscopic characterization revealed a distinct UV-visible spectrum, tentatively assigned to the T3 center, whereas the EPR spectra confirmed the presence of T1, T2 and T3 copper centers. Enzymatic studies demonstrated limited laccase and oxygen-dependent ferroxidase activities. On the other hand, enzymatic assays performed in cell extracts from Escherichia coli and Shewanella oneidensis, harbouring the recombinant HAO-like domain, exhibited a robust hydroxylamine reductase activity using methyl viologen as the electron donor. Our results showed that the BpMHAO potentially plays a role in the anammox process/reactions by converting hydroxylamine into hydrazine. This feature can be relevant to anammox bacteria either by i) mitigating unwanted hydroxylamine, obtained by incorrect formation of this compound, by converting it into hydrazine and enabling its use in the anammox reaction or ii) using hydroxylamine from the outside medium as a substitute for ammonium, delivering hydrazine directly to the last step of the cycle, short-circuiting its first steps.

Kakadu plum (Terminalia ferdinandiana), an Australian native fruit, is among the richest known dietary sources of vitamin C and hydrolysable tannins, yet its capacity to protect the intestinal epithelium against oxidative stress remains largely unexplored. This study optimised the extraction of bioactive compounds from freeze-dried Kakadu plum powder and evaluated their antioxidant activity using both chemical and cellular antioxidant in vitro assay. Phenolic compounds were extracted using three solvents (water, 80% ethanol, and 80% methanol) combined with shaking, ultrasound, or microwave assistance. Solvent, rather than processing technique, was the dominant determinant of antioxidant capacity: ethanol and methanol maximised total phenolic content, total flavonoid content, and DPPH radical-scavenging activity, whereas water extracts showed the highest ferric-reducing antioxidant power. Twenty-four phenolic compounds identified by HPLC-ESI-QTOF-MS/MS were mapped by network pharmacology to nine core oxidative-stress targets, and cross-species molecular docking predicted conserved binding of key phenolics to canine orthologs of PTGS2 and MMP2. In an H2O2-induced oxidative-stress in vitro cell model using canine small intestinal epithelial cells, both water (less than 25 ug/mL) and ethanol (less than 250 ug/mL) extracts significantly suppressed intracellular reactive oxygen species (ROS) in a dose-dependent manner, with the ethanol extract effective across a wider concentration range. This work demonstrated that Kakadu plum extract could be a promising natural, multi-target antioxidant ingredient for canine intestinal health, and provided a reference for future in vivo research.

Highly reactive aldehydes are generated during metabolic processes in the body, and their detoxification is essential for maintaining cellular homeostasis. Hexadecenal, a long-chain fatty aldehyde, is formed during the sphingolipid degradation pathway from the lipid mediator sphingosine-1-phosphate (S1P). However, the cytotoxicity resulting from dysregulation of hexadecenal metabolism is still unclear. To elucidate the effects of impaired hexadecenal metabolism, we analyzed the function of ALDH3A2, an aldehyde dehydrogenase in humans. Our results revealed that ALDH3A2 enzymatic activity is crucial for the suppression of DNA damage, particularly interstrand DNA crosslinks, upon S1P exposure. Furthermore, we demonstrated that hexadecenal accumulation promotes cell death accompanied by the activation of cellular stress responses and morphological abnormalities in the endoplasmic reticulum. These findings suggest that ALDH3A2 functions as a metabolic safeguard to suppress DNA damage and cell death in response to the enhanced metabolic flux of hexadecenal.

Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant utilized as a preservative in many products from foods to cosmetics. Population-based studies have detected BHT in the vast majority (>90%) of human specimens (serum, urine, fingernails) as well as home dust samples. Thus, BHT constitutes a ubiquitous environmental contaminant without obvious adverse health effects to humans. In contrast, exposure of mice to a single intraperitoneal dose of BHT triggers distal epithelial damage including loss of gas-exchanging alveolar type 1 epithelial cells, thus providing an invaluable tool to study alveolar repair and transient fibrosis. Presently, the molecular basis of BHT lung toxicity remains unknown. To address this, mouse lung single cell transcriptomic data were used to identify cytochrome P450 2B10 (CYP2B10) in AT1 cells as a BHT-activating enzyme. In cell culture systems, expression of CYP2B10 leads to marked BHT sensitization consistent with bioactivation of BHT into a toxic quinone methide. Targeted and proteome-wide experiments identify BHT-induced protein alkylation, DNA damage response, stress kinase activation and intrinsic apoptosis in a CYP2B10-dependent manner. Structure-activity relationship studies reveal the necessity of para-methyl and ortho-t-butyl groups necessary for BHT bioactivation. Our findings elucidate the molecular basis by which BHT is converted from an innocuous antioxidant into a highly toxic quinone methide and identify the first mammalian enzyme known to catalyze BHT bioactivation. Further, CYP2B10-catalyzed BHT bioactivation may provide a strategy for future targeted cell ablation technologies or environmental remediation of BHT.

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

RationaleOxidative stress, resulting from an imbalance between the production of reactive oxygen species (ROS) and antioxidant defence mechanisms, disrupts cellular redox homeostasis and contributes to damage of biomolecules, including nucleic acids, proteins and lipids. Such disturbances can impair intracellular signalling pathways and have been implicated in the pathophysiology of female reproductive disorders. Nitroblue Tetrazolium (NBT) is a distinctive dye that assesses cellular redox activity in neutrophils through the detection of superoxide anion (O2-). However, the relationship between neutrophil-derived oxidative stress and women infertility remains to be fully elucidated. Aim of the study: We aimed to investigate the role of neutrophil oxidative stress in women infertility using a novel Nitroblue Tetrazolium (NBT) method developed in our laboratory. Materials and methodsBlood and serum specimens were obtained from a total of 100 Libyan women, comprising healthy fertile women (n = 21; controls) and infertile women (n = 79). Superoxide anion (O2-) generation in neutrophils was assessed using the novel NBT method, while malondialdehyde (MDA), a marker of lipid peroxidation, was determined using the thiobarbituric acid reactive substances (TBARS) assay. ResultsA significant increase in both NBT-reactivity levels and the percentage of NBT-positive neutrophils was observed in infertile women compared with healthy fertile controls (P < 0.0001). In addition, MDA levels were significantly higher in infertile women than in the control group, indicating enhanced lipid peroxidation. MDA levels were positively correlated with NBT-reactivity levels (r = 0.410, P = 0.0001) and the percentage of NBT-positive neutrophils (r = 0.21, P = 0.047). A significant positive correlation was also observed between NBT-reactivity levels and the percentage of NBT-positive neutrophils (r = 0.510, P = 0.0001), demonstrating a close association between neutrophil oxidative activity and lipid peroxidation in women infertility. ConclusionThe present study demonstrates the utility of a novel NBT method for detecting reactive oxygen species (ROS), a marker of oxidative stress, in neutrophils from both blood and serum specimens. The findings demonstrate increased neutrophil-derived oxidative stress in infertile women and suggest that this method may have potential as a diagnostic tool for the assessment of women infertility. ImpactThis study provides evidence that neutrophil-derived oxidative stress is significantly increased in women infertility and demonstrates the application of a novel Nitroblue Tetrazolium (NBT) assay for assessing oxidative stress in both blood and serum specimens. The significant associations between NBT-reactivity, NBT-positive neutrophils and malondialdehyde levels provide further insight into the contribution of oxidative stress to female reproductive pathophysiology. These findings support the potential utility of the novel NBT method as a simple and reliable diagnostic approach for evaluating oxidative stress in women infertility and provide a foundation for future studies investigating oxidative stress biomarkers in reproductive medicine.

The Spike protein (S-protein) of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV2) exhibits important and negative effects on human cells. Accordingly, the adverse effects of COVID-19 mRNA vaccines could be due to the presence of circulating spike and need to be mitigated. One of the biological activities of the Spike protein (and, indirectly, of the Spike-producing vaccines) is the high induction of apoptosis and pro-inflammatory gene expression in different cellular systems. We have recently demonstrated that Aged Garlic Extract (AGE) and its components S-allyl-Cysteine (SAC) and S1-propenyl-I-Cysteine (S1PC) are potent inhibitors of the expression of pro-inflammatory genes induced by treatment of the bronchial epithelial IB3-1 cells with the Spike RNA-based BNT162b2 vaccine. The main purpose of the present study was to determine (a) whether treatment of the IB3-1 bronchial epithelia cells with the BNT162b2 vaccine is associated with activation of the apoptotic pathway and (b) whether AGE, SAC and S1PC have any effect of therapeutic relevance on this experimental model system. We have exposed IB3-1 cells to increasing amounts of the BNT162b2 vaccine and, after 3 days of culture, apoptosis was assessed using the Annex V and the Caspase 3/7 apoptosis assays. The results obtained demonstrated highly significant increase of the percentage of apoptotic cells after treatment of IB3-1 cells with the BNT162b2 vaccine. The percentage of apoptotic cells was found to be significantly reduced in cell populations treated with the BNT162b2 vaccine in the presence of AGE, SAC and S1PC. The main conclusion of the results of our study is that Aged Garlic Extracts, and its components S-allyl-Cysteine and S1-propenyl-l-Cysteine are able to mitigate the pro-apoptotic effects of the Spike-producing BNT162b2 vaccine using the bronchial epithelial IB3-1 cell line as experimental model system. The efficacy of the reversion of BNT162b2 induced apoptosis suggests that AGE, SAC and S1PC should be considered for the development of protocols aimed at counteracting apoptosis induced by the Spike mRNA-based COVID-19 vaccines. This might be of potential clinical relevance, since one of the biological adverse effects of Spike protein (and, indirectly, of the Spike-producing vaccines) is the high induction of apoptosis in different cellular systems.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

Life evolved under broad-spectrum sunlight (300-3500 nm), but most modern light-emitting diodes (LEDs) emit within a restricted visible range (350-650 nm) leaving lighting environments that are short-wavelength dominant. We ask if chronic 450nm exposure dominant in LEDs alters metabolism and organ integrity in ageing mice environmentally exposed at 13 mW/cm{superscript 2} for 5 h/day over 40 weeks, at an intensity within the range of human environmental exposures. Mice exhibited progressive weight gain despite reduced food intake, with altered glucose tolerance and insulin sensitivity over time. White adipose tissue mass increased, while brown adipose tissue weight and UCP1 protein levels remained unchanged. Relative organ weights (heart, liver, kidney, testis) were each reduced by ~10%, with histopathology revealing fat deposition, hepatocellular degeneration, renal tubular damage, steatosis, and impaired testicular architecture. These findings demonstrate that chronic 450 nm light exposure is associated with metabolic alterations and multiorgan pathology in mice.

Purpose: Age is the leading factor for age-related macular degeneration (AMD), in which retinal pigmented epithelium (RPE) atrophy is a hallmark. The nuclear factor-erythroid 2-related factor 2 (Nrf2), a major anti-stress transcription factor, decreases in AMD, suggesting bioenergetic stress interacts with age during AMD pathogenesis. We profiled visual function and gene expression of the retina and RPE-choroid with Nrf2 deletion and aging. Methods: We tested 3mo and 14mo Nrf2 null (Nrf2 KO) and Nrf2 WT mice for optokinetic tracking (OKT) and dark-adapted electroretinography (ERG) responses. Eyes were harvested for retina outer nuclear layer (ONL) thickness analysis and retina and RPE-choroid cup RNA isolation for RNAseq profiling. Results: Visual acuity was reduced with aging with lower values in the Nrf2 KO vs Nrf2 WT comparison at 14mo. We observed aged-related reduction of a-, b-, and c-wave ERG amplitudes in both WT and KO mice. Age-related c-wave decline was exacerbated by Nrf2 loss. By RNAseq, two-way ANOVA of RPE-choroid samples revealed differentially expressed genes (DEGs) with age and genotype main effects, as well as DEGs with age and genotype interaction effects. Age DEGs were over-represented in pathways related to inflammation and phagocytosis. Genotype DEGs were associated with alterations in antioxidant and inflammatory processes. Retina RNAseq showed age and genotype DEGs. Conclusions: Ablation of Nrf2 exacerbates age-related declines suggesting that decrease in Nrf2 with aging has functional consequences for inflammation/resiliency. We provide retina and RPE-choroid bulk-RNAseq datasets from the same cohort of mice that can be interrogated to better understand degeneration of retina/RPE-choroid.